The characteristics of capillary remodeling in cerebellar neurodegenerative diseases revealed through layered imaging and stereoscopic analysis

The characteristics of capillary remodeling in cerebellar neurodegenerative diseases revealed through layered imaging and stereoscopic analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The characteristics of capillary remodeling in cerebellar neurodegenerative diseases revealed through layered imaging and stereoscopic analysis Yayun Wang, Hui Liu, Ziwei Ni, Yuxuan Liu, Xintong Deng, Yun-Qiang Huang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5607179/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Neurodegenerative diseases refer to a group of clinical conditions characterized by progressive neuronal loss, resulting in impaired brain structural integrity and functional abnormalities. These diseases can lead to widespread cerebrovascular remodeling; however, the spatial remodeling features of capillaries with diameters ≤ 10 μm remain poorly understood, particularly with regard to changes in the relationship between neurons and capillaries. In this study, we first developed a double-fluorescent transgenic mouse model of cerebellar neurodegenerative disease (CBND), the PCKO Tomato Vessel Green mouse, in which Purkinje cells (PCs) in the cerebellum express red fluorescent protein, while the cerebrovascular system in the cerebellum expresses green fluorescent protein (with no differentiation between arteries and veins). Subsequently, we employed whole-brain clearing combined with the Amira/Imaris system to conduct three-dimensional layered imaging and computational analysis of the cerebrovascular network in both adult control and PCKO Tomato Vessel Green mice. A total of 181744 PCs and cerebrovascular vessels with a total length of 17.7363 meters, 266175 segments, and a total volume of 0.5314 mm³ were analyzed. Compared to the Control mice, PCKO Tomato Vessel Green mice exhibited a 93% reduction in count of PCs, a 97% reduction in total volume of PCs, a 69% reduction in cerebellar volume, a 58% decrease in total cerebrovascular vessel length, and a 52% decrease in total cerebrovascular vascular volume. Depth analysis of vessels with diameters ≤ 10 μm revealed a 55%, 58%, 58%, and 52% reduction in capillary volume, chord length, curved length, and tortuosity, respectively, with no statistical differences in node count or φ and θ directional values. Further volume fraction (VF) analysis revealed a 59% increase in capillary-cerebellum VF, while the PC-vessel VF, PC-capillary VF, and PC-noncapillary VF decreased by 95%, 95%, and 96%, respectively. Additionally, the shortest distance between PCs and cerebrovascular vessels decreased by 58%, while vessel-cerebellum VF and noncapillary-cerebellum VF showed no statistical differences. Our results indicated that while capillaries with diameters ≤ 10 μm were significantly lost, their vascular topology remained stable, with the distance between PCs and cerebrovascular vessels decreasing from 16 μm to 7 μm. This remodeling process is central to the pathogenesis of cerebrovascular changes in CBND. Furthermore, the increase in capillary-cerebellum VF and the decrease in PC-vessel VF may serve as biological markers for the early diagnosis of CBND. These findings provide a foundation for the early diagnosis and development of targeted therapies for CBND. Short abstract Cerebrovascular remodeling caused by neurodegenerative diseases can be used for early diagnosis, but its characteristics are unclear. Our research group first constructed PCKO Tomato Vessel Green mice with cerebellar neurodegenerative disease, and then adopted whole cerebellar transparency combined with Amira/Imaris system. A total of 181744 Purkinje cells with a total length of 17.7363 m, a total number of 266175 segments and a total volume of 0.5314 mm 3 were analyzed by three-dimensional stratified imaging and computational analysis. A total of 3.15 TB of data revealed that capillaries with diameters ≤ 10 μm were significantly lost although the vascular topology remained stable. Additionally, the distance between Purkinje cells and blood vessels decreased from 16 μm to 7 μm, identifying this as a central feature of neurovascular remodeling in cerebellar neurodegenerative disease. The increase of capillary-cerebellum volume fraction and the decrease of Purkinje cell-vessel volume fraction can be used as biological markers for the early diagnosis of neurodegenerative diseases. Biological sciences/Neuroscience/Neuro–vascular interactions Health sciences/Diseases/Neurological disorders/Neurodegenerative diseases Cerebellar neurodegenerative disease Purkinje cells Capillaries Topological structure Volume fraction. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Highlights Whole cerebellum clearing combined with the Amira / Imaris system was used to conduct three-dimensional layered imaging and computational analysis of the cerebellar vascular network in adult control mice and double-fluorescent transgenic mice with cerebellar neurodegenerative disease (CBND). The results showed significant loss of capillaries with diameters ≤ 10 μm, while their vascular topology remained stable. Additionally, the distance between Purkinje cells and blood vessels decreased from 16 μm to 7 μm, which was central to cerebrovascular remodeling in CBND. The findings indicated that an increase in capillary-cerebellum volume fraction and a decrease in Purkinje cell-vessel volume fraction could serve as biological markers for the early diagnosis of CBND. Introduction The function of the human brain is highly dependent on the vascular system, with the brain constituting only 2% of body weight but consuming 20% of cardiac output and 20% of oxygen and glucose 1 – 4 . The human cerebral blood vessels consist of a complex three-dimensional (3D) cerebrovascular network spanning 644 km, ensuring that the distance between each neuron and the nearest capillary is no more than 22–27 µm, for timely and effective oxygen delivery 1 , 5 . The relationship between the brain and blood vessels in mice follows the same pattern 5 , 6 . Neurodegenerative diseases are a group of clinical disorders characterized by progressive loss of neurons leading to impaired brain structural integrity and abnormal function, including Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia, Huntington's disease, traumatic brain injury, stroke, multiple sclerosis (MS), and amyotrophic lateral sclerosis, etc., which can cause extensive cerebral vascular remodeling 7 – 10 . In the AD mouse model, super-resolution ultrasound imaging showed that the length of cerebral blood vessels was shortened and the flow rate decreased 11 . Meanwhile, imaging results showed a significant decline in cerebrovascular reactivity and blood flow regulation in AD patients 12 – 14 . In addition, in patients with mild cognitive impairment, AD, dementia, MS, and PD, retinal imaging studies revealed vascular pathological changes such as reduced capillary blood flow, vascular Aβ deposition, and impaired blood-retinal barrier 15 , 16 . An increasing body of evidence suggests that dysfunction of the vascular system, especially dysregulation of the microvasculature, is expected to be a biomarker for the early diagnosis of neurodegenerative diseases. Neurovascular coupling (NVC) is the close interaction between neurons and the vascular system in the brain, with capillaries playing a key role 17 , 18 . Specifically, pericytes and microglia surrounding the capillaries could regulate the diameter and blood flow of capillaries through molecular mechanisms such as intercellular tunneling nanotubes and P2RY12 receptors, maintaining the stability of the cerebral microcirculation 19 . Optogenetic studies in mouse models also showed that although the contraction of pericytes was slow, it would consistently reduce the diameter of capillaries and decrease blood flow, which had profound effects on long-term cerebral blood flow regulation 20 . Although these findings have emphasized the core role of capillaries in NVC, serving not only as a platform for the exchange of substances between blood and nerve cells but also as a critical node for signal regulation 21 , it remains unclear how neurons at risk of progressive loss in the state of neurodegenerative diseases interact with their surrounding capillaries. However, most of existing researches have largely focused on the morphological and functional changes of larger blood vessels in the brain, while there is short of comprehensive exploration of changes at the capillary level. The reason for this lies in the lack of 3D imaging and computational analysis techniques for the fine structure of the cerebrovascular network. At present, the study of cerebrovascular network has usually used vascular casting, tissue clearing, microscopic imaging, topological data analysis and other methods to visualize and assess structural differences in the cerebrovascular system at the single microvascular level 1 , 22 – 27 . Furthermore, single-cell and spatial transcriptomics methods have revealed molecular differences between various cells in the brain, providing insights into the regional heterogeneity of the vascular system 28 , 29 . Recent studies have also used 3D bioprinting to construct various microvascular models for comprehensive analysis of vascular function 30 – 32 . These attempts have tried to analyze the remodeling of capillary network with a diameter of 10 µm, but they still fail to provide information on topological changes of capillaries, such as nodes, bends, and tortuosity. Consequently, there is a significant lack of research on the spatial location relationship between neurons and capillaries. Our group has previously successfully generated Drp1 (Dynamin-related protein 1) knockout mice in cerebellar Purkinje cells (PCs), which have typical characteristics of cerebellar neurodegenerative disease (CBND). To further address this issue, our team constructed a double-fluorescent CBND mouse model with targeted knockout (KO) of the mitochondrial fission protein Drp1 in PCs, named PCKO Tomato Vessel Green mice due to their expression of the red fluorescent protein tdTomato in PCs and green fluorescent protein ZsGreen in the cerebellar vascular system (arteries and veins couldn’t be distinguished). Subsequently, we used whole cerebellum clearing combined with the Amira/Imaris system to perform 3D hierarchical imaging and computational analysis on the cerebellar vascular network of adult Control mice and PCKO Tomato Vessel Green mice. A total of three levels of indicators were analyzed. The first-level indicators were the overall situation, including 5 indicators such as the number of PCs, total volume of PCs, total volume of the cerebellum, total length of cerebellar blood vessels, and total volume of cerebellar blood vessels. The second-level indicators distinguished noncapillaries with a diameter of greater than 10 µm and capillaries with a diameter of less than 10 µm, each including 7 indicators such as volume, chord length, curved length, tortuosity, number of nodes, φ direction value, and θ direction value. The third-level indicators were volume fraction (VF) and neuron-vascular distance, including 7 indicators such as capillary-cerebellum VF, PC-vessel VF, PC-capillary VF, PC-noncapillary VF, vessel-cerebellum VF, noncapillary-cerebellum VF, and the shortest distance between PCs and blood vessels. Our results indicated that capillaries with a diameter ≤ 10 µm were significantly lost, but their vascular topological structure remained stable. Moreover, the nearest distance between PCs and blood vessels decreased from 16.6 µm to 7.0 µm, which represented the core of cerebrovascular remodeling in CBND. Additionally, the increased capillary-cerebellum VF and the decreased PC-vessel VF might serve as biological markers for the early diagnosis of CBND. These findings provide valuable insights for the early diagnosis of CBND and the development of targeted therapies. Results Construction of a double-fluorescent transgenic mouse of PCKO Tomato Vessel Green to provide a tool for the study of CBND A double-fluorescent transgenic mouse model of CBND, PCKO Tomato Vessel Green , was constructed using Cre-LoxP technology. This model featured progressive loss of cerebellar PCs expressing red fluorescence, while the molecular layer vessels expressed green fluorescence, providing a tool for detailed 3D cerebrovascular network studies. As shown in Fig. 1 a, the research group first compared Pcp2 Cre mice purchased from the Jackson Laboratory (cerebellar PCs carrying Cre gene 33 ) with B6/JGpt-H11 em1Cin (CAG−LoxP−ZsGreen−Stop−LoxP−tdTomato) /Gpt (B6-G/R) mice purchased from the GemPharmatech Co., Ltd (the mice all endothelial cells express green fluorescence ZsGreen and carry LoxP units 34 ) hybrid. Figure 1 b showed that after validation by genotype identification, the resulting transgenic mice were designated PC Tomato Vessel Green transgenic mice. The confocal microscopy results in the top row in Fig. 1 c showed that PC Tomato Vessel Green transgenic mice cut Cre gene to make PCs carry red fluorescence tdTomato, while blood vessels still expressed green fluorescence ZsGreen. As shown in Fig. 1 a, PC Tomato Vessel Green mice were then cross-bred with Drp1 f/f mice purchased from Jackson's laboratory (the mouse had mitochondrial division protein Drp1 with LoxP units on both sides). Transgenic mice (named PCKO Tomato Vessel Green ) targeting PC-specific Drp1 knockout were obtained. Figure 1 b showed that Pcp2 Cre gene produced 567 bp bands, Loxp in Drp1 flox/flox produced 291 bp bands, and B6-G/R mouse Loxp gene produced 1465 bp bands, which verified the gene expression correctness of the two transgenic mice. The confocal microscopy results in the lower row in Fig. 1 c showed that PCKO Tomato Vessel Green mice's cerebellar PCs expressed red fluorescence while molecular layer vessels expressed green fluorescence, providing a tool for the fine study of 3D cerebellar vascular network. The cerebellum and PCs status of adult (2 - month - old) PC Tomato Vessel Green mice (Control) and PCKO Tomato Vessel Green mice (KO) were further compared. Figure 1 c showed that the cerebellum of PCKO Tomato Vessel Green mice still had the appearance of 10 lobules in the vermis. Figure 1 d analysis of the morphological data of vermis on the median sagittal surface showed that compared to Control mice, the cerebellar area in PCKO Tomato Vessel Green mice was reduced by 43%, from 7652719 ± 453169 µm² in Control mice to 4340231 ± 93977 µm² in KO mice ( P < 0.0001 ). The area of PCs somas decreased by 19%, from 261.80 ± 33.54 µm² in Control mice to 211.40 ± 75.13 µm² in KO mice ( P = 0.0003 ). The density of PCs per unit area was reduced by 72%, from 3.94 ± 1.06 cells / µm² in Control mice to 1.10 ± 0.42 cells/µm² in KO mice ( P < 0.0001 ). Additionally, the thickness of the granule cell layer (GCL) decreased by 21%, from 418.00 ± 71.58 µm in Control mice to 329.00 ± 59.04 µm in KO mice ( P = 0.0020 ), while the thickness of the molecular layer (MCL) was reduced by 40%, from 148.40 ± 23.76 µm to 88.62 ± 13.62 µm ( P < 0.0001 ). These results indicated that PCKO Tomato Vessel Green mice were double-fluorescent transgenic mice with cerebellar neurodegeneration. Verification of the applicability of PCKO Tomato Vessel Green in fine study of 3D cerebellar vascular network remodeling in CBND Whether the expression of green fluorescent protein in PCKO Tomato Vessel Green double-fluorescent transgenic mice can accurately label all blood vessels is the key of this experiment. According to previous literature reports, the vascular marker lectin staining could label all blood vessels in the cerebellum, including large vessels with a larger lumen diameter and capillaries with a diameter less than 10 µm 35 – 37 . Therefore, we performed lectin immunofluorescence staining on PC Tomato Vessel Green mice, the maternal mouse of PCKO Tomato Vessel Green , and then counted its double labeling with green fluorescent protein ZsGreen. Figure 2 a showed the superposition results of green ZsGreen, red Lectin, and blue DAPI under confocal microscope. Figure 2 b was locally enlarged, and the results showed that red Lectin clearly delineated the movement of large blood vessels (shown by white arrows) and internal small blood vessels (shown by white arrows) between the molecular layer MCL of the lobule of 4/5Cb and the MCL of the 6Cb lobule. Green fluorescent protein and red Lectin were almost completely co-labeled. DAPI showed a large number of cells in the GCL layer. The co-labeling efficiency diagram of Fig. 2 c showed the co-labeling in the third row of Fig. 2 b, and the results showed that green fluorescent protein and red Lectin almost completely overlapped. These results indicated that the double-fluorescent transgenic mice of CBND, PCKO Tomato Vessel Green mice, constructed by us, provided a tool for the fine study of 3D cerebellar vascular network remodeling of CBND. Construction of transparent cerebellum of PCKO Tomato Vessel Green mice In order to further quantify the changes of cerebellar vascular network in CBND model mice, the transparent cerebellum of PCKO Tomato Vessel Green mice was constructed. Figure 3 a showed in detail the transparency process of mice in the control group (n = 3) and the experimental group (n = 3), which involved up to 14 days. First, the tissue was cleaned, the fat removal solution was added for 6 days, and the above process was repeated on day 7–12. After the lipid removal was completed, refractive index matching was performed for two days, and the tissue was placed on a transparent substrate to obtain the transparent result. In Fig. 3 b, Imaris software was used to collect 3D fluorescence imaging of transparent tissues from the upper view (first row), the lateral view (second row), the posterior upper view (third row) and the posterior view (fourth row). The left side of each group showed the whole brain results, the right side showed the local cerebellar magnifying results, and the main blood vessels passing through the cerebellum were labeled. The results showed that the red pseudo-color marked PCs were significantly lost in the cerebellum of PCKO Tomato Vessel Green , and the green pseudo-color marked blood vessels maintained the anatomical line and structure of the trunk in the cerebellum of PCKO Tomato Vessel Green . In order to investigate the changes of cerebellar vasculature and PCs in mice, next, we further remodeled the cerebellar tissues in 3D with the help of Amira software. Figure 4 showed the results of the above view. We found that the width of the cerebellar earthworm section of the PCKO Tomato Vessel Green mice was narrowed, and at the same time, the PCs in the earthworm section were severely lost, and the PCs in both hemispheres were also lost, but less lost compared to their own earthworm part. We also observed the changes in the vascular trunk, and found that the vascular trunk of PCKO Tomato Vessel Green mice was shorter but the overall morphology was relatively stable, and the density of capillaries on both sides of the trunk, especially in the 6Cb-SCA, was reduced. Further magnifying the results of the rear view, Fig. 5 showed that the number of PCs in the vermis region of the cerebellum of PCKO Tomato Vessel Green mice was significantly reduced, and the width of the vermis was also significantly narrowed. In contrast, relatively few PCs were lost in the Sim, Crus1, and Crus2 areas on both sides of the cerebellum. At the same time, the cerebellar vascular network of PCKO Tomato Vessel Green mice showed that the overall morphology of main blood vessels was relatively stable, and the capillary density on both sides of the main body, especially 3Cb-superior cerebellar artery (SCA), was significantly reduced. We further presented the results of the lateral view and labeled the different lobules of blood vessels and PCs. Figure 6 showed that the overall area of PCs distribution in PCKO Tomato Vessel Green mice was noticeably reduced. In addition, the integrity of the cerebellar vascular network was substantially changed in KO mice: the main vessel became shorter and the capillary density was significantly reduced, especially in the posterior inferior cerebellar artery supply area (PICA) and the SCA supply area of the 6th lobule (6Cb - SCA). In contrast, in Control mice, PCs and cerebellar vascular network were evenly distributed with good structural integrity, main blood vessels and their branches extended to various functional areas, and capillaries were densely distributed. Primary indicators of CBND mice, including PCs count, PCs volume, cerebellar volume, total cerebellar vascular length, and total cerebellar vascular volume To further quantify the changes in the cerebellar vascular network in the CBND model mice, we employed tissue clearing technology combined with 3D reconstruction and light-sheet microscopy to analyze the vascular structures and PCs across the entire cerebellar region, focusing on primary indicators. The cell bodies and dendrites of PCs are located in the molecular layer (MCL) and Purkinje cells layer (PCL) of the cerebellum. We aimed to explore the changes of blood vessels in the progressive loss of neurons, so we divided the cerebellar vessels into the PCL and the MCL, and focused our analysis on the cerebellar vessels. Figure 7 b revealed that in contrast to PC Tomato Vessel Green mice, the average number of PCs in PCKO Tomato Vessel Green mice declined from 56470 ± 19787 to 4111 ± 3621, representing a significant reduction of 93% ( P = 0.0108 ). This notable reduction in PC indicated a considerable loss of cells in the cerebellum of PCKO Tomato Vessel Green mice. Figure 7 a presented the morphological outcomes of the total cerebellar volume. The results indicated that the average total cerebellar volume of PCKO Tomato Vessel Green mice decreased from 217.13 ± 20.35 mm³ to 66.57 ± 16.35 mm³ compared with the Control mice. A significant reduction of 69% ( P = 0.0006 ) was observed, suggesting that the deletion of Drp1 led to significant cerebellar atrophy. Simultaneously, significant differences also emerged in the cerebellar vascular network, and Fig. 7 c showed that the average total vascular length in PCKO Tomato Vessel Green mice was significantly reduced by 58%, from 4149.06 ± 819.26 mm in Control mice to 1763.03 ± 348.03 mm in KO mice ( P = 0.0097 ). This implied that PCKO Tomato Vessel Green mice impaired vascularization and obvious atrophy or reduction of the cerebellar vascular network, which might be related to the effect of Drp1 gene knockout on angiogenesis or maintenance. The statistics of the total volume fraction of the cerebellar vessels in Fig. 7 d demonstrated that there was no statistical difference. This suggested that the deletion of Drp1 had a significant impact on the cerebellar structure and the distribution and extensibility of the cerebellar vascular network. However, the total vascular volume and vascular VF did not change significantly, possibly indicating that the structure of the remaining vessels maintained a relatively constant VF. Secondary indicators of CBND mice, including vascular diameter and volume distribution By defining nodes and segments, the cerebellar vascular network was segmented into independent segments, providing a foundation for subsequent analyses of volume, length, and radius 25 , 38 . Capillaries typically have a diameter of 10 µm or less 1 , 39 , and the subdivision of vascular architecture in Fig. 8 a classified vessels based on diameter as capillary (≤ 10 µm) or noncapillary (> 10 µm) 40 . To display the 3D structure more visually, we utilized Amira/Imaris software to reconstruct the fluorescence results of blood vessels and PCs. Figure 8 b indicated that the cerebellar vascular network of PC Tomato Vessel Green mice was tightly structured and regularly distributed. In contrast, the cerebellar vascular network was sparse in PCKO Tomato Vessel Green mice, suggesting that Drp1 deletion led to a significant decrease in cerebellar vessel density. We further conducted statistics on vessel radius and Fig. 8 c - d revealed that PCKO Tomato Vessel Green mice exhibited significant changes in the radius distribution of cerebellar vessels. We normalized the overall absolute frequency of the Control mice by 50% dimensionality reduction and found that the Control mice showed a very similar interval distribution to that of PCKO Tomato Vessel Green mice, and most of the vessel segments were concentrated in small-radius vessels. We statistically analyzed the number of vascular segments within different range intervals. Figure. 8f showed that, compared to the control group, the vascular frequency in the 1–3 µm short radius range in PCKO Tomato Vessel Green mice decreased from 46145 ± 7940 to 21811 ± 4578, a reduction of 53% ( P = 0.0100 ). In the 3–5 µm short radius range, the vascular frequency decreased from 9490 ± 1554 to 4451 ± 687, also a reduction of 53% ( P = 0.0068 ).These changes suggested that the development or maintenance of small radius blood vessels, especially capillaries, was seriously affected, which directly impacted the regional blood perfusion and oxygen transport ability of the cerebellum, and might led to insufficient energy supply and metabolic disorders in neurons. The statistical analysis of the average radius of blood vessels in Fig. 8 e showed that there was no significant difference in the mean radius of the blood vessels between the two groups, probably because, despite the reduction of the small radius of capillaries was reduced, the larger radius vessels (e.g. trunk vessels or arterioles) might have been relatively preserved and compensated to some extent, resulting in no significant change in mean radius. Further analyzing the volume of the vascular segments, Fig. 8 g showed that the vast majority of the vascular segments were small in size, concentrating in the range of 0 to 6000 µm³.The distribution frequency curves of the PCKO Tomato Vessel Green mice as a whole were located below those of the PC Tomato Vessel Green mice, especially in the small volume segments. In Figure. 8h , the overall absolute frequency of PC Tomato Vessel Green mice was normalized by 50% dimensionality reduction, and it was found that the two fitting curves were highly consistent, which revealed the systematic reduction of the density of the cerebellar vascular network by Drp1 gene knockout. Figure 8 j statistically analyzed the distribution of vascular segments in different volume ranges, and the results showed that in a smaller volume range (0–6000 µm³), the frequency of vascular segments in PCKO Tomato Vessel Green mice was significantly lower than that in PC Tomato Vessel Green mice. Specifically, the frequency of vascular segments in PCKO Tomato Vessel Green mice was reduced from 47244 ± 7918 to 22693 ± 4799, a 52% reduction ( P = 0.0101 ), from 6277 ± 1100 to 2714 ± 520, a 57% reduction ( P = 0.0071 ), and from 2369 ± 726 to 1070 ± 316, a 55% reduction ( P = 0.0467 ), in the segmental volume ranges of 0–2000 µm³, 2000–4000 µm³, and 4000–6000 µm³, respectively. These results showed that the number of blood vessels in these small volume segments was significantly reduced in PCKO Tomato Vessel Green mice. However, in the larger volume range (6000 µm³ and above), there was no significant difference in vessel segment volume frequency between PCKO Tomato Vessel Green mice and Control mice. Statistical analysis of the mean volume of blood vessels was performed in Fig. 8 i, and the results showed that there was no statistically significant difference in the mean value between Control mice and PCKO Tomato Vessel Green mice. It might be that the small volume vessel segment was smaller in volume in the overall vascular structure, and the reduction in number had a weaker pulling effect on the average volume. This further showed that the contribution of capillaries to the total blood vascular volume was small, but the effect was huge. Third level indicators of CBND mice including vascular segment length and curvature distribution Reduced blood vessel length is associated with brain function degradation 25 , 41 , 42 . We analyzed the length and distribution of blood vessels in PC Tomoato Vessel Green mice and PCKO Tomoato Vessel Green mice. Figure 9 a showed the model diagram of blood vessel length, and Fig. 9 b showed the 3D fluorescence diagram showing the length of blood vessels. Figure 9 c and g showed that the frequency of PCKO Tomoato Vessel Green mice in the whole length (chord length/curved length) was significantly lower than that of Control mice. In Fig. 9 d and h , the overall absolute frequency of mice in the control group was standardized by 50% dimensionality reduction, and the curve of its shape was very similar to that of PCKO Tomoato Vessel Green mice. This observation suggested that although the absolute frequency of PCKO Tomoato Vessel Green mice was low, some degree of cerebellar vascular network structure was retained. Figure 9 f and j made a statistical analysis of the distribution of the length of blood vessel segments. The absolute frequency of Control mice was higher than that of PCKO Tomoato Vessel Green mice in almost all the length intervals of blood vessels, and the difference between the two groups showed an increasing trend, especially in the longer intervals. Specifically, compared with PC Tomoato Vessel Green mice, the chord length distribution frequency of PCKO Tomoato Vessel Green mice decreased by 49% from 28562 ± 3796 at 0–40 µm to 14707 ± 3312 ( P = 0.0089 ). With the increase of blood orchestra length, the frequency of vessel segment distribution decreased from 59 ± 8 to 12 ± 3, which decreased by 80% ( P = 0.0006 ) at 360–400 µm. The curved length showed the same trend. Compared with PC Tomoato Vessel Green mice, the distribution frequency of curved length decreased by 49% from 23926 ± 3594 to 12142 ± 2887 at 0–40 µm ( P = 0.0114 ). With the increase of blood curved length, the frequency of vessel segment distribution decreased by 83% from 143 ± 29 to 24 ± 3 at 360–400 µm ( P = 0.0022 ). The above results suggested that vascular growth and repair mechanisms might be inhibited or incompletely activated following Drp1 deletion, resulting in reduced overall vascularization, particularly in medium and long length vessels. However, the presence of a relatively concentrated distribution within short length intervals might indicate that PCKO Tomoato Vessel Green mice retained minimal cerebellar vascular network function to maintain minimal tissue blood supply. Next, we statistically analyzed the mean lengths of the vessel segments, and Fig. 9 e and i indicated that the mean lengths were not statistically different, which might be due to the fact that the number of vessel segments in the short length interval dominated overall and was relatively stable in PCKO Tomoato Vessel Green mice, resulting in a decrease in the number of long segments of the vessels with little effect on the mean value. Changes in vascular tortuosity have been shown to correlate with the severity of cerebrovascular diseases 41 , 43 – 45 . Therefore, we analyzed the tortuosity and distribution of blood vessels in PC Tomoato Vessel Green and PCKO Tomoato Vessel Green mice. Figure 9 k was the schematic diagram of vascular tortuosity, and the number of segments in each range of vascular tortuosity was counted in Fig. 9 l and o . It was found that the absolute frequency of vascular tortuosity in PCKO Tomoato Vessel Green mice in most tortuosity ranges was significantly reduced to about half of that in the control group. Figure 9 m normalized the absolute frequency of PC Tomato Vessel Green mice by 50% dimensionality reduction, and it was found that the two fitting curves were highly consistent. This result indicated that degenerative lesions led to an overall decrease in the density of vascular distribution. Figure 9 n calculated the average tortuosity of blood vessels in PC Tomoato Vessel Green mice and PCKO Tomoato Vessel Green mice. The results showed that there was no significant difference in the average tortuosity of blood vessels between the two groups, indicating that the lesions might be more prone to reduce the density of vessels with different tortuosity. It did not directly affect the curved structure of the blood vessels. This meant that the microstructure of the cerebellar vascular network changed significantly in distribution density during neurodegeneration, but its tortuosity remained relatively constant. The fourth level indicators of CBND mice including density of vascular segments and the topological structure The branching angle of blood vessels can help reveal the random branching pattern of blood vessels, which is crucial for understanding the development of blood vessels and optimizing the cerebrovascular network structure 46 . To explore the effect of progressive neuronal loss on the branching angle of vascular segments, we analyzed the orientation of the vascular segments of PC Tomato Vessel Green and PCKO Tomato Vessel Green . Figure 10 a showed a schematic diagram of branching angles. Figure 10 b and c with f and g showed that DRP1 knockdown-induced cerebellar neurodegeneration resulted in a general reduction in vascular distribution across angular intervals in PCKO Tomato Vessel Green mice but did not show significant frequency peaks or dramatic fluctuating angular preferences. Figure 10 e and i showed that the angular distribution of the within each angle interval, the absolute frequency of PCKO Tomato Vessel Green mice was significantly reduced by about half compared to controls. This consistent decrease suggested that CBND broadly affected the spatial distribution of the vasculature and were not concentrated in a particular direction, implying a significant perturbation of the overall spatial organization of the cerebellar vasculature. Phi (φ) values are azimuthal angles, describing the angle of the vascular segment in the XY plane with respect to the Z-axis in three dimensions, ranging from 0–360°. Theta (θ) values are elevation angles, indicating the offset of the vascular segment in the Z-axis direction, describing the angle of the vascular segment with respect to the XY plane, ranging from 0–90°. Notably, despite the significant differences between the angular intervals, Fig. 10 d and h showed that the differences in the means and values between the two groups of mice were not statistically significant. Figure 10 j and k demonstrated the angle of vascular segments in different interval ranges in PC Tomato Vessel Green and PCKO Tomato Vessel Green mice by Imaris software. The above results indicated that the overall directionality of the vessels was not significantly altered after progressive neuronal loss, suggesting that the microstructure of the cerebellar vessels underwent extensive but balanced reorganization in CBND. The density of branch points and the topological and functional properties of vessels were also used to assess the health of cerebrovascular network and disease progression 47 – 50 . Figure 11 a showed a schematic diagram of the cerebellar vascular network nodes, including branch nodes and terminal nodes. Using Amira software, Fig. 11 b showed partial cerebellar vascular network in the cerebellum of PC Tomato Vessel Green and PCKO Tomato Vessel Green mice. In Fig. 11 c and d , the 3D reconstruction of branch and terminal nodes was performed using Imaris software, respectively. The statistics of total nodes, branch nodes, and terminal nodes in Fig. 11 e - g showed that there was no significant difference in branching nodes between PC Tomato Vessel Green and PCKO Tomato Vessel Green mice. This further indicated that although CBND had extensive effects on vascular distribution and density, it did not change the overall connectivity characteristics and branching structure of the cerebellar vascular network. However, changes at other levels of detail, such as the distribution density of specific vessel segments or microcirculation regulation, might be further affected by the lesion. Criticality of microcirculation to the metabolic demands of cerebellar regions To explore the relationship between neurons and blood vessels after neurodegenerative lesions, we performed a presentation of PC-vessel VF. Using Amira software, Fig. 12 displayed the 3D fluorescence results of PC Tomato Vessel Green and PCKO Tomato Vessel Green cerebellar MCL blood vessels and PCs. Vascular VF is an important measure of tissue blood supply status, metabolic requirements and microcirculation efficiency 1 , 4 , 51 – 53 . Figure 13 a and b used Imaris software to perform 3D reconstruction of the MCL blood vessels and PCs in the region of the SCA supply area of the cerebellar 4/5 lobule (4/5Cb - SCA). We found that compared with PC Tomato Vessel Green mice, PCKO Tomato Vessel Green mice more PCs entered the vascular interior, capillaries showed a relatively obvious decrease in density, and the reduction of PCs number was more significant than that of capillaries. Figure 13 c showed that compared with PC Tomato Vessel Green mice, the distance between PCs and blood vessels in PCKO Tomato Vessel Green mice decreased from 16.540 ± 7.317 µm to 7.151 ± 9.067 µm, with a 58% reduction ( P < 0.0001 ). Figure 13 d showed that compared with PC Tomato Vessel Green mice, PCKO Tomato Vessel Green mice PC-vessel VF decreased from 1039.000 ± 362.000 to 48.440 ± 39.950. This was a 95% reduction ( P = 0.0092 ). Figure 13 e showed that PC-capillary VF decreased from 1904.000 ± 634.600 to 95.040 ± 80.930, with a 95% reduction ( P = 0.0081 ). Figure 13 f showed that PC-noncapillary VF decreased from 2292.000 ± 839.700 to 101.300 ± 82.860, with a 96% reduction ( P = 0.0109 ). Figure 13 g showed that there was no statistically significant difference in noncapillary vessels - CB VF, which further verified that Drp1 gene knockout did not change the overall VF of large diameter vessels, especially in the noncapillary VF with a diameter of > 10 µm. Figure 13 h showed that, compared with PC Tomato Vessel Green mice, capillary - CB VF of PCKO Tomato Vessel Green mice increased from 0.029 ± 0.009 to 0.047 ± 0.001, an increase of 59% ( P = 0.0263 ), which suggested that cerebellar lesions were concentrated at the microcirculatory level, while the large vascular system might still support global blood flow. These results suggested that the distance between Purkinje cells and blood vessels decreased from 16 µm to 7 µm, which might be a central feature of neurovascular remodeling in cerebellar neurodegenerative disease. Panoramic display of capillary remodeling features in CBND To visually compare the brain tissue of PC Tomato Vessel Green mice and PCKO Tomato Vessel Green mice, a 3D reconstruction was performed and shown in a three-minute Video 1 (the two-dimensional code was shown in Graphical abstract ). The results showed that the brain tissues of normal mice and CBND mice were labeled with tdTomato for PCs and ZsGreen for blood vessels. The 0–4" results showed that the overall volume of CBND brain tissue was smaller than that of normal brain tissue, and the area of cerebellar region was reduced in the sagittal direction. 5–30" showed that the cerebellar volume of CBND mice was significantly reduced after reconstructing the cerebellar volume. On 31" − 1'10" display, the coronal section area of brain tissue in CBND mice was reduced when the images were observed in the coronal direction. 1'11" − 1'15" showed that the volume of blood vessels in the region constituting the cerebellum was also reduced. In 1'16" − 2' display, when the images were magnified, it was observed that the noncapillary volume in the cerebellar region of the CBND mice was reduced, and the capillary volume was severely reduced. 2' − 2'45" showed that the number of PCs in the cerebellum of CBND mice was reduced, accompanied by cell body swelling. The 2'50" − 3' display image in the horizontal direction showed that the horizontal section area of brain tissue in CBND mice was reduced. Discussions Neurodegenerative diseases are characterized by progressive loss of neurons leading to impaired structural integrity and dysfunction of the brain, which can cause extensive cerebral vascular remodeling 7 – 10 . As an important component of the microcirculation and the blood-brain barrier, capillaries have been confirmed to be associated with vascular remodeling in various models (e.g., the ischemic stroke mouse model 46 , the traumatic brain injury rat model 54 ), playing a significant role in the development of neurodegenerative diseases 55 – 58 . Our results first confirm that capillary loss is central to the vascular remodeling caused by multiple neuronal degenerations. Erlen Lugo-Hernandez et al. used solvent-based clearing and light-sheet microscopy to 3D visualize and quantify the microvasculature throughout the ischemic mouse brain, and the results showed a significant loss of capillaries with diameters ≤ 10 µm, which was useful for studying microvascular damage and remodeling after stroke 24 . Additionally, Hannah C Bennett et al. found that in an aging mouse model, blood-brain barrier damage led to a general reduction in vessel length and branch density, as well as more tortuous arterioles, indicating that the vascular network is sparser and more remodeled in the aged brain 41 . These studies support our view that capillary loss is central to the vascular remodeling caused by neuronal degeneration. However, there are also studies that differ from our perspective. Matthew V Russo et al. studied a mouse model of mild traumatic brain injury, and found that the meningeal vasculature can regenerate after mild traumatic brain injury, orchestrated by different myeloid cell subsets over time 59 . This reflects that vascular remodeling may be the result of multiple factors acting together. We propose capillary VF as an early diagnostic indicator for CBND. Obtaining a 3D analysis of the true vascular VF can more accurately quantify the cerebrovascular network and also allows for better comparison between different studies 1 . A similar study has supported our idea, as Grace Rosen et al. compared 41 male brain donors with chronic traumatic encephalopathy and found that the ratio of vascular branch density and VF of sulcus and gyrus was greater than the control group 60 . Traditional diagnostic methods largely rely on a combination of mental status examination and conventional imaging techniques (such as MRI, PET, etc.), while also detecting biomarkers related to neuronal degeneration (such as Aβ, tau, etc.) 61 – 63 . However, by the time significant neuronal degeneration has occurred, these methods offer limited value for disease treatment. Recently, various new imaging tests have been developed to evaluate the structure and function of the vasculature, such as ultrasound localization microscopy, three-photon microscopy, etc., with resolutions reaching up to tens of micrometers 64 – 66 . Moreover, in terms of materials, advanced nanomaterials have been employed in the diagnosis of neurodegenerative diseases, through in vivo imaging and in vitro sensors 67 . Given that we propose using capillary VF as an early diagnostic indicator for CBND, rather than biomarkers associated with neuronal degeneration, this provides motivation for the development of higher resolution detection methods. Early diagnosis of neurodegenerative diseases at the capillary level with high resolution can spare patients from invasive procedures such as blood tests, reduce the risk of infection, save medical costs, and has greater clinical significance. Although we have found a significant loss of capillaries in CBND, interestingly, despite the widespread impact of cerebellar neurodegenerative lesions on vascular distribution and density, the overall connectivity characteristics and branching structure of the cerebellar vascular network remain unchanged. This suggests that the maintenance of vascular topological homeostasis is a feature of vascular remodeling caused by CBND. Therefore, early changes in capillaries should be a particular focus. The vascular network includes arteries, veins, and capillaries. During vascular development, embryonic stem cells first differentiate into mesodermal progenitor cells, which then produce endothelial cells 39 . Next, endothelial cells differentiate into arterial or venous endothelium by regulating VEGF concentration, with various molecules participating in the regulation 39 , 68 , 69 . This indicates that there are morphological, cellular composition, molecular expression, and signaling differences between capillaries and arteries or veins 70 , 71 . However, current high-resolution imaging tests used clinically to assess blood vessels, such as 7T MRI with a resolution of about 0.5 mm 72 , 73 , cannot yet display the early changes of capillaries in neurodegenerative diseases. Based on the many differences in the presence of blood vessels of different radii, we believe that it is risky to infer the degree of neurodegeneration from changes at the noncapillary level of blood vessels. It is likely that changes in the morphology and function of noncapillary vessels have already indicated a massive death of neurons, resulting in irreversible consequences, significantly delaying the treatment of the disease. Our results showed that the distance between PCs and blood vessels decreased from 16 µm to 7 µm, which seemed to indicate a more sufficient blood supply for PCs. However, this was actually a subsequent result of cerebellar volume reduction, i.e., cerebellar atrophy. At the same time, our results also showed that PC-capillary VF reduced by 94%, suggesting that the reduction in PC volume was more significant than the reduction in capillary volume, and the structural remodeling of capillaries was not sufficient to prevent further loss of PCs. It may mean that simply targeting blood vessels treatments cannot be effective in delaying the progression of neurodegenerative diseases. The mechanism of NVC plays an important role in this process, through which neural activity regulates changes in cerebral blood flow to meet the oxygen and nutrient needs of brain cells 17 , 18 . It is not only essential for maintaining homeostasis in brain function, but also plays a key role in the occurrence and development of a variety of brain diseases 74 , 75 . Therefore, the treatment of neurodegenerative diseases should take into account the dual effects of nerves and blood vessels. Robert Zivadinov et al. found that extracranial venous angioplasty was ineffective in treating MS 76 . Current research has also largely focused on neuroprotective and regenerative therapies. Amandine Virlogeux et al. found that improving vesicular transport of brain-derived neurotrophic factors could improve the behavioral phenotype and neuropathology of Huntington's disease model mice 77 . Hikari Tanaka et al. found that using AAV vectors to restore YAP protein levels successfully inhibited neuronal necrosis in the early stages of AD, effectively preventing cognitive impairment and extracellular Aβ aggregation in AD model mice 78 . However, there are also studies have found that treatments targeting blood vessels can improve NDs. Robert D Bell et al. have suggested that serum response factors and cardiomyocyte proteins in cerebral vascular smooth muscle cells act as transcriptional switches, controlling Aβ clearance from the brain and the progression of AD 79 . In summary, by knocking out Drp1 in the cerebellar PCs of mice, we constructed a model of CBND with direct neuronal damage. We found that although vascular remodeling was manifested in the early stage of CBND, with capillaries showing signs of adaptive reorganization, and the microcirculatory system attempting to maintain local blood flow stability to alleviate structural changes caused by the lack of PCs. However, this cannot delay the progression of PCs loss. This may be because the reduction in the number of capillaries, as the most basic structure that provides oxygen and nutrients to tissues, places remaining cells in the PCs region under ischemic and hypoxic conditions, increasing the metabolic stress on PCs and further leading to cellular damage. Therefore, we believe that targeting the vasculature alone cannot fundamentally solve the problem of neuronal loss, but can only temporarily alleviate the metabolic stress on neurons. The focus of treatment for neurodegenerative diseases should still be on the protection and regeneration of neurons. Our research provides a basis for the development of early diagnostic strategies and specific therapies for neurodegenerative diseases. Although capillaries can remodel autonomously under the condition of CBND, this is not sufficient to halt the progression of the disease caused by neuronal loss. The core of disease treatment should focus on the improvement of neurons, while structural remodeling of capillaries and the vascular VF per unit area can serve as diagnostic indicators for the early stages of clinical diseases. Limitations First, our study has revealed morphological changes in blood vessels of different diameters in CBND, but it did not distinguish between arteries and veins, requiring further research to clarify. Second, the total number of blood vessel segments measured exceeded 265,000, and the total number of PCs exceeded 180,000. However, compared to the vast number of blood vessels and cells in the brain, future studies will need to measure a larger sample size. Third, it is likely to be regional heterogeneity in the remodeling of the cerebellar vascular network caused by CBND, but this needs to be further clarified. Fourth, due to the structural differences between the human brain and the mouse brain, our results need to be validated and further optimized in human clinical samples. Fifth, with the development of emerging technologies, it may be possible in the future to make more precise distinctions for capillaries with diameters ≤ 10 µm. Sixth, the medium by which capillaries couple with neurons and the specific molecular mechanisms that lead to the remodeling of the capillary network require further research. Methods Animals and ethics statements B6/JGpt-H11 em1Cin(CAG−LoxP−ZsGreen−Stop−LoxP−tdTomato)/Gpt (B6-G/R) mice were purchased from GemPharmatech Co., Ltd (E2101190037); Pcp2 cre mice were purchased from Jackson Laboratory (Stock No: 004146, America), and Drp1 fl/fl mice were purchased from Seye (serial number: CKOAIS191230RT5, China). Pcp2 Cre mice were crossed with B6-G/R (B6/JGpt−H11em1Cin(CAG−LoxP−ZsGreen−Stop−LoxP−tdTomato)/Gpt) mice to generate PC Tomato Vessel Green mice. PC Tomato Vessel Green mice were crossed with Drp1 f/f mice to generate PCKO Tomato Vessel Green mice. PC Tomato Vessel Green mice were used as controls. All experimental animals were 8 weeks old. All animal protocols were approved by the Ethics Committee of the Air Force Medical University and followed our institutional guidelines for the use of laboratory animals. Mouse genotyping The mouse genotype was identified by polymerase chain reaction (PCR) with genomic DNA obtained from the tails. Primers were shown in Table 1. The PCR program used was as follows: 94°C for 3 min, then 35 cycles of 94°C for 30 s for denaturation, 62°C for 35 s for annealing, and 72°C 45 s for elongation. PCKO Tomato Vessel Green mice were subjected to agarose gel electrophoresis and three bands were obtained: a 292 bp Drp1 f/f band, a 567 bp Pcp2 Cre band, and a 1465 bp B6-G/R band. Tissue transparency Mouse brain tissues were treated with 4% paraformaldehyde overnight at 4°C to fix the samples. Mouse brain tissues were washed three times with PBS. We then performed tissue transparency using CUBIC reagent solutions A, B, and C (Nuohai, Cat#210701 Nuohai Life Sciences Co., LTD.). Solutions A and B were used for defatting and solution C for refractive index matching. First, brain tissue was gently shaken with CUBIC reagent solutions A and B at 37°C for 12 days. Then, brain tissue was washed with PBS for 6 h and gently shaken at 37°C. After washing, brain tissues were embedded in solution C at 20°C until they appeared completely transparent. Transparent imaging and processing of results Raw data for the 3D image analysis work was captured by LiTScan (Light Innovation Technology Limited) and converted to AM format for compatibility with Amira software (Thermo Fisher Scientific). The image samples were resampled to adjust the resolution by setting the appropriate voxel size using the Resample module. In the pre-processing stage, a background detection correction module with specific filters was used to remove background noise. Subsequently, structural enhancement filters, specifically 3D filters, were used to enhance the structural features. An interactive thresholding module was then applied to segment the region of interest based on a specified intensity range, and then the segmented region was manually labelled and dots were removed to eliminate defects. Lymphatic vessels, blood vessels and lymph nodes were segmented based on raw data. Equipment (Light-sheet Microscope); Software: LiTScan 3.3.0 (Light Innovation Technology Limited); Lenses (Objectives): 4X, N.A. = 0.28, WD = 28 mm 10×, N.A. = 0.6, WD = 8 mm. Imaris 10.2 was used to manually mark the target area and establish a surface model, and the vascular and neuronal signal channels in the labeled area were masked out respectively. Then, the median filtering was performed on the extracted vascular channels to make the signal of the intravascular cavity uniform. The filament was used to reconstruct the vascular channel after the filter, and the autopath mode was used for automatic identification. Based on the established model, the statistics of vessel length and volume could be extracted. The model of spots was used to identify the neuronal signals after the mask, and the background subtraction mode was selected, which could also accurately identify the signals with large differences in brightness. Immunofluorescence staining and imaging 4% PFA-treated mouse brain tissue was used overnight to fix the samples and moved in 30% sucrose solution until the tissue sank, and the tissue was sectioned after extended sagittal-free cuts to 30 µm. Sections were blocked with 10% fetal bovine serum and 0.3% Triton for 30 min at room temperature, Lectin − 647 (Nuohai, NH − 240525 - DL649, 1:1000) was added, incubated at 4°C for 18 h, and the nuclei of the cells were stained with 4',6 - diamidino − 2 - phenylindole using a Lycra Stellaris 5 laser confocal microscope. (laser lines: 405, 488, 561 and 637 nm) and 10x objective lens in Navigator mode to obtain the above immunofluorescence staining images. The fluorescence signals of blood vessels and Lectin were obtained by excitation at 405, 488, 561 and 637 nm, respectively, at 40x effective magnification. The acquired images were processed using Image J software. Global cerebellar neuronal vascular network morphology and vascular network topology analysis Because the cell bodies and dendrites of PCs are located in the cerebellar MCL and the PCL, we focused on this area for the analysis of cerebellar vessels, that is, the PCL was used as the boundary, and the cerebellar vessels of the PCL and the MCL were divided for further analysis. The vessels were divided into capillary (diameter ≤ 10 µm), noncapillary (diameter > 10 µm), and integral (diameter was not distinguished). The whole was a combination of capillary and noncapillary vessels. Total cerebellar volume, total number of PCs, total PCs volume, total length of blood vessels, and total volume of blood vessels were calculated. The distribution frequency of chord length of the whole vessel segment was calculated and the fitting curved was drawn. The overall absolute frequency of PC Tomato Vessel Green mice group was normalized by 50%-dimension reduction, and the frequency distribution trend of the two groups was observed. The chord length of the vessel segments was the straight-line distance between the origin and end of the vessel in each segment. The distribution frequency of the overall vascular segment curved length was calculated and the fitting curved was drawn. The overall absolute frequency of PC Tomato Vessel Green mice group was normalized by 50%-dimension reduction, and the frequency distribution trend of the two groups was observed. Vessel segment curved length referred to the true path length from the origin to the end point along the natural curved of the vessel. The distribution frequency of the mean radius of the whole blood vessel was calculated and the fitting curved was drawn. The overall absolute frequency of PC Tomato Vessel Green mice group was normalized by 50%-dimension reduction, and the frequency distribution trend of the two groups was observed. The average radius of a vessel was usually measured by measuring radius values at multiple locations on a certain vessel segment and then taking the average of these radii. The whole vascular node and its middle terminal branch were calculated. Vascular nodes included two important geometric and functional locations in the vasculature: terminals and branch points. Vessel terminal referred to the last point along the direction of the vessel along the center line of the vessel, when the vessel no longer bifurcates. Vascular branch nodes in the vascular system referred to the key locations where larger blood vessels split into smaller vessels. The distribution frequency of φ value of the whole vascular segment was calculated and the fitting curved was drawn. The overall absolute frequency of PC Tomato Vessel Green mice group was normalized by 50%-dimension reduction, and the frequency distribution trend of the two groups was observed. The φ value was the azimuth angle, which described the angle between the vessel segment in the XY plane and the Z axis in 3D space, and we analyzed the φ value in the range of 0 to 360°. The distribution frequency of θ value of the whole vascular segment was calculated and the fitting curved was drawn. The overall absolute frequency of PC Tomato Vessel Green mice group was normalized by 50%-dimension reduction, and the frequency distribution trend of the two groups was observed. The θ value was the elevation angle, which indicated the offset of the vessel segment in the z-axis direction and was commonly used to describe the angle between the vessel segment and the XY plane, and we analyzed the θ values in the 0–90° range. The VF was calculated including the vessel-cerebellum VF (vascular volume/cerebellar volume), capillary-cerebellum VF (capillary volume/cerebellar volume), noncapillary-cerebellum VF (non - capillary volume/cerebellar volume), PC-vessel VF (PC volume/vascular volume), PC-capillary VF (PC volume/capillary volume), PC-noncapillary VF (PC volume/noncapillary volume). Data processing and visual analysis In this study, Python programming language was used for data processing and image plotting for different experimental groups (PC Tomato Vessel Green and PCKO Tomato Vessel Green groups). Data read. Through the Pandas library, the data of the control group and the PC Tomato Vessel Green mouse group were first read. We wrote a data reading function, read_name (), which iteratively read each file and clears missing values (NaN) to ensure data integrity and consistency. Data division and statistical calculation. In order to study the volume distribution of different groups, we combined all the data and divided the volume data into boxes. The container division range was defined as V min - V max , the container width was x, and the specific container division range was as follows. Through the numpy. histogram () function, the sample frequency in each sub-box was calculated using the following formula: bins = {V min , V min + x, V min + 2x, ..., V max }. Average frequency and standard deviation in each subbox: Ci was the binning frequency of the i th sample, and n was the total number of samples. After data processing, we used the Matplotlib library to plot the volume data of the control and PCKO Tomato Vessel Green mice groups. Data visualization. To further analyze the volume distribution trends, we smoothed the data using cubic spline interpolation (make_interp_spline () function). For each experimental group, we defined the following interpolation function: \(\:\text{M}\text{e}\text{a}\text{n}\hspace{0.25em}\text{C}\text{o}\text{u}\text{n}\text{t}=\frac{1}{n}\sum\:_{i=1}^{n}{C}_{i}\) . Bi (x) was the B-spline basis function, ci was the interpolation coefficient, and x was the center point of the bining of the volume. By using this interpolation function, a more continuous and smooth volume distribution curved between control and PCKO Tomato Vessel Green mice was generated. Regions of standard deviation were shown by adding and subtracting standard deviations from the mean curves, respectively, and were filled with color. Data output and saving. All processed data, including volumetric frequency statistics for the control and PCKO Tomato Vessel Green mouse groups, raw frequency counts of experimental samples from each group, and centroid information for each subbox, were saved as Excel files. Statistical analysis In this paper, all data are expressed as mean ± SD, and error lines indicate SD. all statistics were analyzed using GraphPad Prism 9. Unpaired t-test and Mann-Whitney U test were used to compare the two groups. P < 0.05 was considered statistically significant. Abbreviations 3D Three-Dimensional AD Alzheimer’s Disease Cb Cerebellar Lobules CBND Cerebellar Neurodegenerative Disease CDN Cerebellar Dentate Nucleus Drp1 Dynamin-related protein 1 GCL Granular Cell Layer MCL Molecular Layer MRI Magnetic Resonance Imaging MS Multiple Sclerosis NVC Neurovascular Coupling P2Y12 Purinergic Receptor P2Y12 PCs Purkinje Cells PCL Purkinje Cell Layer PD Parkinson’s Disease PFL Paraflocculus PICA Posterior Inferior Cerebellar Artery SCA Superior Cerebellar Artery Sim Simplex Lobules VF Volume Fraction Declarations Data availability The Data for each figure can be found in the Source Data files. Acknowledgements We would like to thank the optical imaging platform of Chinese Institute for Brain Research, Beijing, China and Shenzhen Guangyuan Lit Company for the technical support of this experiment. Thanks to Shujiao Li and Jingjing Tie for providing some transgenic mice. This work was supported by the National Natural Science Foundation of China (82201627) by Feifei Wu, the Basic Research Program of Natural Science of Shaanxi Province (2022JQ820) by Feifei Wu, the Basic Research Program of Natural Science of Shaanxi Province (2024JCZDXM60) by Yanling Yang, the New Clinical Technology of Xi-Jing Hospital (2023XJSY27) by Yanling Yang, Military Medicine Promotion Program of Air Force Military Medical University (2020SWAQ04) by Yayun Wang and Shaanxi Provincial Innovation Capability Support Program (2023CXPT33) by Yayun Wang. Author contributions Yayun Wang and Yanling Yang designed the study protocol and interpreted the experimental results. Hui Liu was responsible for the breeding of transgenic mice, most of the basic experiments and the production of videos. Yunqiang Huang and Changlei Zhu assisted in basic experiments. Shujiao Li and Jingjing Tie were responsible for providing some transgenic mice. Ziwei Ni was responsible for the processing of experimental data. Xintong Deng was responsible for the drawing of the image; Xueyin Pu was responsible for the guidance of the image; Yayun Wang, Yuxuan Liu, and Ziwei Ni wrote the manuscript, which was revised by all the authors; Feifei Wu provided the funding. Competing interests The authors declare no conflict of interest. This research project has been approved by the relevant ethics committee or institution and is conducted in strict compliance with ethical guidelines. Throughout the study, the rights and privacy of all participants are maintained and guaranteed, while ensuring the confidentiality of their personal information. References Wälchli, T. et al. Hierarchical imaging and computational analysis of three-dimensional vascular network architecture in the entire postnatal and adult mouse brain. Nat Protoc. 16 4564–4610 (2021) Mink, J. W., Blumenschine, R. J. & Adams, D. B. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5607179","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":392362797,"identity":"09afcfc2-2830-4461-9a29-97955f79157f","order_by":0,"name":"Yayun 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University","correspondingAuthor":false,"prefix":"","firstName":"Xueyin","middleName":"","lastName":"Pu","suffix":""},{"id":392362808,"identity":"67321bc2-22cb-4ca2-8873-d8966d97335e","order_by":11,"name":"Yan-Ling Yang","email":"","orcid":"https://orcid.org/0000-0001-8246-1756","institution":"Air Force Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yan-Ling","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-12-09 08:30:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5607179/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5607179/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79128657,"identity":"77742161-0bf8-4e6c-b4cf-fcdaef3a3354","added_by":"auto","created_at":"2025-03-24 18:06:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":293199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStrategy for constructing the cerebellar neurodegenerative disease double-fluorescent transgenic mouse model PCKO\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTomato\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eVessel\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eGreen\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and measurements based on fluorescent confocal imaging results.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003ePcp2\u003csup\u003eCre\u003c/sup\u003e mice were bred with B6-G/R\u003csup\u003e（B6/JGpt-H11em1Cin（CAG-LoxP-ZsGreen-Stop-LoxP-tdTomato）/Gpt）\u003c/sup\u003e mice to generate PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice were crossed with Drp1\u003csup\u003ef/f\u003c/sup\u003e mice to generate PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. PCls were labeled tdTomato and blood vessels were labeled ZsGreen. \u003cstrong\u003eb. \u003c/strong\u003eRepresentative PCR results from transgenic mice. Pcp2\u003csup\u003ecre\u003c/sup\u003e mice showed 567 bp; Drp1\u003csup\u003eLoxp\u003c/sup\u003e showed 292 bp; B6/RLoxp was shown to be 1465 bp. \u003cstrong\u003ec. \u003c/strong\u003eConfocal images of immunofluorescence staining for expression in total sagittal brain sections from PC\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice and PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice (Bar = 1 mm). PCs (red pseudo color), blood vessels (green pseudo color). \u003cstrong\u003ed. \u003c/strong\u003eThe area of the cerebellum shown in the fluorescence pictures. Control, 7652719 ± 453169 μm\u003csup\u003e2\u003c/sup\u003e; KO，4340231 ± 93977 μm\u003csup\u003e2 \u003c/sup\u003e(\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt; 0.0001\u003c/strong\u003e).\u003cstrong\u003e e. \u003c/strong\u003eThe area of PCs somas was shown in the fluorescence image. Control, 261.80 ± 33.54 μm\u003csup\u003e2\u003c/sup\u003e; KO, 311.40 ± 75.13 μm\u003csup\u003e2 \u003c/sup\u003e(\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0003\u003c/strong\u003e). \u003cstrong\u003ef. \u003c/strong\u003eNumber of PCs per unit area as shown in the fluorescence picture. Control, 3.94± 1.06; KO, 1.13 ± 0.42 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt; 0.0001\u003c/strong\u003e).\u003cstrong\u003e g and h.\u003c/strong\u003e Cerebellar GCL and MCL thickness. GCL thickness: Control, 418.00 ± 71.58 μm; KO, 329.00 ± 59.04 μm (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.002\u003c/strong\u003e). MCL thickness: Control, 148.4 ± 23.76 μm; KO, 88.62 ± 13.62 μm (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt; 0.0001\u003c/strong\u003e). The data were presented as means ± SD. \u003cem\u003eP\u003c/em\u003e was calculated by unpaired two-tailed t-test (d - h), n = 3 for Control; n = 3 for KO animals were used. **\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01; ***\u003cem\u003eP \u0026lt; \u003c/em\u003e0.001; ****\u003cem\u003eP \u0026lt; \u003c/em\u003e0.0001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/00d7b8a4491f57467fcc6571.png"},{"id":79128660,"identity":"40ed5b92-6c53-49f3-b3bf-fd14108cf3b5","added_by":"auto","created_at":"2025-03-24 18:06:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":320101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe efficiency of vascular labeling in PC\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTomato\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eVessel\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eGreen\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice was verified by Lectin immunofluorescence staining.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eConfocal overlapping images of immunofluorescence staining for expression in total sagittal brain sections of PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice (Bar = 100 μm). \u003cstrong\u003eb.\u003c/strong\u003e Results of local magnification of three rois in the molecular layer in the left panel. Blood vessel ZsGreen (green pseudo-color); Blood vessel marker Lectin (red pseudo-color); PCs tdTomato (blue false-color). The co-localization results of ZsGreen and Lectin were shown in the bottom left. Cb: Cerebellar Lobules; ML: Molecular Layer; MCL: Molecular Cell Layer.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/f86a856ffecd5ba66c69b305.png"},{"id":79129389,"identity":"9ec0f0e7-c2c7-462a-a2c4-102ea05a725c","added_by":"auto","created_at":"2025-03-24 18:14:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":545582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransparency of the cerebellum in double-fluorescent transgenic mice PCKO\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTomato\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eVessel\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eGreen\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eTissue transparency process of PC\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice and PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, including fixation, 1st degreasing, 2nd degreasing, and refractive index matching. \u003cstrong\u003eb. \u003c/strong\u003e3D fluorescence images of whole brain and cerebellum tissues after tissue transparency (bar = 3000 μm), upper view, the lateral view, the posterior upper view and the posterior view. The white box was the whole brain and the blue box was the cerebellum. The whole brain orientation was shown on the left side of the figure. Cb: Cerebellar Lobules; SCA: Superior Cerebellar Artery; A: Anterior; P: Posterior; L: Left; R: Right; D: Dorsal; V: Ventral.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/3b2bb034a18e32fb9964c148.png"},{"id":79129800,"identity":"c91622b3-4187-478a-a13d-e6654ec4ad8e","added_by":"auto","created_at":"2025-03-24 18:22:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":371067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUpper view of the cerebellum in the transparent double-fluorescent transgenic mouse PCKO\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTomato\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eVessel\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eGreen\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe left was a PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mouse, and the right was a 3D reconstruction of the cerebellar vascular network of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, showing the distribution of blood vessels and PCs in different regions. The top row showed an overlapping image of PCs and blood vessels. The central micrograph showed the macroscopic localization of the cerebellar anatomy. Middle row: Only tdTomato signal was displayed, marking the distribution of PCs. Bottom row: Only ZsGreen signal was displayed, marking the distribution of blood vessels and their spatial positioning. (bar = 3000 μm). Cb: Cerebellar Lobules; SCA: Superior Cerebellar Artery. A: Anterior; P: Posterior; L: Left; R: Right.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/10564649ea361ea7c50a3ad4.png"},{"id":79130256,"identity":"4746b828-9430-418f-810f-d820c416d6db","added_by":"auto","created_at":"2025-03-24 18:30:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":396911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePosterior view of the cerebellum in the transparent double-fluorescent transgenic mouse PCKO\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTomato\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eVessel\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eGreen\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe left was a PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mouse, and the right was a 3D reconstruction of the cerebellar vascular network of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, showing the distribution of blood vessels and PCs in different regions. The top row showed an overlapping image of PCs and blood vessels. The central micrograph showed the macroscopic localization of the cerebellar anatomy. Middle row: Only tdTomato signal was displayed, marking the distribution of PCs. Bottom row: Only the ZsGreen signal was displayed, marking the distribution of blood vessels and their spatial positioning (bar = 3000 μm). Cb: Cerebellar Lobules; SCA: Superior Cerebellar Artery; Sim: Simplex Lobule; PFL: Paraflocculus; D: Dorsal; V: Ventral; L: Left; R: Right.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/c16db8c172f292ca034b01a4.png"},{"id":79128668,"identity":"8d4a8bd3-bb31-4ef9-9f6e-97a000430951","added_by":"auto","created_at":"2025-03-24 18:06:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":215453,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLateral view of the cerebellum in the transparent double-fluorescent transgenic mouse PCKO\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eTomato\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eVessel\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eGreen\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe left was a PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mouse, and the right was a 3D reconstruction of the cerebellar vascular network of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, showing the distribution of blood vessels and PCs in different regions. The top row showed an overlapping image of PCs and blood vessels. The central micrograph showed the macroscopic localization of the cerebellar anatomy. Middle row: Only tdTomato signal was displayed, marking the distribution of PCs. Bottom row: Only the ZsGreen signal was displayed, marking the distribution of blood vessels and their spatial positioning (bar = 3000 μm). Cb: Cerebellar Lobules; SCA: Superior Cerebellar Artery; PICA: Posterior Inferior Cerebellar Artery. D: Dorsal; V: Ventral; A: Anterior; P: Posterior.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/24d8ab4b4ba423796a4388eb.png"},{"id":79128662,"identity":"f9c01759-7faa-41e5-9ef9-ecc91b077ec5","added_by":"auto","created_at":"2025-03-24 18:06:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":81420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLayered imaging and primary indicator assessment in cerebellar neurodegeneration double-fluorescent transgenic mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eTotal cerebellar volume. Control, 217.13 ± 20.35 mm\u003csup\u003e3\u003c/sup\u003e; KO, 66.57 ± 16.35 mm\u003csup\u003e3\u003c/sup\u003e. \u003cstrong\u003eb. \u003c/strong\u003eTotal number of PCs. Control, 56470 ± 19787; KO, 4111 ± 3621. \u003cstrong\u003ec. \u003c/strong\u003eTotal length of blood vessels. Control, 4149.06 ± 819.26 mm; KO, 1763.03 ± 348.03 mm. \u003cstrong\u003ed. \u003c/strong\u003eVessel-cerebellum volume fraction. The cerebellar volume, PCs number and total blood vessel length in KO group were significantly lower than those in the control group, and the fraction of cerebellar volume had no significant difference. The data were presented as means ± SD. \u003cem\u003eP\u003c/em\u003e was calculated by unpaired two-tailed t-test, n = 3 for Control; n = 3 for KO animals were used. * \u003cem\u003eP \u0026lt; \u003c/em\u003e0.05; **\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01; ***\u003cem\u003eP \u0026lt; \u003c/em\u003e0.001; ns: not significant.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/bcd24e1fbfa9646635ee90b7.png"},{"id":79128674,"identity":"41843bc9-9af4-4400-bf12-b184d5c0ab82","added_by":"auto","created_at":"2025-03-24 18:06:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":311028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVascular diameter and volume distribution results in cerebellar neurodegeneration double-fluorescent transgenic mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eSchematic diagram of vascular grading. Thick vessels in red on both sides were large arteries with diameters greater than 10 μm, thick vessels in blue in the middle were veins with diameters greater than 10 μm. The small vessels within the dotted line at the arteriovenous junction were capillaries with diameters less than or equal to 10. \u003cstrong\u003eb.\u003c/strong\u003e Cerebellar 4/5Cb of PC\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice and PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice ROI of PC-vessel 3D network reconstruction with capillaries (green), PCs (blue), and magnified results of the area in the box (bar = 200 μm and bar = 100 μm) in the lower panels.\u003cstrong\u003e c - j.\u003c/strong\u003e Computational analyses were carried out using global morphometry. \u003cstrong\u003ec. \u003c/strong\u003eQuantitative fitting of the mean radius of the vascular segments, only 0 - 21 μm was selected for presentation (bin width 2 μm, number of bins 11). \u003cstrong\u003ed. \u003c/strong\u003eThe average radius of the vascular segments was selected for presentation by combining the overall absolute frequency of the mean radius of the vascular segments in the PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mouse group was found after 50% dimensionality reduction normalization, and only 0 - 21 μm was selected for presentation (bin width 2 μm, number of bins 11). \u003cstrong\u003ee. \u003c/strong\u003eResults of the quantitative analysis of the mean of the overall mean radius of the vascular segments. \u003cstrong\u003ef. \u003c/strong\u003eResults of the quantitative analysis of the hierarchy of the mean radii of the vascular segments. 1 - 3 m: Control. 46145 ± 7948; KO, 21811 ± 4578 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0100\u003c/strong\u003e). 3 - 5 μm: Control, 9490±1554; KO, 4451 ± 687 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0068\u003c/strong\u003e). \u003cstrong\u003eg.\u003c/strong\u003e Quantitative fitting of the vascular segment volume, only 0 - 20,000 μm\u003csup\u003e3\u003c/sup\u003e was selected for presentation (bin width 1000 μm\u003csup\u003e3\u003c/sup\u003e, number of bins 315). \u003cstrong\u003eh. \u003c/strong\u003eResults of quantitative analyses of overall vascular segment mean radius by combining the overall absolute frequency of vascular segment volume in the PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen \u003c/sup\u003emice group was normalized by 50% downscaling and only 0 - 20000 μm\u003csup\u003e3\u003c/sup\u003e was selected for presentation (bin width 1000 μm\u003csup\u003e3\u003c/sup\u003e, number of bins 315). \u003cstrong\u003ei. \u003c/strong\u003eResults of quantitative analysis of the mean of overall vascular segment volume. \u003cstrong\u003ej.\u003c/strong\u003e Results of the quantitative analysis of the graded vascular segment volume. 0 - 2000 μm³: Control, 47244 ± 7918; KO, 22693 ± 4799 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0101\u003c/strong\u003e). 2000 - 4000 μm³: Control, 6277 ± 1100; KO, 2714 ± 520 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0071\u003c/strong\u003e). 4000 - 6000 μm³: Control, 2369 ± 726; KO, 1070 ± 316 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0467\u003c/strong\u003e). The data were presented as means ± SD. \u003cem\u003eP\u003c/em\u003e were calculated by unpaired two-tailed t-test or Mann-Whitney U test, n = 3 for Control; n = 3 for KO animals were used. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ns: not significant. Cb: Cerebellar Lobules; SCA: Superior Cerebellar Artery.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/8f90c14b7e017cfd92af876a.png"},{"id":79129409,"identity":"6e5cb41e-5b6f-4bdf-8188-ad3af778f3fa","added_by":"auto","created_at":"2025-03-24 18:14:26","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":426457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003evascular chord length, curved length, and tortuosity distribution results in cerebellar neurodegeneration double-fluorescent transgenic mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eSchematic diagram of vessel segment chord length and curved length. Vascular segment chord length was the straight-line distance between the start and end points of each vessel segment. Vascular segment curved length was the true path length from the starting point to the end point along the natural curved of the vessel. The ratio of vascular segment curved length to chord length was the vascular segment tortuosity. \u003cstrong\u003eb.\u003c/strong\u003e Fluorescence microscopy images showing structural features of PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e vessels. Measurements of curved length (le) and chord length (ls) were labelled. (bar = 100 μm). Asterisks marked the curved length of the vascular segments in PC\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice and PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, and dashed lines marked the chord length of the vascular segments in PC\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice and PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. \u003cstrong\u003ec - o. \u003c/strong\u003eGlobal morphometric measurements were used computational analyses were performed. \u003cstrong\u003ec.\u003c/strong\u003e Frequency distribution plots were quantitatively fitted to the vessel segment chord lengths, and only 0 - 400 μm were selected for display. The red dashed line marked the chord lengths where the frequency of the vascular segment distribution was at its peak (bin width 10 μm, number of bins 87). \u003cstrong\u003ed. \u003c/strong\u003eOverall absolute frequency of the vascular segment chord lengths of the PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice group was normalized by 50% downscaling to show only 0 - 400 μm (bin width 10 μm, number of bins 87). \u003cstrong\u003ee. \u003c/strong\u003eThe frequency distribution of the vascular segment chord lengths was quantified by the global morphometric method. \u003cstrong\u003ef. \u003c/strong\u003eResults of quantitative analysis of chord length grading of vascular segments. 0 - 40 μm: Control, 28562 ± 3796; KO, 14707 ± 3312 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0089\u003c/strong\u003e). 40 - 80 μm: Control, 18621 ± 4102; KO, 8953 ± 1599 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0190\u003c/strong\u003e). 80 - 120 μm: Control, 7018 ± 2200; KO, 2906 ± 576 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0351\u003c/strong\u003e). 120 - 160 μm: Control, 2964 ± 816; KO, 1173 ± 234(\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0216\u003c/strong\u003e). 160 - 200 μm: Control, 1327 ± 270; KO, 481 ± 133(\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0082\u003c/strong\u003e). 200 - 240 μm: Control, 711 ± 94; KO, 204 ± 62 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0015\u003c/strong\u003e). 240 - 280 μm: Control, 435 ± 56; KO, 91 ± 17 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0005\u003c/strong\u003e). 280 - 320 μm: Control, 241 ± 12; KO, 40 ± 11 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt; 0.0001\u003c/strong\u003e). 320 - 360 μm: Control, 123 ± 9; KO, 15 ± 5 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt; 0.0001\u003c/strong\u003e). 360 - 400 μm: Control, 59 ± 8; KO, 12 ± 3 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0006\u003c/strong\u003e). \u003cstrong\u003eg. \u003c/strong\u003eFrequency distribution plots were quantitatively fitted to the vascular segment curved lengths, with only 0 - 400 μm selected for display. The red dashed line marked the curved lengths where the frequency of the vascular segment distribution was at its peak (bin width 10 μm, number of bins 91). \u003cstrong\u003eh.\u003c/strong\u003e Overall absolute frequency of the vascular segment curved lengths of the PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice group normalized by 50% downscaling, only 0 - 400 μm were selected for presentation (bin width 10 μm, number of bins 91). \u003cstrong\u003ei.\u003c/strong\u003e Results of the quantitative analysis of the mean of the overall vascular segment curved lengths. Control, 68.74 ± 5.93 μm; KO, 61.75 ± 2.89 μm (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.1403\u003c/strong\u003e). \u003cstrong\u003ej. \u003c/strong\u003eResults of quantitative analysis of curved length grading of vascular segments. 0 - 40 μm: Control, 23926 ± 3594；KO, 12142 ± 2887 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0114\u003c/strong\u003e). 40 - 80 μm: Control, 19513 ± 3163; KO, 9716 ± 1681 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0091\u003c/strong\u003e). 80 - 120 μm: Control, 8165 ± 2177; KO, 3645 ± 718 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0269\u003c/strong\u003e). 120 - 160 μm: Control, 3862 ± 1182; KO, 1624 ± 344 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0346\u003c/strong\u003e). 160 - 200 μm: Control, 1942 ± 576; KO, 741 ± 170 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0257\u003c/strong\u003e). 200 - 240 μm: Control, 1083 ± 299; KO, 370 ± 104 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0174\u003c/strong\u003e). 240 - 280 μm: Control, 661 ± 137; KO, 173 ± 47 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0043\u003c/strong\u003e). 280 - 320 μm: Control, 398 ± 78; KO, 82 ± 26 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0027\u003c/strong\u003e). 320 - 360 μm: Control, 254 ± 22; KO, 43 ± 12 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0001\u003c/strong\u003e). 360 - 400 μm: Control, 143 ± 29; KO, 24 ± 3 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0022\u003c/strong\u003e). \u003cstrong\u003ek. \u003c/strong\u003eSchematic representation of vascular tortuosity.\u003cstrong\u003e l.\u003c/strong\u003e Frequency distribution plots quantitatively fitted to vascular segment tortuosity, only 1 - 5 selected for presentation (bin width 0.5 μm, number of bins 283). \u003cstrong\u003em.\u003c/strong\u003e Overall absolute frequency of vascular segment tortuosity in the PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice group normalized to a 50% downscaled frequency, only 1 - 5 selected for presentation (bin width 0.5 μm, number of bins 283). \u003cstrong\u003en.\u003c/strong\u003e Results of the quantitative analysis of the mean overall vascular segment tortuosity. o. Results of the quantitative analysis of the graded vascular segment tortuosity. Control, 1.202 ± 0.009; KO, 1.212 ± 0.015 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.394\u003c/strong\u003e). \u003cstrong\u003eo. \u003c/strong\u003eResults of quantitative analysis of vascular segment tortuosity grading. 1 - 1.4: Control, 52410 ± 7564; KO, 24481 ± 4794 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0057\u003c/strong\u003e). 1.4 - 1.8: Control, 4356 ± 783; KO, 2375 ± 542 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0227\u003c/strong\u003e). 1.8 - 2.2: Control, 1093 ± 111; KO, 552 ± 144 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0068\u003c/strong\u003e). 2.2 - 2.6: Control, 477 ± 46; KO, 256 ± 64 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0081\u003c/strong\u003e). 2.6 – 3.0: Control, 243 ± 9; KO, 125 ± 28 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0023\u003c/strong\u003e). 3 - 3.4: Control, 152 ± 14; KO, 79 ± 15 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0036\u003c/strong\u003e). 3.4 - 3.8: Control, 62 ± 5; KO, 34 ± 11 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0164\u003c/strong\u003e). 3.8 - 4.2: Control, 103 ± 33; KO, 48 ± 15 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0602\u003c/strong\u003e). 4.2 - 4.6: Control, 66 ± 2; KO, 32 ± 15 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0168\u003c/strong\u003e). 4.6 – 5.0: Control, 24 ± 3; KO, 11 ± 3 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0039\u003c/strong\u003e). The data were presented as means ± SD. \u003cem\u003eP\u003c/em\u003e were calculated by unpaired two-tailed t-test (\u003cstrong\u003ef, i, j, n, o\u003c/strong\u003e) or Mann-Whitney U test (\u003cstrong\u003ee\u003c/strong\u003e), n = 3 for Control; n = 3 for KO animals were used.\u0026nbsp; *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001; ns: not significant.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/cac51aa3940aa8e59f7616c7.png"},{"id":79128688,"identity":"d60fc51d-255b-4fad-aec2-0b44cf80a2aa","added_by":"auto","created_at":"2025-03-24 18:06:26","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":661450,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResults of vascular distribution density and Phi and Theta distribution in double fluorescent transgenic mice with cerebellar neurodegeneration.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eSchematic diagram of vessel segment angles. phi value was the azimuth angle, which described the angle of the vessel segment in the XY plane with respect to the Z-axis in three dimensions. Theta value was the elevation angle, which was used to describe the angle of the vessel segment with respect to the XY plane. \u003cstrong\u003eb-i.\u003c/strong\u003e Computational analyses were carried out by using global morphometry. \u003cstrong\u003eb. \u003c/strong\u003eFrequency distribution plots were quantitatively fitted to theta values of the vascular segments.\u0026nbsp; (bin width 10 μm, number of bins 91).\u003cstrong\u003ec. \u003c/strong\u003eOverall absolute frequency of theta values of vascular segments in the PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice group was normalized by 50% dimensionality reduction. \u003cstrong\u003ed. \u003c/strong\u003eResults of quantitative analysis of the mean overall vascular segment theta values. \u003cstrong\u003ee. \u003c/strong\u003eResults of graded quantitative analysis of theta values in vascular segments. 0 - 15°: Control, 5729 ± 286; KO，3137 ± 850 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0075\u003c/strong\u003e). 15 - 30°: Control, 11375 ± 474; KO, 5895 ± 1119 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0015\u003c/strong\u003e). 30 - 45°: Control, 11379 ± 1542; KO, 5518 ± 1127 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0060\u003c/strong\u003e). 45 - 60°: Control, 10172 ± 1646; KO, 4658 ± 1006 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0078\u003c/strong\u003e). 60 - 75°: Control, 8544 ± 2064; KO, 3810 ± 858 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0214\u003c/strong\u003e). 75 - 90°: Control, 12928 ± 2903; KO, 5578 ± 1713 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0195\u003c/strong\u003e). \u003cstrong\u003ef.\u003c/strong\u003e Quantitative fitting of phi value of vascular segment by frequency distribution map. \u003cstrong\u003eg.\u003c/strong\u003e The overall absolute frequency of phi in PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was normalized by 50% dimensionality reduction. \u003cstrong\u003eh. \u003c/strong\u003eQuantitative analysis results of the average phi value of the whole vascular segment. Control, 183.10 ± 1.74°; KO, 181.70 ± 1.28° (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.3133\u003c/strong\u003e). \u003cstrong\u003ei. \u003c/strong\u003eResults of quantitative analysis of graded phi values of vascular segments. 0 - 45°: Control, 8428 ± 1227; KO, 4003 ± 821 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0066\u003c/strong\u003e). 45 - 90°: Control, 6733 ± 738; KO, 3285 ± 541 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0028\u003c/strong\u003e). 90 - 135°: Control, 7028 ± 1024; KO, 3438 ± 600 (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 0.0063\u003c/strong\u003e). 135 - 180°: Control, 6857 ± 927; KO, 3331 ± 761 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0070\u003c/strong\u003e). 180 - 225°: Control, 6528 ± 920; KO, 3086 ± 682 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0065\u003c/strong\u003e). 225 - 270°: Control, 7998 ± 1230; KO, 3541 ± 658 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0052\u003c/strong\u003e). 270 - 315°: Control, 8094 ± 862; KO, 3935 ± 867 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0041\u003c/strong\u003e). 315 - 360°; Control, 8461 ± 1335; KO, 3977 ± 931 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0088\u003c/strong\u003e). \u003cstrong\u003ej and k.\u003c/strong\u003e 3D network reconstruction of cerebellar vasculature in PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice over the range 0 - 360° (bar = 100 μm and bar = 200 μm). The data were presented as means ± SD. \u003cem\u003eP\u003c/em\u003e were calculated by unpaired two-tailed t-test (\u003cstrong\u003ed and e, h and i\u003c/strong\u003e). \u003cstrong\u003ee. \u003c/strong\u003en = 3 for Control; n = 3 for KO animals were used. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ns: not significant.\u003c/p\u003e","description":"","filename":"010.png","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/b42fda5c6e0f60146344586d.png"},{"id":79128665,"identity":"a593c979-70b2-431b-a2ef-9d139ac6ba3f","added_by":"auto","created_at":"2025-03-24 18:06:25","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":546065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of vascular branching nodes and terminal nodes in double-fluorescent transgenic mice with cerebellar neurodegeneration.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e The nodes were divided into branching nodes (hollow circles) and terminal nodes (solid blue circles), which represented the intersection points and ends of blood vessel branches respectively. \u003cstrong\u003eb-d.\u003c/strong\u003e 3D reconstruction of the nodes of the small cerebral vascular network in PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. \u003cstrong\u003eb. \u003c/strong\u003eTotal nodes of local cerebellum of mice. \u003cstrong\u003ec. \u003c/strong\u003eBranching nodes in local regions of mouse cerebellum. \u003cstrong\u003ed. \u003c/strong\u003eTerminal nodes of local cerebellum region of mice. \u003cstrong\u003ee-g. \u003c/strong\u003eResults of statistical quantitative analysis of nodes. Number of nodes: Control, 13617 ± 2030; KO, 10137 ± 1879 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0948\u003c/strong\u003e). Number of terminal nodes: Control, 5287 ± 1187; KO, 3527 ± 793 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0997\u003c/strong\u003e). Number of branching nodes: Control, 8330 ± 880; KO, 6609 ± 1120 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.2000\u003c/strong\u003e). The data were presented as means ± SD. \u003cem\u003eP\u003c/em\u003e were calculated by unpaired two-tailed t-test (\u003cstrong\u003ee and f\u003c/strong\u003e) or Mann-Whitney U test (\u003cstrong\u003eg\u003c/strong\u003e).\u003cstrong\u003e \u003c/strong\u003en = 3 for Control; n = 3 for KO animals were used. ns: not significant.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/e0072054319872d9083ece74.png"},{"id":79129411,"identity":"61e02075-d6ea-457d-bd36-78742fde314f","added_by":"auto","created_at":"2025-03-24 18:14:26","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":613110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D fluorescent reconstruction of PCs and vessels in cerebellar neurodegeneration double-fluorescent transgenic mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reconstruction of cerebellar 3 - 7 lobular vessels and PCs in PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen \u003c/sup\u003emice was demonstrated. On the left was the upper view of the whole brain, in the middle was the enlarged ROI image of the cerebellum, the green fluorescence image was the blood vessel, and the red fluorescence image was the PCs. On the far right was an enlarged image of the ROI region of the middle cerebellar fluorescence image. bar = 500 μm and bar = 200 μm. PCL: Purkinje Cell Layer; GCL: Granular Cell Layer; MCL: Molecular Cell Layer;\u0026nbsp; Cb: Cerebellar Lobules; SCA: Superior Cerebellar Artery.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/f2dd1bce5282e1e2336d7a8f.png"},{"id":79130259,"identity":"45696cf1-cf14-46e1-9fe1-189075c929a7","added_by":"auto","created_at":"2025-03-24 18:30:26","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":521391,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatial relationship between PCs and vascular structure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea and b.\u003c/strong\u003e PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, the results of 3D reconstruction of MCL blood vessels and PCs in the region of the superior cerebellar artery supply area (4/5Cb-SCA) of the cerebellar 4/5 lobule. The purple was the large blood vessels, the green was the capillaries, and the blue was the PCs. The following figure showed the enlarged results in the box (bar = 200 μm and bar = 50 μm). \u003cstrong\u003ec.\u003c/strong\u003e Distance between PCs and blood vessel. These results were analyzed by Mann-Whitney test. Control, 16.540 ± 7.317 μm; KO, 7.151 ± 9.067 μm (\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt;0.0001\u003c/strong\u003e). n = 3 mice per group, ****P \u0026lt; 0.0001. \u003cstrong\u003ed-h.\u003c/strong\u003e Results of quantitative analysis of vascular VF. The data were presented as means ± SD. \u003cem\u003eP\u003c/em\u003e were calculated by unpaired two-tailed t-test. PC-Vessel VF: Control, 1039.000 ± 362.000; KO, 48.440 ± 39.950 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0092\u003c/strong\u003e). PC-Capillary VF: Control, 1904.000 ± 634.600; KO, 95.040 ± 80.930 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0081\u003c/strong\u003e). PC-Noncapillary VF: Control, 2292.000 ± 839.700; KO, 101.300 ± 82.860 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0109\u003c/strong\u003e). Noncapillary-Cerebellum VF: Control, 0.025 ± 0.007; KO, 0.038 ± 0.015 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e0.2514\u003c/strong\u003e\u003c/em\u003e). Capillary-Cerebellum VF: Control, 0.029 ± 0.009; KO, 0.047 ± 0.001 (\u003cem\u003e\u003cstrong\u003eP \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e= 0.0263\u003c/strong\u003e). n = 3 for Control; n = 3 for KO animals were used. *\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05; **\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01; ****\u003cem\u003eP \u0026lt; \u003c/em\u003e0.0001; ns: not significant. Cb: Cerebellar Lobules; SCA: Superior Cerebellar Artery; VF: Volume Fraction.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/a6fb3150f9aad76959b3a0a7.png"},{"id":79130688,"identity":"5657d0a1-969c-403a-bcab-4b940653c9bb","added_by":"auto","created_at":"2025-03-24 18:38:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7705778,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/3da6abd9-28cf-4b1d-b78b-1fc37fd2d8da.pdf"},{"id":79128656,"identity":"c2ebf364-f9df-45de-99fd-c8258f1c99b6","added_by":"auto","created_at":"2025-03-24 18:06:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":57897,"visible":true,"origin":"","legend":"","description":"","filename":"Video1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/8f3c5a63a1384e7a0faf25be.docx"},{"id":79129390,"identity":"92f9f4e6-762a-49af-a416-58f0f4200754","added_by":"auto","created_at":"2025-03-24 18:14:25","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":152097,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-5607179/v1/e6ed18c52f721d5569f1cc87.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The characteristics of capillary remodeling in cerebellar neurodegenerative diseases revealed through layered imaging and stereoscopic analysis","fulltext":[{"header":"Highlights","content":"\u003col\u003e\n \u003cli\u003eWhole cerebellum clearing combined with the\u0026nbsp;Amira / Imaris system was used to conduct three-dimensional layered imaging and computational analysis of the cerebellar vascular network in adult control mice and double-fluorescent transgenic mice with cerebellar neurodegenerative disease (CBND).\u003c/li\u003e\n \u003cli\u003eThe results showed significant loss of capillaries with diameters ≤ 10 μm, while their vascular topology remained stable. Additionally, the distance between Purkinje cells and blood vessels decreased from 16 μm to 7 μm, which was central to cerebrovascular remodeling in CBND.\u003c/li\u003e\n \u003cli\u003eThe findings indicated that an increase in capillary-cerebellum volume fraction and a decrease in Purkinje cell-vessel volume fraction could serve as biological markers for the early diagnosis of CBND.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe function of the human brain is highly dependent on the vascular system, with the brain constituting only 2% of body weight but consuming 20% of cardiac output and 20% of oxygen and glucose \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. The human cerebral blood vessels consist of a complex three-dimensional (3D) cerebrovascular network spanning 644 km, ensuring that the distance between each neuron and the nearest capillary is no more than 22\u0026ndash;27 \u0026micro;m, for timely and effective oxygen delivery \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. The relationship between the brain and blood vessels in mice follows the same pattern \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNeurodegenerative diseases are a group of clinical disorders characterized by progressive loss of neurons leading to impaired brain structural integrity and abnormal function, including Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia, Huntington's disease, traumatic brain injury, stroke, multiple sclerosis (MS), and amyotrophic lateral sclerosis, etc., which can cause extensive cerebral vascular remodeling \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. In the AD mouse model, super-resolution ultrasound imaging showed that the length of cerebral blood vessels was shortened and the flow rate decreased\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Meanwhile, imaging results showed a significant decline in cerebrovascular reactivity and blood flow regulation in AD patients \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. In addition, in patients with mild cognitive impairment, AD, dementia, MS, and PD, retinal imaging studies revealed vascular pathological changes such as reduced capillary blood flow, vascular Aβ deposition, and impaired blood-retinal barrier \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. An increasing body of evidence suggests that dysfunction of the vascular system, especially dysregulation of the microvasculature, is expected to be a biomarker for the early diagnosis of neurodegenerative diseases.\u003c/p\u003e \u003cp\u003eNeurovascular coupling (NVC) is the close interaction between neurons and the vascular system in the brain, with capillaries playing a key role \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Specifically, pericytes and microglia surrounding the capillaries could regulate the diameter and blood flow of capillaries through molecular mechanisms such as intercellular tunneling nanotubes and P2RY12 receptors, maintaining the stability of the cerebral microcirculation \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Optogenetic studies in mouse models also showed that although the contraction of pericytes was slow, it would consistently reduce the diameter of capillaries and decrease blood flow, which had profound effects on long-term cerebral blood flow regulation \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Although these findings have emphasized the core role of capillaries in NVC, serving not only as a platform for the exchange of substances between blood and nerve cells but also as a critical node for signal regulation \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, it remains unclear how neurons at risk of progressive loss in the state of neurodegenerative diseases interact with their surrounding capillaries.\u003c/p\u003e \u003cp\u003eHowever, most of existing researches have largely focused on the morphological and functional changes of larger blood vessels in the brain, while there is short of comprehensive exploration of changes at the capillary level. The reason for this lies in the lack of 3D imaging and computational analysis techniques for the fine structure of the cerebrovascular network. At present, the study of cerebrovascular network has usually used vascular casting, tissue clearing, microscopic imaging, topological data analysis and other methods to visualize and assess structural differences in the cerebrovascular system at the single microvascular level \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Furthermore, single-cell and spatial transcriptomics methods have revealed molecular differences between various cells in the brain, providing insights into the regional heterogeneity of the vascular system \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Recent studies have also used 3D bioprinting to construct various microvascular models for comprehensive analysis of vascular function \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. These attempts have tried to analyze the remodeling of capillary network with a diameter of 10 \u0026micro;m, but they still fail to provide information on topological changes of capillaries, such as nodes, bends, and tortuosity. Consequently, there is a significant lack of research on the spatial location relationship between neurons and capillaries.\u003c/p\u003e \u003cp\u003eOur group has previously successfully generated Drp1 (Dynamin-related protein 1) knockout mice in cerebellar Purkinje cells (PCs), which have typical characteristics of cerebellar neurodegenerative disease (CBND). To further address this issue, our team constructed a double-fluorescent CBND mouse model with targeted knockout (KO) of the mitochondrial fission protein Drp1 in PCs, named PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice due to their expression of the red fluorescent protein tdTomato in PCs and green fluorescent protein ZsGreen in the cerebellar vascular system (arteries and veins couldn\u0026rsquo;t be distinguished). Subsequently, we used whole cerebellum clearing combined with the Amira/Imaris system to perform 3D hierarchical imaging and computational analysis on the cerebellar vascular network of adult Control mice and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. A total of three levels of indicators were analyzed. The first-level indicators were the overall situation, including 5 indicators such as the number of PCs, total volume of PCs, total volume of the cerebellum, total length of cerebellar blood vessels, and total volume of cerebellar blood vessels. The second-level indicators distinguished noncapillaries with a diameter of greater than 10 \u0026micro;m and capillaries with a diameter of less than 10 \u0026micro;m, each including 7 indicators such as volume, chord length, curved length, tortuosity, number of nodes, φ direction value, and θ direction value. The third-level indicators were volume fraction (VF) and neuron-vascular distance, including 7 indicators such as capillary-cerebellum VF, PC-vessel VF, PC-capillary VF, PC-noncapillary VF, vessel-cerebellum VF, noncapillary-cerebellum VF, and the shortest distance between PCs and blood vessels. Our results indicated that capillaries with a diameter\u0026thinsp;\u0026le;\u0026thinsp;10 \u0026micro;m were significantly lost, but their vascular topological structure remained stable. Moreover, the nearest distance between PCs and blood vessels decreased from 16.6 \u0026micro;m to 7.0 \u0026micro;m, which represented the core of cerebrovascular remodeling in CBND. Additionally, the increased capillary-cerebellum VF and the decreased PC-vessel VF might serve as biological markers for the early diagnosis of CBND. These findings provide valuable insights for the early diagnosis of CBND and the development of targeted therapies.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eConstruction of a double-fluorescent transgenic mouse of PCKO\u003c/b\u003e \u003csup\u003e \u003cb\u003eTomato\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eVessel\u003c/b\u003e \u003csup\u003e \u003cb\u003eGreen\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eto provide a tool for the study of CBND\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA double-fluorescent transgenic mouse model of CBND, PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e, was constructed using Cre-LoxP technology. This model featured progressive loss of cerebellar PCs expressing red fluorescence, while the molecular layer vessels expressed green fluorescence, providing a tool for detailed 3D cerebrovascular network studies.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the research group first compared Pcp2\u003csup\u003eCre\u003c/sup\u003e mice purchased from the Jackson Laboratory (cerebellar PCs carrying Cre gene\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e) with B6/JGpt-H11\u003csup\u003eem1Cin (CAG\u0026minus;LoxP\u0026minus;ZsGreen\u0026minus;Stop\u0026minus;LoxP\u0026minus;tdTomato) /Gpt\u003c/sup\u003e (B6-G/R) mice purchased from the GemPharmatech Co., Ltd (the mice all endothelial cells express green fluorescence ZsGreen and carry LoxP units\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e) hybrid. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb showed that after validation by genotype identification, the resulting transgenic mice were designated PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e transgenic mice. The confocal microscopy results in the top row in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec showed that PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e transgenic mice cut Cre gene to make PCs carry red fluorescence tdTomato, while blood vessels still expressed green fluorescence ZsGreen.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice were then cross-bred with Drp1\u003csup\u003ef/f\u003c/sup\u003e mice purchased from Jackson's laboratory (the mouse had mitochondrial division protein Drp1 with LoxP units on both sides). Transgenic mice (named PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e) targeting PC-specific Drp1 knockout were obtained. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb showed that Pcp2\u003csup\u003eCre\u003c/sup\u003e gene produced 567 bp bands, Loxp in Drp1\u003csup\u003eflox/flox\u003c/sup\u003e produced 291 bp bands, and B6-G/R mouse Loxp gene produced 1465 bp bands, which verified the gene expression correctness of the two transgenic mice. The confocal microscopy results in the lower row in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec showed that PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice's cerebellar PCs expressed red fluorescence while molecular layer vessels expressed green fluorescence, providing a tool for the fine study of 3D cerebellar vascular network.\u003c/p\u003e \u003cp\u003eThe cerebellum and PCs status of adult (2 - month - old) PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice (Control) and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice (KO) were further compared. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec showed that the cerebellum of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice still had the appearance of 10 lobules in the vermis. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed analysis of the morphological data of vermis on the median sagittal surface showed that compared to Control mice, the cerebellar area in PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was reduced by 43%, from 7652719 \u0026plusmn; 453169 \u0026micro;m\u0026sup2; in Control mice to 4340231 \u0026plusmn; 93977 \u0026micro;m\u0026sup2; in KO mice (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e). The area of PCs somas decreased by 19%, from 261.80 \u0026plusmn; 33.54 \u0026micro;m\u0026sup2; in Control mice to 211.40\u0026thinsp;\u0026plusmn;\u0026thinsp;75.13 \u0026micro;m\u0026sup2; in KO mice (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0003\u003c/b\u003e). The density of PCs per unit area was reduced by 72%, from 3.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06 cells / \u0026micro;m\u0026sup2; in Control mice to 1.10 \u0026plusmn; 0.42 cells/\u0026micro;m\u0026sup2; in KO mice (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e). Additionally, the thickness of the granule cell layer (GCL) decreased by 21%, from 418.00 \u0026plusmn; 71.58 \u0026micro;m in Control mice to 329.00 \u0026plusmn; 59.04 \u0026micro;m in KO mice (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0020\u003c/b\u003e), while the thickness of the molecular layer (MCL) was reduced by 40%, from 148.40 \u0026plusmn; 23.76 \u0026micro;m to 88.62 \u0026plusmn; 13.62 \u0026micro;m (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e). These results indicated that PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice were double-fluorescent transgenic mice with cerebellar neurodegeneration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVerification of the applicability of PCKO\u003c/b\u003e \u003csup\u003e \u003cb\u003eTomato\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eVessel\u003c/b\u003e \u003csup\u003e \u003cb\u003eGreen\u003c/b\u003e \u003c/sup\u003e \u003cb\u003ein fine study of 3D cerebellar vascular network remodeling in CBND\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhether the expression of green fluorescent protein in PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e double-fluorescent transgenic mice can accurately label all blood vessels is the key of this experiment. According to previous literature reports, the vascular marker lectin staining could label all blood vessels in the cerebellum, including large vessels with a larger lumen diameter and capillaries with a diameter less than 10 \u0026micro;m \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Therefore, we performed lectin immunofluorescence staining on PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, the maternal mouse of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e, and then counted its double labeling with green fluorescent protein ZsGreen. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea showed the superposition results of green ZsGreen, red Lectin, and blue DAPI under confocal microscope. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb was locally enlarged, and the results showed that red Lectin clearly delineated the movement of large blood vessels (shown by white arrows) and internal small blood vessels (shown by white arrows) between the molecular layer MCL of the lobule of 4/5Cb and the MCL of the 6Cb lobule. Green fluorescent protein and red Lectin were almost completely co-labeled. DAPI showed a large number of cells in the GCL layer. The co-labeling efficiency diagram of Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec showed the co-labeling in the third row of Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, and the results showed that green fluorescent protein and red Lectin almost completely overlapped. These results indicated that the double-fluorescent transgenic mice of CBND, PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, constructed by us, provided a tool for the fine study of 3D cerebellar vascular network remodeling of CBND.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of transparent cerebellum of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eIn order to further quantify the changes of cerebellar vascular network in CBND model mice, the transparent cerebellum of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was constructed. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea showed in detail the transparency process of mice in the control group (n\u0026thinsp;=\u0026thinsp;3) and the experimental group (n\u0026thinsp;=\u0026thinsp;3), which involved up to 14 days. First, the tissue was cleaned, the fat removal solution was added for 6 days, and the above process was repeated on day 7\u0026ndash;12. After the lipid removal was completed, refractive index matching was performed for two days, and the tissue was placed on a transparent substrate to obtain the transparent result. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, Imaris software was used to collect 3D fluorescence imaging of transparent tissues from the upper view (first row), the lateral view (second row), the posterior upper view (third row) and the posterior view (fourth row). The left side of each group showed the whole brain results, the right side showed the local cerebellar magnifying results, and the main blood vessels passing through the cerebellum were labeled. The results showed that the red pseudo-color marked PCs were significantly lost in the cerebellum of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e, and the green pseudo-color marked blood vessels maintained the anatomical line and structure of the trunk in the cerebellum of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to investigate the changes of cerebellar vasculature and PCs in mice, next, we further remodeled the cerebellar tissues in 3D with the help of Amira software. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e showed the results of the above view. We found that the width of the cerebellar earthworm section of the PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was narrowed, and at the same time, the PCs in the earthworm section were severely lost, and the PCs in both hemispheres were also lost, but less lost compared to their own earthworm part. We also observed the changes in the vascular trunk, and found that the vascular trunk of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was shorter but the overall morphology was relatively stable, and the density of capillaries on both sides of the trunk, especially in the 6Cb-SCA, was reduced.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther magnifying the results of the rear view, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e showed that the number of PCs in the vermis region of the cerebellum of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was significantly reduced, and the width of the vermis was also significantly narrowed. In contrast, relatively few PCs were lost in the Sim, Crus1, and Crus2 areas on both sides of the cerebellum. At the same time, the cerebellar vascular network of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice showed that the overall morphology of main blood vessels was relatively stable, and the capillary density on both sides of the main body, especially 3Cb-superior cerebellar artery (SCA), was significantly reduced.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further presented the results of the lateral view and labeled the different lobules of blood vessels and PCs. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e showed that the overall area of PCs distribution in PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was noticeably reduced. In addition, the integrity of the cerebellar vascular network was substantially changed in KO mice: the main vessel became shorter and the capillary density was significantly reduced, especially in the posterior inferior cerebellar artery supply area (PICA) and the SCA supply area of the 6th lobule (6Cb - SCA). In contrast, in Control mice, PCs and cerebellar vascular network were evenly distributed with good structural integrity, main blood vessels and their branches extended to various functional areas, and capillaries were densely distributed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePrimary indicators of CBND mice, including PCs count, PCs volume, cerebellar volume, total cerebellar vascular length, and total cerebellar vascular volume\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further quantify the changes in the cerebellar vascular network in the CBND model mice, we employed tissue clearing technology combined with 3D reconstruction and light-sheet microscopy to analyze the vascular structures and PCs across the entire cerebellar region, focusing on primary indicators. The cell bodies and dendrites of PCs are located in the molecular layer (MCL) and Purkinje cells layer (PCL) of the cerebellum. We aimed to explore the changes of blood vessels in the progressive loss of neurons, so we divided the cerebellar vessels into the PCL and the MCL, and focused our analysis on the cerebellar vessels. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb revealed that in contrast to PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, the average number of PCs in PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice declined from 56470 \u0026plusmn; 19787 to 4111 \u0026plusmn; 3621, representing a significant reduction of 93% (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0108\u003c/b\u003e). This notable reduction in PC indicated a considerable loss of cells in the cerebellum of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea presented the morphological outcomes of the total cerebellar volume. The results indicated that the average total cerebellar volume of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice decreased from 217.13 \u0026plusmn; 20.35 mm\u0026sup3; to 66.57 \u0026plusmn; 16.35 mm\u0026sup3; compared with the Control mice. A significant reduction of 69% (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0006\u003c/b\u003e) was observed, suggesting that the deletion of Drp1 led to significant cerebellar atrophy. Simultaneously, significant differences also emerged in the cerebellar vascular network, and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec showed that the average total vascular length in PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was significantly reduced by 58%, from 4149.06 \u0026plusmn; 819.26 mm in Control mice to 1763.03 \u0026plusmn; 348.03 mm in KO mice (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0097\u003c/b\u003e). This implied that PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice impaired vascularization and obvious atrophy or reduction of the cerebellar vascular network, which might be related to the effect of Drp1 gene knockout on angiogenesis or maintenance. The statistics of the total volume fraction of the cerebellar vessels in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed demonstrated that there was no statistical difference. This suggested that the deletion of Drp1 had a significant impact on the cerebellar structure and the distribution and extensibility of the cerebellar vascular network. However, the total vascular volume and vascular VF did not change significantly, possibly indicating that the structure of the remaining vessels maintained a relatively constant VF.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSecondary indicators of CBND mice, including vascular diameter and volume distribution\u003c/h3\u003e\n\u003cp\u003eBy defining nodes and segments, the cerebellar vascular network was segmented into independent segments, providing a foundation for subsequent analyses of volume, length, and radius\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Capillaries typically have a diameter of 10 \u0026micro;m or less\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, and the subdivision of vascular architecture in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea classified vessels based on diameter as capillary (\u0026le;\u0026thinsp;10 \u0026micro;m) or noncapillary (\u0026gt;\u0026thinsp;10 \u0026micro;m)\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. To display the 3D structure more visually, we utilized Amira/Imaris software to reconstruct the fluorescence results of blood vessels and PCs. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb indicated that the cerebellar vascular network of PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was tightly structured and regularly distributed. In contrast, the cerebellar vascular network was sparse in PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, suggesting that Drp1 deletion led to a significant decrease in cerebellar vessel density. We further conducted statistics on vessel radius and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec \u003cb\u003e- d\u003c/b\u003e revealed that PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice exhibited significant changes in the radius distribution of cerebellar vessels. We normalized the overall absolute frequency of the Control mice by 50% dimensionality reduction and found that the Control mice showed a very similar interval distribution to that of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, and most of the vessel segments were concentrated in small-radius vessels. We statistically analyzed the number of vascular segments within different range intervals. \u003cb\u003eFigure. 8f\u003c/b\u003e showed that, compared to the control group, the vascular frequency in the 1\u0026ndash;3 \u0026micro;m short radius range in PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice decreased from 46145 \u0026plusmn; 7940 to 21811 \u0026plusmn; 4578, a reduction of 53% (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0100\u003c/b\u003e). In the 3\u0026ndash;5 \u0026micro;m short radius range, the vascular frequency decreased from 9490\u0026thinsp;\u0026plusmn;\u0026thinsp;1554 to 4451 \u0026plusmn; 687, also a reduction of 53% (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0068\u003c/b\u003e).These changes suggested that the development or maintenance of small radius blood vessels, especially capillaries, was seriously affected, which directly impacted the regional blood perfusion and oxygen transport ability of the cerebellum, and might led to insufficient energy supply and metabolic disorders in neurons. The statistical analysis of the average radius of blood vessels in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee showed that there was no significant difference in the mean radius of the blood vessels between the two groups, probably because, despite the reduction of the small radius of capillaries was reduced, the larger radius vessels (e.g. trunk vessels or arterioles) might have been relatively preserved and compensated to some extent, resulting in no significant change in mean radius.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther analyzing the volume of the vascular segments, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eg showed that the vast majority of the vascular segments were small in size, concentrating in the range of 0 to 6000 \u0026micro;m\u0026sup3;.The distribution frequency curves of the PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice as a whole were located below those of the PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, especially in the small volume segments. In \u003cb\u003eFigure. 8h\u003c/b\u003e, the overall absolute frequency of PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was normalized by 50% dimensionality reduction, and it was found that the two fitting curves were highly consistent, which revealed the systematic reduction of the density of the cerebellar vascular network by Drp1 gene knockout. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ej statistically analyzed the distribution of vascular segments in different volume ranges, and the results showed that in a smaller volume range (0\u0026ndash;6000 \u0026micro;m\u0026sup3;), the frequency of vascular segments in PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was significantly lower than that in PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. Specifically, the frequency of vascular segments in PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was reduced from 47244 \u0026plusmn; 7918 to 22693 \u0026plusmn; 4799, a 52% reduction (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0101\u003c/b\u003e), from 6277 \u0026plusmn; 1100 to 2714 \u0026plusmn; 520, a 57% reduction (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0071\u003c/b\u003e), and from 2369 \u0026plusmn; 726 to 1070 \u0026plusmn; 316, a 55% reduction (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0467\u003c/b\u003e), in the segmental volume ranges of 0\u0026ndash;2000 \u0026micro;m\u0026sup3;, 2000\u0026ndash;4000 \u0026micro;m\u0026sup3;, and 4000\u0026ndash;6000 \u0026micro;m\u0026sup3;, respectively. These results showed that the number of blood vessels in these small volume segments was significantly reduced in PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. However, in the larger volume range (6000 \u0026micro;m\u0026sup3; and above), there was no significant difference in vessel segment volume frequency between PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice and Control mice. Statistical analysis of the mean volume of blood vessels was performed in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ei, and the results showed that there was no statistically significant difference in the mean value between Control mice and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. It might be that the small volume vessel segment was smaller in volume in the overall vascular structure, and the reduction in number had a weaker pulling effect on the average volume. This further showed that the contribution of capillaries to the total blood vascular volume was small, but the effect was huge.\u003c/p\u003e\n\u003ch3\u003eThird level indicators of CBND mice including vascular segment length and curvature distribution\u003c/h3\u003e\n\u003cp\u003eReduced blood vessel length is associated with brain function degradation\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. We analyzed the length and distribution of blood vessels in PC\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice and PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea showed the model diagram of blood vessel length, and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb showed the 3D fluorescence diagram showing the length of blood vessels. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec \u003cb\u003eand g\u003c/b\u003e showed that the frequency of PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice in the whole length (chord length/curved length) was significantly lower than that of Control mice. In Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed \u003cb\u003eand h\u003c/b\u003e, the overall absolute frequency of mice in the control group was standardized by 50% dimensionality reduction, and the curve of its shape was very similar to that of PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. This observation suggested that although the absolute frequency of PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was low, some degree of cerebellar vascular network structure was retained. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ef \u003cb\u003eand j\u003c/b\u003e made a statistical analysis of the distribution of the length of blood vessel segments. The absolute frequency of Control mice was higher than that of PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice in almost all the length intervals of blood vessels, and the difference between the two groups showed an increasing trend, especially in the longer intervals. Specifically, compared with PC\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, the chord length distribution frequency of PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice decreased by 49% from 28562 \u0026plusmn; 3796 at 0\u0026ndash;40 \u0026micro;m to 14707 \u0026plusmn; 3312 (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0089\u003c/b\u003e). With the increase of blood orchestra length, the frequency of vessel segment distribution decreased from 59 \u0026plusmn; 8 to 12 \u0026plusmn; 3, which decreased by 80% (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0006\u003c/b\u003e) at 360\u0026ndash;400 \u0026micro;m. The curved length showed the same trend. Compared with PC\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, the distribution frequency of curved length decreased by 49% from 23926 \u0026plusmn; 3594 to 12142 \u0026plusmn; 2887 at 0\u0026ndash;40 \u0026micro;m (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0114\u003c/b\u003e). With the increase of blood curved length, the frequency of vessel segment distribution decreased by 83% from 143 \u0026plusmn; 29 to 24 \u0026plusmn; 3 at 360\u0026ndash;400 \u0026micro;m (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0022\u003c/b\u003e). The above results suggested that vascular growth and repair mechanisms might be inhibited or incompletely activated following Drp1 deletion, resulting in reduced overall vascularization, particularly in medium and long length vessels. However, the presence of a relatively concentrated distribution within short length intervals might indicate that PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice retained minimal cerebellar vascular network function to maintain minimal tissue blood supply. Next, we statistically analyzed the mean lengths of the vessel segments, and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ee \u003cb\u003eand i\u003c/b\u003e indicated that the mean lengths were not statistically different, which might be due to the fact that the number of vessel segments in the short length interval dominated overall and was relatively stable in PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, resulting in a decrease in the number of long segments of the vessels with little effect on the mean value.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eChanges in vascular tortuosity have been shown to correlate with the severity of cerebrovascular diseases\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Therefore, we analyzed the tortuosity and distribution of blood vessels in PC\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e and PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ek was the schematic diagram of vascular tortuosity, and the number of segments in each range of vascular tortuosity was counted in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003el \u003cb\u003eand o\u003c/b\u003e. It was found that the absolute frequency of vascular tortuosity in PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice in most tortuosity ranges was significantly reduced to about half of that in the control group. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003em normalized the absolute frequency of PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice by 50% dimensionality reduction, and it was found that the two fitting curves were highly consistent. This result indicated that degenerative lesions led to an overall decrease in the density of vascular distribution. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003en calculated the average tortuosity of blood vessels in PC\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice and PCKO\u003csup\u003eTomoato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. The results showed that there was no significant difference in the average tortuosity of blood vessels between the two groups, indicating that the lesions might be more prone to reduce the density of vessels with different tortuosity. It did not directly affect the curved structure of the blood vessels. This meant that the microstructure of the cerebellar vascular network changed significantly in distribution density during neurodegeneration, but its tortuosity remained relatively constant.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe fourth level indicators of CBND mice including density of vascular segments and the topological structure\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe branching angle of blood vessels can help reveal the random branching pattern of blood vessels, which is crucial for understanding the development of blood vessels and optimizing the cerebrovascular network structure\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. To explore the effect of progressive neuronal loss on the branching angle of vascular segments, we analyzed the orientation of the vascular segments of PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea showed a schematic diagram of branching angles. Figure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb \u003cb\u003eand c with f and g\u003c/b\u003e showed that DRP1 knockdown-induced cerebellar neurodegeneration resulted in a general reduction in vascular distribution across angular intervals in PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice but did not show significant frequency peaks or dramatic fluctuating angular preferences. Figure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ee \u003cb\u003eand i\u003c/b\u003e showed that the angular distribution of the within each angle interval, the absolute frequency of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was significantly reduced by about half compared to controls. This consistent decrease suggested that CBND broadly affected the spatial distribution of the vasculature and were not concentrated in a particular direction, implying a significant perturbation of the overall spatial organization of the cerebellar vasculature. Phi (φ) values are azimuthal angles, describing the angle of the vascular segment in the XY plane with respect to the Z-axis in three dimensions, ranging from 0\u0026ndash;360\u0026deg;. Theta (θ) values are elevation angles, indicating the offset of the vascular segment in the Z-axis direction, describing the angle of the vascular segment with respect to the XY plane, ranging from 0\u0026ndash;90\u0026deg;. Notably, despite the significant differences between the angular intervals, Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ed \u003cb\u003eand h\u003c/b\u003e showed that the differences in the means and values between the two groups of mice were not statistically significant. Figure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ej \u003cb\u003eand k\u003c/b\u003e demonstrated the angle of vascular segments in different interval ranges in PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice by Imaris software. The above results indicated that the overall directionality of the vessels was not significantly altered after progressive neuronal loss, suggesting that the microstructure of the cerebellar vessels underwent extensive but balanced reorganization in CBND.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe density of branch points and the topological and functional properties of vessels were also used to assess the health of cerebrovascular network and disease progression\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR48 CR49\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003ea showed a schematic diagram of the cerebellar vascular network nodes, including branch nodes and terminal nodes. Using Amira software, Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eb showed partial cerebellar vascular network in the cerebellum of PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. In Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003ec \u003cb\u003eand d\u003c/b\u003e, the 3D reconstruction of branch and terminal nodes was performed using Imaris software, respectively. The statistics of total nodes, branch nodes, and terminal nodes in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003ee \u003cb\u003e- g\u003c/b\u003e showed that there was no significant difference in branching nodes between PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. This further indicated that although CBND had extensive effects on vascular distribution and density, it did not change the overall connectivity characteristics and branching structure of the cerebellar vascular network. However, changes at other levels of detail, such as the distribution density of specific vessel segments or microcirculation regulation, might be further affected by the lesion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCriticality of microcirculation to the metabolic demands of cerebellar regions\u003c/h3\u003e\n\u003cp\u003eTo explore the relationship between neurons and blood vessels after neurodegenerative lesions, we performed a presentation of PC-vessel VF. Using Amira software, Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e displayed the 3D fluorescence results of PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e cerebellar MCL blood vessels and PCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eVascular VF is an important measure of tissue blood supply status, metabolic requirements and microcirculation efficiency \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e \u0026ndash; \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003ea \u003cb\u003eand b\u003c/b\u003e used Imaris software to perform 3D reconstruction of the MCL blood vessels and PCs in the region of the SCA supply area of the cerebellar 4/5 lobule (4/5Cb - SCA). We found that compared with PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice more PCs entered the vascular interior, capillaries showed a relatively obvious decrease in density, and the reduction of PCs number was more significant than that of capillaries. Figure\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003ec showed that compared with PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, the distance between PCs and blood vessels in PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice decreased from 16.540 \u0026plusmn; 7.317 \u0026micro;m to 7.151 \u0026plusmn; 9.067 \u0026micro;m, with a 58% reduction (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003ed showed that compared with PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice PC-vessel VF decreased from 1039.000 \u0026plusmn; 362.000 to 48.440 \u0026plusmn; 39.950. This was a 95% reduction (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0092\u003c/b\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003ee showed that PC-capillary VF decreased from 1904.000 \u0026plusmn; 634.600 to 95.040 \u0026plusmn; 80.930, with a 95% reduction (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0081\u003c/b\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003ef showed that PC-noncapillary VF decreased from 2292.000 \u0026plusmn; 839.700 to 101.300 \u0026plusmn; 82.860, with a 96% reduction (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0109\u003c/b\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eg showed that there was no statistically significant difference in noncapillary vessels - CB VF, which further verified that Drp1 gene knockout did not change the overall VF of large diameter vessels, especially in the noncapillary VF with a diameter of \u0026gt;\u0026thinsp;10 \u0026micro;m. Figure\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eh showed that, compared with PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, capillary - CB VF of PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice increased from 0.029 \u0026plusmn; 0.009 to 0.047 \u0026plusmn; 0.001, an increase of 59% (\u003cb\u003eP\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0263\u003c/b\u003e), which suggested that cerebellar lesions were concentrated at the microcirculatory level, while the large vascular system might still support global blood flow. These results suggested that the distance between Purkinje cells and blood vessels decreased from 16 \u0026micro;m to 7 \u0026micro;m, which might be a central feature of neurovascular remodeling in cerebellar neurodegenerative disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePanoramic display of capillary remodeling features in CBND\u003c/h3\u003e\n\u003cp\u003eTo visually compare the brain tissue of PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice, a 3D reconstruction was performed and shown in a three-minute Video 1 (the two-dimensional code was shown in \u003cb\u003eGraphical abstract\u003c/b\u003e). The results showed that the brain tissues of normal mice and CBND mice were labeled with tdTomato for PCs and ZsGreen for blood vessels. The 0\u0026ndash;4\" results showed that the overall volume of CBND brain tissue was smaller than that of normal brain tissue, and the area of cerebellar region was reduced in the sagittal direction. 5\u0026ndash;30\" showed that the cerebellar volume of CBND mice was significantly reduced after reconstructing the cerebellar volume. On 31\" \u0026minus;\u0026thinsp;1'10\" display, the coronal section area of brain tissue in CBND mice was reduced when the images were observed in the coronal direction. 1'11\" \u0026minus;\u0026thinsp;1'15\" showed that the volume of blood vessels in the region constituting the cerebellum was also reduced. In 1'16\" \u0026minus;\u0026thinsp;2' display, when the images were magnified, it was observed that the noncapillary volume in the cerebellar region of the CBND mice was reduced, and the capillary volume was severely reduced. 2' \u0026minus;\u0026thinsp;2'45\" showed that the number of PCs in the cerebellum of CBND mice was reduced, accompanied by cell body swelling. The 2'50\" \u0026minus;\u0026thinsp;3' display image in the horizontal direction showed that the horizontal section area of brain tissue in CBND mice was reduced.\u003c/p\u003e"},{"header":"Discussions","content":"\u003cp\u003eNeurodegenerative diseases are characterized by progressive loss of neurons leading to impaired structural integrity and dysfunction of the brain, which can cause extensive cerebral vascular remodeling\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. As an important component of the microcirculation and the blood-brain barrier, capillaries have been confirmed to be associated with vascular remodeling in various models (e.g., the ischemic stroke mouse model\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, the traumatic brain injury rat model\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e), playing a significant role in the development of neurodegenerative diseases\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR56 CR57\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Our results first confirm that capillary loss is central to the vascular remodeling caused by multiple neuronal degenerations. Erlen Lugo-Hernandez et al. used solvent-based clearing and light-sheet microscopy to 3D visualize and quantify the microvasculature throughout the ischemic mouse brain, and the results showed a significant loss of capillaries with diameters\u0026thinsp;\u0026le;\u0026thinsp;10 \u0026micro;m, which was useful for studying microvascular damage and remodeling after stroke\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Additionally, Hannah C Bennett et al. found that in an aging mouse model, blood-brain barrier damage led to a general reduction in vessel length and branch density, as well as more tortuous arterioles, indicating that the vascular network is sparser and more remodeled in the aged brain\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. These studies support our view that capillary loss is central to the vascular remodeling caused by neuronal degeneration. However, there are also studies that differ from our perspective. Matthew V Russo et al. studied a mouse model of mild traumatic brain injury, and found that the meningeal vasculature can regenerate after mild traumatic brain injury, orchestrated by different myeloid cell subsets over time\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. This reflects that vascular remodeling may be the result of multiple factors acting together.\u003c/p\u003e \u003cp\u003eWe propose capillary VF as an early diagnostic indicator for CBND. Obtaining a 3D analysis of the true vascular VF can more accurately quantify the cerebrovascular network and also allows for better comparison between different studies\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. A similar study has supported our idea, as Grace Rosen et al. compared 41 male brain donors with chronic traumatic encephalopathy and found that the ratio of vascular branch density and VF of sulcus and gyrus was greater than the control group\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Traditional diagnostic methods largely rely on a combination of mental status examination and conventional imaging techniques (such as MRI, PET, etc.), while also detecting biomarkers related to neuronal degeneration (such as Aβ, tau, etc.)\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. However, by the time significant neuronal degeneration has occurred, these methods offer limited value for disease treatment. Recently, various new imaging tests have been developed to evaluate the structure and function of the vasculature, such as ultrasound localization microscopy, three-photon microscopy, etc., with resolutions reaching up to tens of micrometers\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Moreover, in terms of materials, advanced nanomaterials have been employed in the diagnosis of neurodegenerative diseases, through in vivo imaging and in vitro sensors\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Given that we propose using capillary VF as an early diagnostic indicator for CBND, rather than biomarkers associated with neuronal degeneration, this provides motivation for the development of higher resolution detection methods. Early diagnosis of neurodegenerative diseases at the capillary level with high resolution can spare patients from invasive procedures such as blood tests, reduce the risk of infection, save medical costs, and has greater clinical significance.\u003c/p\u003e \u003cp\u003eAlthough we have found a significant loss of capillaries in CBND, interestingly, despite the widespread impact of cerebellar neurodegenerative lesions on vascular distribution and density, the overall connectivity characteristics and branching structure of the cerebellar vascular network remain unchanged. This suggests that the maintenance of vascular topological homeostasis is a feature of vascular remodeling caused by CBND. Therefore, early changes in capillaries should be a particular focus. The vascular network includes arteries, veins, and capillaries. During vascular development, embryonic stem cells first differentiate into mesodermal progenitor cells, which then produce endothelial cells\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Next, endothelial cells differentiate into arterial or venous endothelium by regulating VEGF concentration, with various molecules participating in the regulation\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. This indicates that there are morphological, cellular composition, molecular expression, and signaling differences between capillaries and arteries or veins\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. However, current high-resolution imaging tests used clinically to assess blood vessels, such as 7T MRI with a resolution of about 0.5 mm\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, cannot yet display the early changes of capillaries in neurodegenerative diseases. Based on the many differences in the presence of blood vessels of different radii, we believe that it is risky to infer the degree of neurodegeneration from changes at the noncapillary level of blood vessels. It is likely that changes in the morphology and function of noncapillary vessels have already indicated a massive death of neurons, resulting in irreversible consequences, significantly delaying the treatment of the disease.\u003c/p\u003e \u003cp\u003eOur results showed that the distance between PCs and blood vessels decreased from 16 \u0026micro;m to 7 \u0026micro;m, which seemed to indicate a more sufficient blood supply for PCs. However, this was actually a subsequent result of cerebellar volume reduction, i.e., cerebellar atrophy. At the same time, our results also showed that PC-capillary VF reduced by 94%, suggesting that the reduction in PC volume was more significant than the reduction in capillary volume, and the structural remodeling of capillaries was not sufficient to prevent further loss of PCs. It may mean that simply targeting blood vessels treatments cannot be effective in delaying the progression of neurodegenerative diseases. The mechanism of NVC plays an important role in this process, through which neural activity regulates changes in cerebral blood flow to meet the oxygen and nutrient needs of brain cells\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. It is not only essential for maintaining homeostasis in brain function, but also plays a key role in the occurrence and development of a variety of brain diseases\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Therefore, the treatment of neurodegenerative diseases should take into account the dual effects of nerves and blood vessels. Robert Zivadinov et al. found that extracranial venous angioplasty was ineffective in treating MS\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Current research has also largely focused on neuroprotective and regenerative therapies. Amandine Virlogeux et al. found that improving vesicular transport of brain-derived neurotrophic factors could improve the behavioral phenotype and neuropathology of Huntington's disease model mice\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Hikari Tanaka et al. found that using AAV vectors to restore YAP protein levels successfully inhibited neuronal necrosis in the early stages of AD, effectively preventing cognitive impairment and extracellular Aβ aggregation in AD model mice\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. However, there are also studies have found that treatments targeting blood vessels can improve NDs. Robert D Bell et al. have suggested that serum response factors and cardiomyocyte proteins in cerebral vascular smooth muscle cells act as transcriptional switches, controlling Aβ clearance from the brain and the progression of AD\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. In summary, by knocking out Drp1 in the cerebellar PCs of mice, we constructed a model of CBND with direct neuronal damage. We found that although vascular remodeling was manifested in the early stage of CBND, with capillaries showing signs of adaptive reorganization, and the microcirculatory system attempting to maintain local blood flow stability to alleviate structural changes caused by the lack of PCs. However, this cannot delay the progression of PCs loss. This may be because the reduction in the number of capillaries, as the most basic structure that provides oxygen and nutrients to tissues, places remaining cells in the PCs region under ischemic and hypoxic conditions, increasing the metabolic stress on PCs and further leading to cellular damage. Therefore, we believe that targeting the vasculature alone cannot fundamentally solve the problem of neuronal loss, but can only temporarily alleviate the metabolic stress on neurons. The focus of treatment for neurodegenerative diseases should still be on the protection and regeneration of neurons.\u003c/p\u003e \u003cp\u003eOur research provides a basis for the development of early diagnostic strategies and specific therapies for neurodegenerative diseases. Although capillaries can remodel autonomously under the condition of CBND, this is not sufficient to halt the progression of the disease caused by neuronal loss. The core of disease treatment should focus on the improvement of neurons, while structural remodeling of capillaries and the vascular VF per unit area can serve as diagnostic indicators for the early stages of clinical diseases.\u003c/p\u003e"},{"header":"Limitations","content":"\u003cp\u003eFirst, our study has revealed morphological changes in blood vessels of different diameters in CBND, but it did not distinguish between arteries and veins, requiring further research to clarify. Second, the total number of blood vessel segments measured exceeded 265,000, and the total number of PCs exceeded 180,000. However, compared to the vast number of blood vessels and cells in the brain, future studies will need to measure a larger sample size. Third, it is likely to be regional heterogeneity in the remodeling of the cerebellar vascular network caused by CBND, but this needs to be further clarified. Fourth, due to the structural differences between the human brain and the mouse brain, our results need to be validated and further optimized in human clinical samples. Fifth, with the development of emerging technologies, it may be possible in the future to make more precise distinctions for capillaries with diameters\u0026thinsp;\u0026le;\u0026thinsp;10 \u0026micro;m. Sixth, the medium by which capillaries couple with neurons and the specific molecular mechanisms that lead to the remodeling of the capillary network require further research.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eAnimals and ethics statements\u003c/h2\u003e\n \u003cp\u003eB6/JGpt-H11\u003csup\u003eem1Cin(CAG\u0026minus;LoxP\u0026minus;ZsGreen\u0026minus;Stop\u0026minus;LoxP\u0026minus;tdTomato)/Gpt\u003c/sup\u003e(B6-G/R) mice were purchased from GemPharmatech Co., Ltd (E2101190037); Pcp2\u003csup\u003ecre\u003c/sup\u003e mice were purchased from Jackson Laboratory (Stock No: 004146, America), and Drp1\u003csup\u003efl/fl\u003c/sup\u003e mice were purchased from Seye (serial number: CKOAIS191230RT5, China). Pcp2\u003csup\u003eCre\u003c/sup\u003e mice were crossed with B6-G/R\u003csup\u003e(B6/JGpt\u0026minus;H11em1Cin(CAG\u0026minus;LoxP\u0026minus;ZsGreen\u0026minus;Stop\u0026minus;LoxP\u0026minus;tdTomato)/Gpt)\u003c/sup\u003e mice to generate PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice were crossed with Drp1\u003csup\u003ef/f\u003c/sup\u003e mice to generate PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice were used as controls. All experimental animals were 8 weeks old. All animal protocols were approved by the Ethics Committee of the Air Force Medical University and followed our institutional guidelines for the use of laboratory animals.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eMouse genotyping\u003c/h2\u003e\n \u003cp\u003eThe mouse genotype was identified by polymerase chain reaction (PCR) with genomic DNA obtained from the tails. Primers were shown in Table\u0026nbsp;1. The PCR program used was as follows: 94\u0026deg;C for 3 min, then 35 cycles of 94\u0026deg;C for 30 s for denaturation, 62\u0026deg;C for 35 s for annealing, and 72\u0026deg;C 45 s for elongation. PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice were subjected to agarose gel electrophoresis and three bands were obtained: a 292 bp Drp1\u003csup\u003ef/f\u003c/sup\u003e band, a 567 bp Pcp2\u003csup\u003eCre\u003c/sup\u003e band, and a 1465 bp B6-G/R band.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eTissue transparency\u003c/h2\u003e\n \u003cp\u003eMouse brain tissues were treated with 4% paraformaldehyde overnight at 4\u0026deg;C to fix the samples. Mouse brain tissues were washed three times with PBS. We then performed tissue transparency using CUBIC reagent solutions A, B, and C (Nuohai, Cat#210701 Nuohai Life Sciences Co., LTD.). Solutions A and B were used for defatting and solution C for refractive index matching. First, brain tissue was gently shaken with CUBIC reagent solutions A and B at 37\u0026deg;C for 12 days. Then, brain tissue was washed with PBS for 6 h and gently shaken at 37\u0026deg;C. After washing, brain tissues were embedded in solution C at 20\u0026deg;C until they appeared completely transparent.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eTransparent imaging and processing of results\u003c/h2\u003e\n \u003cp\u003eRaw data for the 3D image analysis work was captured by LiTScan (Light Innovation Technology Limited) and converted to AM format for compatibility with Amira software (Thermo Fisher Scientific). The image samples were resampled to adjust the resolution by setting the appropriate voxel size using the Resample module. In the pre-processing stage, a background detection correction module with specific filters was used to remove background noise. Subsequently, structural enhancement filters, specifically 3D filters, were used to enhance the structural features. An interactive thresholding module was then applied to segment the region of interest based on a specified intensity range, and then the segmented region was manually labelled and dots were removed to eliminate defects. Lymphatic vessels, blood vessels and lymph nodes were segmented based on raw data. Equipment (Light-sheet Microscope); Software: LiTScan 3.3.0 (Light Innovation Technology Limited); Lenses (Objectives): 4X, N.A. = 0.28, WD\u0026thinsp;=\u0026thinsp;28 mm 10\u0026times;, N.A. = 0.6, WD\u0026thinsp;=\u0026thinsp;8 mm.\u003c/p\u003e\n \u003cp\u003eImaris 10.2 was used to manually mark the target area and establish a surface model, and the vascular and neuronal signal channels in the labeled area were masked out respectively. Then, the median filtering was performed on the extracted vascular channels to make the signal of the intravascular cavity uniform. The filament was used to reconstruct the vascular channel after the filter, and the autopath mode was used for automatic identification. Based on the established model, the statistics of vessel length and volume could be extracted. The model of spots was used to identify the neuronal signals after the mask, and the background subtraction mode was selected, which could also accurately identify the signals with large differences in brightness.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunofluorescence staining and imaging\u003c/h2\u003e\n \u003cp\u003e4% PFA-treated mouse brain tissue was used overnight to fix the samples and moved in 30% sucrose solution until the tissue sank, and the tissue was sectioned after extended sagittal-free cuts to 30 \u0026micro;m. Sections were blocked with 10% fetal bovine serum and 0.3% Triton for 30 min at room temperature, Lectin \u0026minus;\u0026thinsp;647 (Nuohai, NH \u0026minus;\u0026thinsp;240525 - DL649, 1:1000) was added, incubated at 4\u0026deg;C for 18 h, and the nuclei of the cells were stained with 4\u0026apos;,6 - diamidino \u0026minus;\u0026thinsp;2 - phenylindole using a Lycra Stellaris 5 laser confocal microscope. (laser lines: 405, 488, 561 and 637 nm) and 10x objective lens in Navigator mode to obtain the above immunofluorescence staining images. The fluorescence signals of blood vessels and Lectin were obtained by excitation at 405, 488, 561 and 637 nm, respectively, at 40x effective magnification. The acquired images were processed using Image J software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eGlobal cerebellar neuronal vascular network morphology and vascular network topology analysis\u003c/h2\u003e\n \u003cp\u003eBecause the cell bodies and dendrites of PCs are located in the cerebellar MCL and the PCL, we focused on this area for the analysis of cerebellar vessels, that is, the PCL was used as the boundary, and the cerebellar vessels of the PCL and the MCL were divided for further analysis.\u003c/p\u003e\n \u003cp\u003eThe vessels were divided into capillary (diameter\u0026thinsp;\u0026le;\u0026thinsp;10 \u0026micro;m), noncapillary (diameter\u0026thinsp;\u0026gt;\u0026thinsp;10 \u0026micro;m), and integral (diameter was not distinguished). The whole was a combination of capillary and noncapillary vessels.\u003c/p\u003e\n \u003cp\u003eTotal cerebellar volume, total number of PCs, total PCs volume, total length of blood vessels, and total volume of blood vessels were calculated.\u003c/p\u003e\n \u003cp\u003eThe distribution frequency of chord length of the whole vessel segment was calculated and the fitting curved was drawn. The overall absolute frequency of PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice group was normalized by 50%-dimension reduction, and the frequency distribution trend of the two groups was observed. The chord length of the vessel segments was the straight-line distance between the origin and end of the vessel in each segment.\u003c/p\u003e\n \u003cp\u003eThe distribution frequency of the overall vascular segment curved length was calculated and the fitting curved was drawn. The overall absolute frequency of PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice group was normalized by 50%-dimension reduction, and the frequency distribution trend of the two groups was observed. Vessel segment curved length referred to the true path length from the origin to the end point along the natural curved of the vessel.\u003c/p\u003e\n \u003cp\u003eThe distribution frequency of the mean radius of the whole blood vessel was calculated and the fitting curved was drawn. The overall absolute frequency of PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice group was normalized by 50%-dimension reduction, and the frequency distribution trend of the two groups was observed. The average radius of a vessel was usually measured by measuring radius values at multiple locations on a certain vessel segment and then taking the average of these radii.\u003c/p\u003e\n \u003cp\u003eThe whole vascular node and its middle terminal branch were calculated. Vascular nodes included two important geometric and functional locations in the vasculature: terminals and branch points. Vessel terminal referred to the last point along the direction of the vessel along the center line of the vessel, when the vessel no longer bifurcates. Vascular branch nodes in the vascular system referred to the key locations where larger blood vessels split into smaller vessels.\u003c/p\u003e\n \u003cp\u003eThe distribution frequency of \u0026phi; value of the whole vascular segment was calculated and the fitting curved was drawn. The overall absolute frequency of PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice group was normalized by 50%-dimension reduction, and the frequency distribution trend of the two groups was observed. The \u0026phi; value was the azimuth angle, which described the angle between the vessel segment in the XY plane and the Z axis in 3D space, and we analyzed the \u0026phi; value in the range of 0 to 360\u0026deg;.\u003c/p\u003e\n \u003cp\u003eThe distribution frequency of \u0026theta; value of the whole vascular segment was calculated and the fitting curved was drawn. The overall absolute frequency of PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice group was normalized by 50%-dimension reduction, and the frequency distribution trend of the two groups was observed. The \u0026theta; value was the elevation angle, which indicated the offset of the vessel segment in the z-axis direction and was commonly used to describe the angle between the vessel segment and the XY plane, and we analyzed the \u0026theta; values in the 0\u0026ndash;90\u0026deg; range.\u003c/p\u003e\n \u003cp\u003eThe VF was calculated including the vessel-cerebellum VF (vascular volume/cerebellar volume), capillary-cerebellum VF (capillary volume/cerebellar volume), noncapillary-cerebellum VF (non - capillary volume/cerebellar volume), PC-vessel VF (PC volume/vascular volume), PC-capillary VF (PC volume/capillary volume), PC-noncapillary VF (PC volume/noncapillary volume).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eData processing and visual analysis\u003c/h2\u003e\n \u003cp\u003eIn this study, Python programming language was used for data processing and image plotting for different experimental groups (PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e groups).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eData read.\u003c/strong\u003e Through the Pandas library, the data of the control group and the PC\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mouse group were first read. We wrote a data reading function, read_name (), which iteratively read each file and clears missing values (NaN) to ensure data integrity and consistency.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eData division and statistical calculation.\u003c/strong\u003e In order to study the volume distribution of different groups, we combined all the data and divided the volume data into boxes. The container division range was defined as V\u003csub\u003emin\u003c/sub\u003e - V\u003csub\u003emax\u003c/sub\u003e, the container width was x, and the specific container division range was as follows. Through the numpy. histogram () function, the sample frequency in each sub-box was calculated using the following formula: bins = {V\u003csub\u003emin\u003c/sub\u003e, V\u003csub\u003emin\u003c/sub\u003e + x, V\u003csub\u003emin\u003c/sub\u003e + 2x, ..., V\u003csub\u003emax\u003c/sub\u003e}. Average frequency and standard deviation in each subbox:\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\u003cimg 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\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eCi was the binning frequency of the i th sample, and n was the total number of samples. After data processing, we used the Matplotlib library to plot the volume data of the control and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice groups.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eData visualization.\u003c/strong\u003e To further analyze the volume distribution trends, we smoothed the data using cubic spline interpolation (make_interp_spline () function). For each experimental group, we defined the following interpolation function: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{M}\\text{e}\\text{a}\\text{n}\\hspace{0.25em}\\text{C}\\text{o}\\text{u}\\text{n}\\text{t}=\\frac{1}{n}\\sum\\:_{i=1}^{n}{C}_{i}\\)\u003c/span\u003e\u003c/span\u003e. Bi (x) was the B-spline basis function, ci was the interpolation coefficient, and x was the center point of the bining of the volume. By using this interpolation function, a more continuous and smooth volume distribution curved between control and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice was generated. Regions of standard deviation were shown by adding and subtracting standard deviations from the mean curves, respectively, and were filled with color.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eData output and saving.\u003c/strong\u003e All processed data, including volumetric frequency statistics for the control and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mouse groups, raw frequency counts of experimental samples from each group, and centroid information for each subbox, were saved as Excel files.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eIn this paper, all data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, and error lines indicate SD. all statistics were analyzed using GraphPad Prism 9. Unpaired t-test and Mann-Whitney U test were used to compare the two groups. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Abbreviations ","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"514\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e3D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eThree-Dimensional\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAlzheimer’s Disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCerebellar Lobules\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCBND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCerebellar Neurodegenerative Disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCDN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCerebellar Dentate Nucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDrp1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eDynamin-related protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eGranular Cell Layer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMolecular Layer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMRI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMagnetic Resonance Imaging\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMultiple Sclerosis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNVC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNeurovascular Coupling\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eP2Y12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePurinergic Receptor P2Y12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePurkinje Cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePurkinje Cell Layer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eParkinson’s Disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePFL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eParaflocculus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePICA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePosterior Inferior Cerebellar Artery\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSuperior Cerebellar Artery\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSim\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSimplex Lobules\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eVF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eVolume Fraction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Data for each figure can be found in the Source Data files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the optical imaging platform of Chinese Institute for Brain Research, Beijing, China and Shenzhen Guangyuan Lit Company for the technical support of this experiment. Thanks to Shujiao Li and Jingjing Tie for providing some transgenic mice. This work was supported by the National Natural Science Foundation of China (82201627) by Feifei Wu, the Basic Research Program of Natural Science of Shaanxi Province (2022JQ820) by Feifei Wu, the Basic Research Program of Natural Science of Shaanxi Province (2024JCZDXM60) by Yanling Yang, the New Clinical Technology of Xi-Jing Hospital (2023XJSY27) by Yanling Yang, Military Medicine Promotion Program of Air Force Military Medical University (2020SWAQ04) by Yayun Wang and Shaanxi Provincial Innovation Capability Support Program (2023CXPT33) by Yayun Wang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYayun Wang and Yanling Yang designed the study protocol and interpreted the experimental results. Hui Liu was responsible for the breeding of transgenic mice, most of the basic experiments and the production of videos. Yunqiang Huang and Changlei Zhu assisted in basic experiments. Shujiao Li and Jingjing Tie were responsible for providing some transgenic mice. Ziwei Ni was responsible for the processing of experimental data. Xintong Deng was responsible for the drawing of the image; Xueyin Pu was responsible for the guidance of the image; Yayun Wang, Yuxuan Liu, and Ziwei Ni wrote the manuscript, which was revised by all the authors; Feifei Wu provided the funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest. This research project has been approved by the relevant ethics committee or institution and is conducted in strict compliance with ethical guidelines. Throughout the study, the rights and privacy of all participants are maintained and guaranteed, while ensuring the confidentiality of their personal information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eW\u0026auml;lchli, T. et al. Hierarchical imaging and computational analysis of three-dimensional vascular network architecture in the entire postnatal and adult mouse brain. Nat Protoc. 16 4564\u0026ndash;4610 (2021)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMink, J. W., Blumenschine, R. J. \u0026amp; Adams, D. B. Ratio of central nervous system to body metabolism in vertebrates: its constancy and functional basis. Am J Physiol. 241 R203-212 (1981)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZlokovic, B. V. Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders. 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Nat Cell Biol. 11 143\u0026ndash;153 (2009)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cerebellar neurodegenerative disease, Purkinje cells, Capillaries, Topological structure, Volume fraction.","lastPublishedDoi":"10.21203/rs.3.rs-5607179/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5607179/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeurodegenerative diseases refer to a group of clinical conditions characterized by progressive neuronal loss, resulting in impaired brain structural integrity and functional abnormalities. These diseases can lead to widespread cerebrovascular remodeling; however, the spatial remodeling features of capillaries with diameters ≤ 10 μm remain poorly understood, particularly with regard to changes in the relationship between neurons and capillaries. In this study, we first developed a double-fluorescent transgenic mouse model of cerebellar neurodegenerative disease (CBND), the PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mouse, in which Purkinje cells (PCs) in the cerebellum express red fluorescent protein, while the cerebrovascular system in the cerebellum expresses green fluorescent protein (with no differentiation between arteries and veins). Subsequently, we employed whole-brain clearing combined with the Amira/Imaris system to conduct three-dimensional layered imaging and computational analysis of the cerebrovascular network in both adult control and PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice. A total of 181744 PCs and cerebrovascular vessels with a total length of 17.7363 meters, 266175 segments, and a total volume of 0.5314 mm³ were analyzed. Compared to the Control mice, PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice exhibited a 93% reduction in count of PCs, a 97% reduction in total volume of PCs, a 69% reduction in cerebellar volume, a 58% decrease in total cerebrovascular vessel length, and a 52% decrease in total cerebrovascular vascular volume. Depth analysis of vessels with diameters ≤ 10 μm revealed a 55%, 58%, 58%, and 52% reduction in capillary volume, chord length, curved length, and tortuosity, respectively, with no statistical differences in node count or φ and θ directional values. Further volume fraction (VF) analysis revealed a 59% increase in capillary-cerebellum VF, while the PC-vessel VF, PC-capillary VF, and PC-noncapillary VF decreased by 95%, 95%, and 96%, respectively. Additionally, the shortest distance between PCs and cerebrovascular vessels decreased by 58%, while vessel-cerebellum VF and noncapillary-cerebellum VF showed no statistical differences. Our results indicated that while capillaries with diameters ≤ 10 μm were significantly lost, their vascular topology remained stable, with the distance between PCs and cerebrovascular vessels decreasing from 16 μm to 7 μm. This remodeling process is central to the pathogenesis of cerebrovascular changes in CBND. Furthermore, the increase in capillary-cerebellum VF and the decrease in PC-vessel VF may serve as biological markers for the early diagnosis of CBND. These findings provide a foundation for the early diagnosis and development of targeted therapies for CBND.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShort abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCerebrovascular remodeling caused by neurodegenerative diseases can be used for early diagnosis, but its characteristics are unclear. Our research group first constructed PCKO\u003csup\u003eTomato\u003c/sup\u003eVessel\u003csup\u003eGreen\u003c/sup\u003e mice with cerebellar neurodegenerative disease, and then adopted whole cerebellar transparency combined with Amira/Imaris system. A total of 181744 Purkinje cells with a total length of 17.7363 m, a total number of 266175 segments and a total volume of 0.5314 mm\u003csup\u003e3\u003c/sup\u003e were analyzed by three-dimensional stratified imaging and computational analysis. A total of 3.15 TB of data revealed that capillaries with diameters ≤ 10 μm were significantly lost although the vascular topology remained stable. Additionally, the distance between Purkinje cells and blood vessels decreased from 16 μm to 7 μm, identifying this as a central feature of neurovascular remodeling in cerebellar neurodegenerative disease. The increase of capillary-cerebellum volume fraction and the decrease of Purkinje cell-vessel volume fraction can be used as biological markers for the early diagnosis of neurodegenerative diseases.\u003c/p\u003e","manuscriptTitle":"The characteristics of capillary remodeling in cerebellar neurodegenerative diseases revealed through layered imaging and stereoscopic analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 18:06:20","doi":"10.21203/rs.3.rs-5607179/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43472571-3931-4cdf-a35a-270803914191","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":41824226,"name":"Biological sciences/Neuroscience/Neuro\u0026#x2013;vascular interactions"},{"id":41824227,"name":"Health sciences/Diseases/Neurological disorders/Neurodegenerative diseases"}],"tags":[],"updatedAt":"2025-03-24T18:06:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-24 18:06:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5607179","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5607179","identity":"rs-5607179","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}