Mouse brain lymphatic vessels

Mouse brain lymphatic vessels | 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 Research Article Mouse brain lymphatic vessels Amin Tamadon, Alireza Afshar, Nadiar Mussin, Kulyash Zhilisbayeva, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6308497/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 The lymphatic system plays a vital role in immune function and waste removal, yet its involvement in the brain has remained largely unexplored. This study provides compelling evidence of lymphatic vessels in the mouse brain, specifically in the cortex, thalamus, and hippocampus. Using confocal microscopy, western blotting, and real-time PCR, lymphatic vessels were identified by the markers Lyve1, Prox1, and VEGFC. Paraffin-embedded brain slices from wild-type Balb/C mice, stained with antibodies against Lyve1, CD31, and CD34, revealed the presence of these vessels through detailed 2D and 3D imaging. Notably, a coiled 3D structure of lymphatic vessels was observed in the hippocampus, suggesting a complex lymphatic drainage network critical for brain homeostasis. Western blot analysis confirmed the expression of lymphatic markers Lyve1, Prox1, and VEGFC, along with endothelial markers CD31 and CD34. Real-time PCR demonstrated significant mRNA expression of these markers, further supporting their presence. This study uniquely integrates protein ex-pression analysis and gene expression profiling to characterize the brain's lymphatic system. The findings reveal a functional lymphatic system extending from the meninges into deeper brain regions, challenging the long-standing belief that the brain lacks lymphatic vessels. This finding marks a significant advancement in neuroimmunology, providing new insights into immune system involvement in the central nervous system. It highlights potential pathways for clearing macromolecules and immune cells, offering promising therapeutic strategies for brain diseases. Lymphatic System Brain Immunology Lymphatic Vessels Hippocampus Neuroimmunology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The lymphatic system is a network of capillaries that collect lymph and drain it into the venous system for recirculation. In the peripheral lymphatic drainage of the brain, interstitial fluid (ISF) is taken up by specialized junctions in the capillaries and larger collecting vessels before being transferred into the vena cava. However, the lymphatic drainage system in the vertebrate brain has unique structures in the central nervous system (CNS), which have been discovered through physiological and immunological data (Laman and Weller 2013 ). Previous studies have shown that meningeal lymphatic vessels (MLVs) exist in the CNS of rodents, non-human primates, and humans (Antila et al. 2017 ; Aspelund et al. 2015 ; Louveau et al. 2015b ). These vessels drain macromolecules, immune cells, and metabolites from both the cerebrospinal and ISFs into the cervical lymph nodes (Aspelund et al. 2015 ; Louveau et al. 2015b ). The MLVs may play a role in maintaining brain homeostasis. Understanding the drainage and removal of macromolecules from the brain's interstitial space is crucial for elucidating the normal physiology and function of the brain's lymphatic system. A previous study has identified multiple pathways for brain lymphatic drainage, including the basement membrane perivascular pathway, the glymphatic system, the olfactory/cervical lymphatic drainage route, and the meningeal lymphatic network (Sun et al. 2018 ). Despite these advances, the exact function and clearance mechanisms of brain lymphatic drainage remain enigmatic (Sun et al. 2018 ). As it is well known, the lymphatic drainage is essential for removing waste materials and maintaining homeostasis, metabolism, and immunity in the ISF surrounding tissues (Dissing-Olesen et al. 2015 ). Until the late 19th century, it was widely believed that the brain lacked lymphatic vessels. However, Paolo Mascagni was the first to describe the brain lymphatic system (Lukic et al. 2003 ). While there is evidence for dural meningeal lymphatic and glumatic systems, their clearance mechanisms remain ambiguous. The lack of information about deeper lymphatic drainage systems in the brain prompted us to investigate the presence of lymphatic vessel endothelial hyaluronan receptor-1 (Lyve1) signal in the cortex, thalamus, and hippocampus in paraffin-embedded brain slices using a confocal microscope. For much of modern medical history, the brain was considered an immune-privileged organ devoid of a conventional lymphatic system (Louveau et al. 2015a ). This belief stemmed from the inability to detect lymphatic vessels using traditional histological methods and the presence of the blood-brain barrier, which was thought to isolate the brain from peripheral immune surveillance. Early anatomical studies by Paolo Mascagni in the 18th century hinted at the existence of brain lymphatic drainage, but these findings were largely dismissed due to a lack of robust evidence (Da Mesquita et al. 2018 ). The advent of advanced imaging techniques in the late 20th and early 21st centuries, such as confocal microscopy and three-dimensional (3D) reconstruction, began to challenge this dogma (Nowzari et al. 2021 ). However, the identification of lymphatic vessels in the brain has remained contentious, as many markers used to identify lymphatic endothelial cells (e.g., LYVE1, prospero homeobox protein 1 (PROX1)) are also expressed by other cell types, such as perivascular macrophages and blood endothelial cells. These challenges have fueled ongoing debates and necessitated the development of more precise methodologies to definitively characterize the brain's lymphatic system. The present study builds on these historical challenges by employing a combination of advanced imaging, molecular, and biochemical techniques to provide compelling evidence of lymphatic vessels in the mouse brain. This study presents a novel approach to investigate brain lymphatic vessels by utilizing cost-effective and readily available small molecule labeling techniques such as platelet endothelial cell adhesion molecule (CD31) and Lyve1. Additionally, we detected the distribution of lymphatic vessels in the mice hippocampus via western blot and Real-time PCR for lymphatic endothelial receptors (Lyve1, Prox1, and vascular endothelial growth factor C (VEGFC)) and the endothelial cell markers (transmembrane phosphoglycoprotein protein (CD34) and CD31). The unique coiled 3D reconstruction of lymphatic vessels in the mouse hippocampus may have impeded their discovery to date. Advances in our understanding of the pathways and mechanisms of brain lymphatic drainage have revealed crucial CNS functions and led to the development of new clinical therapeutic methods for the treatment of human neurological diseases (Makinen 2019 ). To the best of our knowledge, this is the first study to provide a coiled 3D reconstruction of lymphatic vessels in the paraffin-embedded brain sections of the cortex, striatum, and hippocampus of mice. We also demonstrate the use of western blotting and real-time polymerase chain reaction (PCR) to show the expression of Lyve1, Prox1, and VEGFC in the hippocampus. These findings offer new insights into the complex mechanisms of brain lymphatic drainage and its potential role in maintaining brain homeostasis. 2. Materials and Methods 2.1. Ethical approval statements The present study adhered to established international and national guidelines and regulations governing animal research, including the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and the principles outlined in the Declaration of Helsinki. All experimental procedures were thoroughly reviewed and approved by the Ethical Committee of West Kazakhstan Marat Ospanov Medical University. The protocols ensured the minimization of animal distress, pain, and suffering, in accordance with the 3Rs principles (Replacement, Reduction, and Refinement). Animals were housed under controlled environmental conditions, with appropriate care provided throughout the study duration, as recommended by institutional and international welfare standards. 2.2. Mice brain sampling To investigate the presence of lymphatic vessels in the brain, we stained paraffin-embedded mouse brain sections using the same staining conditions. Wild-type Balb/C/3 mice (3–4 months old) were used for this study. The mice were group-housed (four mice per cage) and provided ad libitum access to food and water. The housing conditions followed a reversed 12-hour light:dark cycle. The experimental procedure involved cardiac perfusion and subsequent brain collection. In details, the mice were anesthetized using chloroform-impregnated cotton, and the heart was carefully dissected. Cardiac perfusion was initiated by incising the right atrium and injecting a phosphate-buffered saline (PBS) solution (0.01 M, pH 7.4) into the left ventricle. The PBS solution slowly entered the circulatory system. Perfusion continued using a syringe containing of 4% formalin until the tissues exhibited pallor. Following perfusion, the brains were meticulously excised. These steps adhered to established protocols and ethical guidelines. 2.3. Mice brain staining and image capturing The samples were then dehydrated in a graded alcohol series, cleared in xylene, and embedded in paraffin wax. The paraffin-embedded tissues were coronally sectioned in 7 µm thickness. The brain sections, having undergone thorough washing, were subjected to immunolabeling using specific primary antibodies. The primary antibodies anti-LYVE1 rabbit antibody (diluted 1:50, Abcam, ab281587, UK), anti-CD34 rabbit antibody (diluted 1:50, Abcam, ab81289, UK), and anti-CD31 rabbit antibody (diluted 1:50, Abcam, ab281583, UK) were dissolved in PBS containing 0.1% Triton X-100 and 0.01% sodium azide. Incubation occurred at 37°C for 48 hours. Subsequently, the sections underwent six washes with PBS-Triton X-100 (each lasting 6 hours) at 37°C. The corresponding secondary antibody goat anti-Rabbit secondary antibody conjugated with Alexa568 (diluted 1:100, Invitrogen, ab6702) were applied in PBS containing 0.1% Triton X-100 and 0.01% sodium azide at 37°C for 48 hours. Following additional washing, the transparent, immune-labeled sections were used for further analysis. Then, the 7 µm thick coronal sections of the brain with Lyve1, CD34, and CD31 were evaluated and captured using a confocal microscope (Olympus BX51, Japan). 2.4. Two-dimensional analysis of mice brains The seven µm coronal brain sections stained with antibodies against Lyve1, CD34, and CD31 were analyzed using a confocal microscope (Olympus BX51, Japan). Images were captured at 20× and 40× magnifications to visualize the distribution and localization of lymphatic vessels in the cortex, thalamus, and hippocampus. The sections were scanned layer by layer to identify vessel patterns and to ensure accurate representation of the immunolabeled markers. Image analysis software (e.g., ImageJ) was used to process and quantify the fluorescence intensity of the labeled markers. This analysis confirmed the presence of lymphatic vessels and highlighted their spatial organization in the brain regions studied. 2.5. 3D analysis of mice brains The fast free-of-acrylamide clearing tissue (FACT) protocol was employed to achieve efficient tissue clearing and high-resolution imaging of brain sections (Xu et al. 2017 ). Briefly, mice were perfused with PBS followed by 4% paraformaldehyde (PFA), and the brains were post-fixed in 4% PFA for 24 hours at 4°C. The fixed brains were embedded in 4% low-melting-point agarose, and coronal sections were cut at a thickness of 200–300 µm using a vibratome. Tissue dehydration was performed using a graded ethanol series (50%, 70%, 80%, 95%, and 100% ethanol), with each step lasting 1 hour at room temperature. The dehydrated sections were then incubated in a clearing solution composed of 88% ethyl cinnamate (ECI) and 12% benzyl alcohol/benzyl benzoate (BABB) for 2–4 hours at room temperature until transparency was achieved. For immunolabeling, the cleared sections were rehydrated in a descending ethanol series and washed in PBS. Blocking was performed using PBS containing 5% normal goat serum and 0.3% Triton X-100 for 2 hours at room temperature. Primary antibodies, including anti-LYVE1 (1:50), anti-CD31 (1:50), and anti-CD34 (1:50), were applied and incubated at 4°C for 48 hours. Following six washes in PBS-T (PBS with 0.1% Triton X-100), secondary antibodies conjugated to Alexa568 (1:100) were applied and incubated at 4°C for 24 hours. The labeled sections were washed six times in PBS-T and re-cleared in the FACT solution for 1–2 hours. Finally, the sections were mounted in FACT solution and imaged using a confocal microscope (Olympus BX51) with Z-stack acquisition at 1–2 µm intervals. 3D reconstructions were generated using Imaris software to analyze the morphology and spatial organization of lymphatic vessels. 2.6. Western blot analysis To perform the Western blot analysis, mice were sacrificed, and the hippocampus was rapidly dissected on ice and stored at -80°C until the biochemical assay. After tissue defreezing, we added lysis buffer to tubes containing hippocampus samples and gently homogenized them. The homogenization buffer consisted of 50 mM Tris buffer (pH 8, 4°C), 8 mM NaCl, 3 mM EDTA, 2 mM sodium deoxycholate (SDS), a protease inhibitor cocktail, and 1% Triton. We then centrifuged the samples at 12,000rpm at 4°C for 10 minutes to obtain the supernatants, which we mixed with equal amounts of loading buffer volume and separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis with 10% gels. We transferred the proteins to polyvinylidenedifluoride (PVDF) membranes, which we then blocked with 5% non-fat milk for 2 hours at room temperature. Next, we incubated the membranes with the following primary antibodies overnight at 4°C: Lyve1 (1:500, R&D, United States), PROX1 (1:500, Elabscience, United States), VEGFC (1:200, Santa, United States), CD31 (1:500, Elabscience, United States), CD34 (1:200, Santa, United States), and β-Actin (1:200, Santa, United States). We subsequently incubated the membranes for 2 hours at room temperature with Mouse IgGκ (1:1000, Santa, United States) and mouse anti-rabbit IgG-HRP (1:1000, Santa, United States) secondary antibodies. We detected the bands using enhanced chemiluminescence (Millipore, United States) and visualized them with an imaging system (BioRad, United States). We used a protein ladder to detect the molecular weight (Protein Ladder, Thermo Scientific, United States). 2.7. Realtime PCR To isolate total RNA from hippocampus tissues, we used the Trizol Reagent (Invitrogen, United States) and determined RNA purity using a Nanodrop 2000 (Wilmington, Delaware, USA). We then synthesized complementary DNA (cDNA) (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) and performed real-time PCR using an Applied Biosystems real-Time PCR System (Life Technologies Corporation, United States) with the Quantifast SYBR Green PCR kit (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) according to the manufacturer’s instructions. We used the expression of β-actin mRNA as the control and provided three replicate tissues for each primer. We analyzed the expression levels of each mRNA using relative quantification and the 2 − ΔΔCt method. 3. Results 3.1. The lymph vessels were shown in mouse brain The results of the current study showed the imaging and tracking of Lyve1 and CD34 in the mouse brain cortex. Analysis of coronal sections labeled for Lyve1 and CD34 confirmed the presence of Lyve1 vessels in the meninges. Our observations of the cortex area labeled for Lyve1 and CD34 revealed that Lyve1 vessels are located adjacent to the blood vessels (Fig. 1 ). Coronal sections confirmed that Lyve1 vessels extend from the meninges to the parenchyma and that lymphatic vessels in the brain parenchyma are visible (Fig. 2 ). Additionally, the lymphatic vessels extend to the hippocampus and thalamus. Lyve1 was expressed in the choroid plexus adjacent to the blood vessels. Moreover, cortex, hippocampus, and thalamus areas labeled for both Lyve1 and CD31 markers showed that Lyve1 was expressed adjacent to CD31. The 3D reconstruction of the brain's functional lymphatic system will be a challenging task. To perform a 3D reconstruction of the vessel surfaces based on the signal thresholds of lymphatic vessels and blood vessels, the Surface plugin of Imaris software was used. Signals and their interactions and relationships detected in the selected areas for 3D reconstruction of blood and lymphatic vessels were analyzed using the Imaris Filament plugin. Moreover, Lyve1, CD34, and CD31 in the hippocampus region of the brain underwent 3D reconstruction. Tracking of Lyve1 and CD34 in the wild mouse brain cortex revealed that Lyve1 was adjacent to CD34, and lymphatic vessels turned over the blood vessels in the cortex area (Videos S1 and S2). Furthermore, CD31 and Lyve1 in the cortex area showed coiled 3D reconstruction of lymphatic vessels that extended to the hippocampus and thalamus areas. 3.2. Western blot analysis To examine the expression of Lyve1, PROX1, VEGFC, CD31, CD34, and β-actin (control) in frozen mice hippocampus samples, we performed Western blot analysis (Fig. 6 A). We identified the presence of Lyve1, PROX1, and VEGFC in the hippocampus, as well as CD31 and CD34. We also isolated total RNA from frozen hippocampus tissue and calculated the expression levels of each mRNA using relative quantification in the hippocampus of wild-type mice (Fig. 6 B). We confirmed the expression of both CD31 and CD34 in the hippocampus. 4. Discussion We have demonstrated lymphatic markers in the hippocampus and thalamus areas of the mouse brain which are entered to brain from meninges. In agreement with our current results, previous experiments have also reported the presence of lymphatic vessels in meninges (Louveau et al. 2015b ; Chang et al. 2023 ; Absinta et al. 2017 ). The MLVs are known to play a crucial role in draining macromolecules and immune cells from the CSF within minutes (Louveau et al. 2018 ). In addition, we have shown that both blood (CD34 and CD31) and lymphatic (Lyve1, Prox1, and VEGFC) markers are expressed in the hippocampus. The presence of three types of vessels in gliomas using immunohistochemical staining for Lyve1, Prox1, and CD34 has been shown (Meng et al. 2020 ). They have observed Lyve1 + lymphatic vessels, CD34 + blood vessels, and Lyve1 + CD34 + blood vessels (Meng et al. 2020 ). They proposed that the three types of vessels are related to the coiled reconstruction of lymphatic vessels. Notably, lymphatic markers were not observed in all sections that turned around a blood vessel in paraffin sections at a thickness of 4 µm. VEGFC/vascular endothelial growth factor receptor 3 (VEGFR3) signaling is one of the main signaling pathways for lymphangiogenesis (Hogan et al. 2009 ), and VEGFC mutants lack complete lymphatic vessels in the brain (van Lessen et al. 2017 ). In contrast, transgenic zebrafish for prox1a, VEGFR4, and Lyve1 have displayed in vivo imaging of lymphangiogenic events in early embryos (van Impel et al. 2014 ). Our experimental results show that VEGFC is expressed in the hippocampus. In this study, we have used a confocal microscope to detect blood vessels followed by lymphatic vessels in the mouse brain. Our results have revealed coiled 3D reconstruction of lymphatic vessels by Lyve1 labeling, indicating that lymphatic vessels turn over blood vessels. Recently, Lessen and colleagues have identified a unique population of cells (Brain lymphatic endothelial cells (BLECs)) in the embryonic and adult zebrafish brain. These cells had a loose connection with endothelial monolayers and absorbed macromolecules at a single-cell level. They also expressed lymphatic molecular markers, and Prox1a and Lyve1 markers were upregulated in these cells. BLECs covered different parts of the brain without forming endothelial tubular structures (van Lessen et al. 2017 ). According to current research, vessel pulsations, intracranial pressure, osmotic gradients, and various transporters are involved in the movement of lymphatic fluids, which are necessary for maintaining brain homeostasis. Recent studies have suggested that lymph is drained from the brain through the paravascular pathway into peripheral lymphatics, with exchange occurring between cerebrospinal fluid and cerebral ISF through astro-glial channels (van Lessen et al. 2017 ). Ultimately, lymph passes through the blood-brain endothelial barrier via the glymphatic mechanism (Xie et al. 2013 ). MLVs have been shown to drain macromolecules from cerebrospinal and ISFs of the CNS into cervical lymph nodes in mice (Da Mesquita et al. 2018 ). Using cellular, molecular, and neuroimaging techniques, anatomical features of vascular, glial, and lymphatic drainage have been demonstrated in rodents and other species (Matsumae et al. 2016 ). Dysfunction of the brain’s lymphatic system has been increasingly implicated in the pathogenesis of neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) (Buccellato et al. 2022 ). In AD, the accumulation of toxic proteins, including amyloid-β (Aβ) and tau, is a hallmark of the disease (Bloom 2014 ). The glymphatic system and MLVs are critical for clearing these proteins from the brain (Li et al. 2022 ). Impaired lymphatic drainage has been shown to exacerbate Aβ deposition and tau pathology, leading to neuronal damage and cognitive decline (Rego et al. 2023 ). Similarly, in PD, the accumulation of α-synuclein aggregates is associated with neurodegeneration (Vidovic and Rikalovic 2022 ). Dysfunctional lymphatic clearance may contribute to the spread of α-synuclein pathology across brain regions, further exacerbating disease progression (Zou et al. 2019 ). Chronic inflammation, a common feature of neurodegenerative diseases, is also linked to impaired lymphatic function (Cai et al. 2024 ). Inefficient clearance of immune cells and inflammatory mediators by the lymphatic system can perpetuate neuroinflammation, creating a vicious cycle that accelerates neuronal loss (Beland et al. 2020 ). These findings underscore the importance of maintaining a functional lymphatic system for brain health and highlight the potential of targeting lymphatic pathways to mitigate neurodegenerative disease progression. The identification of lymphatic vessels in the brain opens new avenues for therapeutic interventions. Enhancing lymphatic function could provide a novel strategy for treating neurodegenerative diseases. For example, upregulation of VEGFC, a key regulator of lymphangiogenesis, has been shown to improve lymphatic drainage and reduce Aβ accumulation in mouse models of AD (Wen et al. 2018 ). Similarly, pharmacological activation of the glymphatic system, through methods such as sleep modulation or aquaporin-4 (AQP4) channel enhancement, has demonstrated promise in improving waste clearance and reducing neuroinflammation (Verghese et al. 2022 ). Additionally, targeting MLVs with gene therapy or small molecules could enhance their function and improve overall brain homeostasis. These approaches could be particularly beneficial in early-stage neurodegenerative diseases, where restoring lymphatic function might prevent or delay the onset of severe pathology. Furthermore, the development of non-invasive imaging techniques to monitor lymphatic function in patients could enable early diagnosis and personalized treatment strategies. Dysfunction of deep cervical lymph nodes has recently received significant attention as it relates to brain function (Thrane et al. 2014 ). Previous studies have indicated that dysfunctional clearance of macromolecules through brain lymphatic pathways is the root cause of various brain disorders, including behavior and sleep disturbances, waste removal problems, AD, chronic stress (Le et al. 2016 ), brain tumors, and traumatic brain injury (van Lessen et al. 2017 ). Brain edema, induced by brain infarction, tumors, and trauma, can cause high mortality rates, and current treatments are often ineffective (Cao et al. 2018 ). The blood-brain barrier limits the movement of macromolecules into the CNS (Chen et al. 2015 ), but lymphatic drainage pathways can bypass these restrictions. The regulation of clearance mechanisms may provide a solution to neurodegenerative diseases (Iliff et al. 2015 ). The development of techniques that allow for visualization of whole tissue has opened the possibility of analyzing entire pieces of tissue. Limitations of these techniques include unavailable transgenic animals, lack of antibody labeling, and high-tech microscopy, which are considered restricting factors for whole tissue imaging. Additionally, this study has a limitation related to a lower depth of imaging of a confocal microscope (7-micron thick paraffin-embedded sections). However, this issue can be addressed by analyzing more sections for imaging. We predict that data gathered from coiled 3D reconstructed lymphatic vessels can provide additional information from animal models of related functions. Furthermore, the development of new techniques and instrumentation could provide the framework for high-throughput analysis of brain lymphatic systems and drug discovery. 5. Conclusions This study provides groundbreaking insights into the intricate lymphatic drainage system of the mouse brain, highlighting its structural and functional complexities. Using advanced imaging techniques such as immunolabeling, confocal microscopy, and 3D reconstruction, we successfully identified the distribution and unique coiled architecture of lymphatic vessels in the hippocampus, cortex, and thalamus. The co-expression of lymphatic-specific markers Lyve1, Prox1, and VEGFC with endothelial markers CD31 and CD34 in these brain regions underscores the presence and potential role of lymphatic pathways in maintaining CNS homeostasis. Our findings contribute to the evolving understanding of brain lymphatic networks by providing the first coiled 3D reconstructions of lymphatic vessels in paraffin-embedded brain sections, advancing the anatomical and functional mapping of the CNS lymphatic system. The results support the hypothesis that MLVs and deeper lymphatic structures collaborate in clearing macromolecules, immune cells, and metabolites, thereby sustaining neural health. Furthermore, the robust expression patterns revealed by Western blotting and Real-Time PCR emphasize the critical involvement of lymphatic markers in brain physiology. These discoveries offer promising implications for future research and clinical applications, particularly in developing therapeutic strategies for neurological disorders that involve impaired lymphatic drainage or accumulation of toxic metabolites. In conclusion, this study paves the way for further exploration of the role of brain lymphatic systems in neurobiology and pathology. By enhancing our comprehension of the lymphatic mechanisms, these findings hold the potential to inform innovative treatments for human neurological diseases, establishing a vital link between anatomical discoveries and translational medical advancements. Declarations Author Contributions: Conceptualization, Amin Tamadon; Data curation, Alireza Afshar, Kulyash Zhilisbayeva and Akmaral Baspakova; Formal analysis, Amin Tamadon; Investigation, Amin Tamadon, Alireza Afshar, Nadiar Mussin, Madina Kurmanalina, Mehdi Mahdipour, Nader Tanideh , Arezoo Khoradmehr and Payam Taheri; Methodology, Amin Tamadon, Alireza Afshar, Nadiar Mussin, Madina Kurmanalina, Raisa Aringazina, Mehdi Mahdipour, Nader Tanideh , Arezoo Khoradmehr, Mostafa Najarasl and Payam Taheri; Project administration, Amin Tamadon; Resources, Nader Tanideh ; Software, Amin Tamadon, Alireza Afshar, Nadiar Mussin, Mostafa Najarasl and Payam Taheri; Validation, Akmaral Baspakova and Raisa Aringazina; Visualization, Kulyash Zhilisbayeva; Writing – original draft, Amin Tamadon and Alireza Afshar; Writing – review & editing, Nadiar Mussin, Kulyash Zhilisbayeva, Madina Kurmanalina, Akmaral Baspakova, Raisa Aringazina, Mehdi Mahdipour, Nader Tanideh , Arezoo Khoradmehr, Mostafa Najarasl and Payam Taheri. Funding: This research received no external funding. Institutional Review Board Statement: The present study adhered to established guidelines and regulations governing animal re-search. All experimental procedures received approval from the ethical committees of West Kazakhstan Marat Ospanov Medical University. Informed Consent Statement: Not applicable. Data Availability Statement: The original contributions presented in this study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author(s). Acknowledgments: The authors would like to express their gratitude to Prof. Dr. Hossein Baharvand and Prof. Dr. Yaser Tahamtani for their support and for providing the necessary equipment. Conflicts of Interest: The authors declare no conflicts of interest. 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J Clin Med 11 (23). doi:10.3390/jcm11236964 Vidovic M, Rikalovic MG (2022) Alpha-Synuclein Aggregation Pathway in Parkinson's Disease: Current Status and Novel Therapeutic Approaches. Cells 11 (11). doi:10.3390/cells11111732 Wen YR, Yang JH, Wang X, Yao ZB (2018) Induced dural lymphangiogenesis facilities soluble amyloid-beta clearance from brain in a transgenic mouse model of Alzheimer's disease. Neural Regen Res 13 (4):709-716. doi:10.4103/1673-5374.230299 Xie L, Kang H, Xu Q, Chen MJ, Liao Y, Thiyagarajan M, O'Donnell J, Christensen DJ, Nicholson C, Iliff JJ, Takano T, Deane R, Nedergaard M (2013) Sleep drives metabolite clearance from the adult brain. Science 342 (6156):373-377. doi:10.1126/science.1241224 Xu N, Tamadon A, Liu Y, Ma T, Leak RK, Chen J, Gao Y, Feng Y (2017) Fast free-of-acrylamide clearing tissue (FACT)-an optimized new protocol for rapid, high-resolution imaging of three-dimensional brain tissue. Sci Rep 7 (1):9895. doi:10.1038/s41598-017-10204-5 Zou W, Pu T, Feng W, Lu M, Zheng Y, Du R, Xiao M, Hu G (2019) Blocking meningeal lymphatic drainage aggravates Parkinson's disease-like pathology in mice overexpressing mutated alpha-synuclein. Transl Neurodegener 8:7. doi:10.1186/s40035-019-0147-y Additional Declarations No competing interests reported. Supplementary Files VideoS1.mp4 VideoS2.mp4 Graphicalabstract.jpg Graphical abstract Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-6308497","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":444434618,"identity":"07d1901b-0d07-45e6-a6cc-a1beee46c1ed","order_by":0,"name":"Amin Tamadon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYFACxgYIfSCBgeEDA0MCcVoOQLUwziBOC0g1VAszDzFa+Kcdbnv8oWabPN/x5GOPbdvs8vjZGxg/fMzBrUXidmK7wYFjtw1nnnmWbpzbllws2XOAWXLmNjzW3E5skzjAdptxw40cM+ncNubEDTcS2Jh58WiRB2v5d9serMWyrZ6wFgOQloNttxPBWhjbDhPWYgjScrbvdjLQL2mSPeeOJ87sOdiM1y9yt9OfSVR8u23bBwwxiR9l1Yn97M0HP3zE530UwMgGJhuIVQ8Cf0hRPApGwSgYBSMFAAAlZV7cG/n1RwAAAABJRU5ErkJggg==","orcid":"","institution":"West Kazakhstan Marat Ospanov State Medical University","correspondingAuthor":true,"prefix":"","firstName":"Amin","middleName":"","lastName":"Tamadon","suffix":""},{"id":444434620,"identity":"1b2b2eac-4802-4b32-b859-dc6bbe4b04b1","order_by":1,"name":"Alireza Afshar","email":"","orcid":"","institution":"West Kazakhstan Marat Ospanov State Medical University","correspondingAuthor":false,"prefix":"","firstName":"Alireza","middleName":"","lastName":"Afshar","suffix":""},{"id":444434623,"identity":"0eefce5c-c868-4e84-8b72-55ab607f59ab","order_by":2,"name":"Nadiar Mussin","email":"","orcid":"","institution":"West Kazakhstan Marat Ospanov State Medical University","correspondingAuthor":false,"prefix":"","firstName":"Nadiar","middleName":"","lastName":"Mussin","suffix":""},{"id":444434624,"identity":"7e9cab7e-e3bb-44d9-8d19-85c55c9260e4","order_by":3,"name":"Kulyash Zhilisbayeva","email":"","orcid":"","institution":"West Kazakhstan Marat Ospanov State Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kulyash","middleName":"","lastName":"Zhilisbayeva","suffix":""},{"id":444434625,"identity":"3af7e792-5758-4a34-8e1a-fbb5021e2ee3","order_by":4,"name":"Madina Kurmanalina","email":"","orcid":"","institution":"West Kazakhstan Marat Ospanov State Medical University","correspondingAuthor":false,"prefix":"","firstName":"Madina","middleName":"","lastName":"Kurmanalina","suffix":""},{"id":444434626,"identity":"de82eed9-abe4-4748-be26-034c458e59d0","order_by":5,"name":"Akmaral Baspakova","email":"","orcid":"","institution":"West Kazakhstan Marat Ospanov State Medical University","correspondingAuthor":false,"prefix":"","firstName":"Akmaral","middleName":"","lastName":"Baspakova","suffix":""},{"id":444434627,"identity":"1dbef0d1-f392-4d30-90b8-c4af0fae0074","order_by":6,"name":"Raisa A. Aringazina","email":"","orcid":"","institution":"West Kazakhstan Marat Ospanov State Medical University","correspondingAuthor":false,"prefix":"","firstName":"Raisa","middleName":"A.","lastName":"Aringazina","suffix":""},{"id":444434629,"identity":"0303ad8b-64de-4d6a-8743-491d98cf004e","order_by":7,"name":"Mehdi Mahdipour","email":"","orcid":"","institution":"Tabriz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mehdi","middleName":"","lastName":"Mahdipour","suffix":""},{"id":444434630,"identity":"2bfabae7-52ec-4413-8eb8-77a6deacfc76","order_by":8,"name":"Nader Tanideh","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Nader","middleName":"","lastName":"Tanideh","suffix":""},{"id":444434631,"identity":"6711faca-234a-4ac2-bd45-44e32022ea00","order_by":9,"name":"Arezoo Khoradmehr","email":"","orcid":"","institution":"Bushehr University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Arezoo","middleName":"","lastName":"Khoradmehr","suffix":""},{"id":444434634,"identity":"e4cac357-0388-4887-b946-5add44752dbd","order_by":10,"name":"Mostafa Najarasl","email":"","orcid":"","institution":"ACECR","correspondingAuthor":false,"prefix":"","firstName":"Mostafa","middleName":"","lastName":"Najarasl","suffix":""},{"id":444434636,"identity":"7e9c6a18-812c-4346-8840-5df84ef8661a","order_by":11,"name":"Payam Taheri","email":"","orcid":"","institution":"ACECR","correspondingAuthor":false,"prefix":"","firstName":"Payam","middleName":"","lastName":"Taheri","suffix":""}],"badges":[],"createdAt":"2025-03-26 04:23:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6308497/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6308497/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81021858,"identity":"b7ae865f-3ea8-4469-8c2e-c32747ece21a","added_by":"auto","created_at":"2025-04-21 09:53:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":512142,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of Lyve1 and CD34 expressing vessels in meninges of a coronal section\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6308497/v1/4bef0309cf052b2107d15c0e.jpg"},{"id":81022624,"identity":"bec9f930-b485-46d3-ae69-bb04b18ac6e1","added_by":"auto","created_at":"2025-04-21 10:01:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":488503,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of Lyve1 and CD34 expressing vessels in cortex areas of a coronal section\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6308497/v1/d4c279bd7ff1826bf3a7aaff.jpg"},{"id":81021865,"identity":"41f50441-5185-481c-b30c-988008c7a00a","added_by":"auto","created_at":"2025-04-21 09:53:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1825748,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of Lyve1-expressing vessels in the meninges and parenchyma of a coronal section. Nissl (left) and anatomical annotations (right) from the Allen Mouse Brain Atlas and Allen Reference Atlas – Mouse Brain (Allen Mouse Brain Atlas 2004), at the same slice position. Images of Lyve1 labeling in whole-mount meninges (scale bars, 700 µm). Lyve1 labeling in a coronal section of whole-mount third ventricle (scale bars, 40 µm).\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6308497/v1/2c602ba0fb08a84aa71e1d7e.jpg"},{"id":81021854,"identity":"a6f185d5-3077-4a4d-b2d1-7bf87b08d9ac","added_by":"auto","created_at":"2025-04-21 09:53:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":560814,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of Lyve1-expressing vessels in the meninges and brain parenchyma areas of a coronal section. Images of Lyve1 labeling in whole-mount meninges. Lyve1 labeling in a coronal section of whole-mount third ventricle (scale bars, 300 µm).\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6308497/v1/a23ad57c0e084e408da0c955.jpg"},{"id":81021915,"identity":"75398f63-574f-4fdf-8f37-40bfe52eeb96","added_by":"auto","created_at":"2025-04-21 09:53:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1011496,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of Lyve1 and CD34 expressing vessels in hippocampus area of a coronal section. a, Nissl (left) and anatomical annotations (right) from the Allen Mouse Brain Atlas and Allen Reference Atlas – Mouse Brain (Allen Mouse Brain Atlas 2004), at the same slice position as b. b, representative images of CD34 and Lyve1 labeling in whole-mount hippocampus (scale bars, 100 µm). c, representation of a coronal section of whole-mount third ventricle (scale bars, 100 µm). d, Nissl (left) and anatomical annotations (right) from the Allen Mouse Brain Atlas and Allen Reference Atlas – Mouse Brain, at the same slice position as e. e, representative images of CD34 and Lyve1 labeling in whole-mount hippocampus (scale bars, 100 µm).\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6308497/v1/c686f292c761c9f150a499ca.jpg"},{"id":81021928,"identity":"3b2ce3c9-7178-4ec9-aa07-7a7adf2bb11d","added_by":"auto","created_at":"2025-04-21 09:53:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":507053,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of lymphatic markers in the hippocampus of wild-type mice by Western blotting and real-time PCR. a, First, we confirmed the expression of Lyve1, ROX1, VEGFC, CD31, and CD34 in the hippocampus. b, then, we identified the relative expression levels of these markers in the hippocampus, using β-Actin as a protein loading control.\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6308497/v1/7b15e6ae83da2ff2d8bec0d3.jpg"},{"id":81132664,"identity":"c6d4406f-c87c-412a-b323-edfd997f5641","added_by":"auto","created_at":"2025-04-22 14:54:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5512522,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6308497/v1/1ec716d1-178f-4eb9-b919-cfbb3f61eaf0.pdf"},{"id":81021939,"identity":"11572462-9b3e-4640-b0c1-ced2bf2aab29","added_by":"auto","created_at":"2025-04-21 09:53:52","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":117697651,"visible":true,"origin":"","legend":"","description":"","filename":"VideoS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6308497/v1/2ae7ef0b577a88a8fb7926f9.mp4"},{"id":81021917,"identity":"17f58058-5e1b-4d29-b6f3-328d77aa5126","added_by":"auto","created_at":"2025-04-21 09:53:47","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":67123135,"visible":true,"origin":"","legend":"","description":"","filename":"VideoS2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6308497/v1/195a8d6a3c6a6808d31c12c1.mp4"},{"id":81021861,"identity":"ae429e81-527a-4ab0-88f5-4470b57f6fc7","added_by":"auto","created_at":"2025-04-21 09:53:43","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":72696,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6308497/v1/d6e654910083c5908306d3d8.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mouse brain lymphatic vessels","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe lymphatic system is a network of capillaries that collect lymph and drain it into the venous system for recirculation. In the peripheral lymphatic drainage of the brain, interstitial fluid (ISF) is taken up by specialized junctions in the capillaries and larger collecting vessels before being transferred into the vena cava. However, the lymphatic drainage system in the vertebrate brain has unique structures in the central nervous system (CNS), which have been discovered through physiological and immunological data (Laman and Weller \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Previous studies have shown that meningeal lymphatic vessels (MLVs) exist in the CNS of rodents, non-human primates, and humans (Antila et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Aspelund et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Louveau et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e). These vessels drain macromolecules, immune cells, and metabolites from both the cerebrospinal and ISFs into the cervical lymph nodes (Aspelund et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Louveau et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e). The MLVs may play a role in maintaining brain homeostasis.\u003c/p\u003e \u003cp\u003eUnderstanding the drainage and removal of macromolecules from the brain's interstitial space is crucial for elucidating the normal physiology and function of the brain's lymphatic system. A previous study has identified multiple pathways for brain lymphatic drainage, including the basement membrane perivascular pathway, the glymphatic system, the olfactory/cervical lymphatic drainage route, and the meningeal lymphatic network (Sun et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Despite these advances, the exact function and clearance mechanisms of brain lymphatic drainage remain enigmatic (Sun et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As it is well known, the lymphatic drainage is essential for removing waste materials and maintaining homeostasis, metabolism, and immunity in the ISF surrounding tissues (Dissing-Olesen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Until the late 19th century, it was widely believed that the brain lacked lymphatic vessels. However, Paolo Mascagni was the first to describe the brain lymphatic system (Lukic et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). While there is evidence for dural meningeal lymphatic and glumatic systems, their clearance mechanisms remain ambiguous. The lack of information about deeper lymphatic drainage systems in the brain prompted us to investigate the presence of lymphatic vessel endothelial hyaluronan receptor-1 (Lyve1) signal in the cortex, thalamus, and hippocampus in paraffin-embedded brain slices using a confocal microscope.\u003c/p\u003e \u003cp\u003eFor much of modern medical history, the brain was considered an immune-privileged organ devoid of a conventional lymphatic system (Louveau et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e). This belief stemmed from the inability to detect lymphatic vessels using traditional histological methods and the presence of the blood-brain barrier, which was thought to isolate the brain from peripheral immune surveillance. Early anatomical studies by Paolo Mascagni in the 18th century hinted at the existence of brain lymphatic drainage, but these findings were largely dismissed due to a lack of robust evidence (Da Mesquita et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The advent of advanced imaging techniques in the late 20th and early 21st centuries, such as confocal microscopy and three-dimensional (3D) reconstruction, began to challenge this dogma (Nowzari et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the identification of lymphatic vessels in the brain has remained contentious, as many markers used to identify lymphatic endothelial cells (e.g., LYVE1, prospero homeobox protein 1 (PROX1)) are also expressed by other cell types, such as perivascular macrophages and blood endothelial cells. These challenges have fueled ongoing debates and necessitated the development of more precise methodologies to definitively characterize the brain's lymphatic system. The present study builds on these historical challenges by employing a combination of advanced imaging, molecular, and biochemical techniques to provide compelling evidence of lymphatic vessels in the mouse brain.\u003c/p\u003e \u003cp\u003eThis study presents a novel approach to investigate brain lymphatic vessels by utilizing cost-effective and readily available small molecule labeling techniques such as platelet endothelial cell adhesion molecule (CD31) and Lyve1. Additionally, we detected the distribution of lymphatic vessels in the mice hippocampus via western blot and Real-time PCR for lymphatic endothelial receptors (Lyve1, Prox1, and vascular endothelial growth factor C (VEGFC)) and the endothelial cell markers (transmembrane phosphoglycoprotein protein (CD34) and CD31). The unique coiled 3D reconstruction of lymphatic vessels in the mouse hippocampus may have impeded their discovery to date. Advances in our understanding of the pathways and mechanisms of brain lymphatic drainage have revealed crucial CNS functions and led to the development of new clinical therapeutic methods for the treatment of human neurological diseases (Makinen \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To the best of our knowledge, this is the first study to provide a coiled 3D reconstruction of lymphatic vessels in the paraffin-embedded brain sections of the cortex, striatum, and hippocampus of mice. We also demonstrate the use of western blotting and real-time polymerase chain reaction (PCR) to show the expression of Lyve1, Prox1, and VEGFC in the hippocampus. These findings offer new insights into the complex mechanisms of brain lymphatic drainage and its potential role in maintaining brain homeostasis.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e \u003cem\u003e2.1. Ethical approval statements\u003c/em\u003e \u003c/p\u003e \u003cp\u003e The present study adhered to established international and national guidelines and regulations governing animal research, including the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and the principles outlined in the Declaration of Helsinki. All experimental procedures were thoroughly reviewed and approved by the Ethical Committee of West Kazakhstan Marat Ospanov Medical University. The protocols ensured the minimization of animal distress, pain, and suffering, in accordance with the 3Rs principles (Replacement, Reduction, and Refinement). Animals were housed under controlled environmental conditions, with appropriate care provided throughout the study duration, as recommended by institutional and international welfare standards.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Mice brain sampling\u003c/h2\u003e \u003cp\u003eTo investigate the presence of lymphatic vessels in the brain, we stained paraffin-embedded mouse brain sections using the same staining conditions. Wild-type Balb/C/3 mice (3\u0026ndash;4 months old) were used for this study. The mice were group-housed (four mice per cage) and provided ad libitum access to food and water. The housing conditions followed a reversed 12-hour light:dark cycle.\u003c/p\u003e \u003cp\u003eThe experimental procedure involved cardiac perfusion and subsequent brain collection. In details, the mice were anesthetized using chloroform-impregnated cotton, and the heart was carefully dissected. Cardiac perfusion was initiated by incising the right atrium and injecting a phosphate-buffered saline (PBS) solution (0.01 M, pH 7.4) into the left ventricle. The PBS solution slowly entered the circulatory system. Perfusion continued using a syringe containing of 4% formalin until the tissues exhibited pallor. Following perfusion, the brains were meticulously excised. These steps adhered to established protocols and ethical guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Mice brain staining and image capturing\u003c/h2\u003e \u003cp\u003eThe samples were then dehydrated in a graded alcohol series, cleared in xylene, and embedded in paraffin wax. The paraffin-embedded tissues were coronally sectioned in 7 \u0026micro;m thickness. The brain sections, having undergone thorough washing, were subjected to immunolabeling using specific primary antibodies. The primary antibodies anti-LYVE1 rabbit antibody (diluted 1:50, Abcam, ab281587, UK), anti-CD34 rabbit antibody (diluted 1:50, Abcam, ab81289, UK), and anti-CD31 rabbit antibody (diluted 1:50, Abcam, ab281583, UK) were dissolved in PBS containing 0.1% Triton X-100 and 0.01% sodium azide. Incubation occurred at 37\u0026deg;C for 48 hours. Subsequently, the sections underwent six washes with PBS-Triton X-100 (each lasting 6 hours) at 37\u0026deg;C. The corresponding secondary antibody goat anti-Rabbit secondary antibody conjugated with Alexa568 (diluted 1:100, Invitrogen, ab6702) were applied in PBS containing 0.1% Triton X-100 and 0.01% sodium azide at 37\u0026deg;C for 48 hours. Following additional washing, the transparent, immune-labeled sections were used for further analysis. Then, the 7 \u0026micro;m thick coronal sections of the brain with Lyve1, CD34, and CD31 were evaluated and captured using a confocal microscope (Olympus BX51, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Two-dimensional analysis of mice brains\u003c/h2\u003e \u003cp\u003eThe seven \u0026micro;m coronal brain sections stained with antibodies against Lyve1, CD34, and CD31 were analyzed using a confocal microscope (Olympus BX51, Japan). Images were captured at 20\u0026times; and 40\u0026times; magnifications to visualize the distribution and localization of lymphatic vessels in the cortex, thalamus, and hippocampus. The sections were scanned layer by layer to identify vessel patterns and to ensure accurate representation of the immunolabeled markers. Image analysis software (e.g., ImageJ) was used to process and quantify the fluorescence intensity of the labeled markers. This analysis confirmed the presence of lymphatic vessels and highlighted their spatial organization in the brain regions studied.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5. 3D analysis of mice brains\u003c/h2\u003e \u003cp\u003eThe fast free-of-acrylamide clearing tissue (FACT) protocol was employed to achieve efficient tissue clearing and high-resolution imaging of brain sections (Xu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Briefly, mice were perfused with PBS followed by 4% paraformaldehyde (PFA), and the brains were post-fixed in 4% PFA for 24 hours at 4\u0026deg;C. The fixed brains were embedded in 4% low-melting-point agarose, and coronal sections were cut at a thickness of 200\u0026ndash;300 \u0026micro;m using a vibratome. Tissue dehydration was performed using a graded ethanol series (50%, 70%, 80%, 95%, and 100% ethanol), with each step lasting 1 hour at room temperature. The dehydrated sections were then incubated in a clearing solution composed of 88% ethyl cinnamate (ECI) and 12% benzyl alcohol/benzyl benzoate (BABB) for 2\u0026ndash;4 hours at room temperature until transparency was achieved. For immunolabeling, the cleared sections were rehydrated in a descending ethanol series and washed in PBS. Blocking was performed using PBS containing 5% normal goat serum and 0.3% Triton X-100 for 2 hours at room temperature. Primary antibodies, including anti-LYVE1 (1:50), anti-CD31 (1:50), and anti-CD34 (1:50), were applied and incubated at 4\u0026deg;C for 48 hours. Following six washes in PBS-T (PBS with 0.1% Triton X-100), secondary antibodies conjugated to Alexa568 (1:100) were applied and incubated at 4\u0026deg;C for 24 hours. The labeled sections were washed six times in PBS-T and re-cleared in the FACT solution for 1\u0026ndash;2 hours. Finally, the sections were mounted in FACT solution and imaged using a confocal microscope (Olympus BX51) with Z-stack acquisition at 1\u0026ndash;2 \u0026micro;m intervals. 3D reconstructions were generated using Imaris software to analyze the morphology and spatial organization of lymphatic vessels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Western blot analysis\u003c/h2\u003e \u003cp\u003eTo perform the Western blot analysis, mice were sacrificed, and the hippocampus was rapidly dissected on ice and stored at -80\u0026deg;C until the biochemical assay. After tissue defreezing, we added lysis buffer to tubes containing hippocampus samples and gently homogenized them. The homogenization buffer consisted of 50 mM Tris buffer (pH 8, 4\u0026deg;C), 8 mM NaCl, 3 mM EDTA, 2 mM sodium deoxycholate (SDS), a protease inhibitor cocktail, and 1% Triton. We then centrifuged the samples at 12,000rpm at 4\u0026deg;C for 10 minutes to obtain the supernatants, which we mixed with equal amounts of loading buffer volume and separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis with 10% gels. We transferred the proteins to polyvinylidenedifluoride (PVDF) membranes, which we then blocked with 5% non-fat milk for 2 hours at room temperature. Next, we incubated the membranes with the following primary antibodies overnight at 4\u0026deg;C: Lyve1 (1:500, R\u0026amp;D, United States), PROX1 (1:500, Elabscience, United States), VEGFC (1:200, Santa, United States), CD31 (1:500, Elabscience, United States), CD34 (1:200, Santa, United States), and β-Actin (1:200, Santa, United States). We subsequently incubated the membranes for 2 hours at room temperature with Mouse IgGκ (1:1000, Santa, United States) and mouse anti-rabbit IgG-HRP (1:1000, Santa, United States) secondary antibodies. We detected the bands using enhanced chemiluminescence (Millipore, United States) and visualized them with an imaging system (BioRad, United States). We used a protein ladder to detect the molecular weight (Protein Ladder, Thermo Scientific, United States).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Realtime PCR\u003c/h2\u003e \u003cp\u003eTo isolate total RNA from hippocampus tissues, we used the Trizol Reagent (Invitrogen, United States) and determined RNA purity using a Nanodrop 2000 (Wilmington, Delaware, USA). We then synthesized complementary DNA (cDNA) (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) and performed real-time PCR using an Applied Biosystems real-Time PCR System (Life Technologies Corporation, United States) with the Quantifast SYBR Green PCR kit (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) according to the manufacturer\u0026rsquo;s instructions. We used the expression of β-actin mRNA as the control and provided three replicate tissues for each primer. We analyzed the expression levels of each mRNA using relative quantification and the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. The lymph vessels were shown in mouse brain\u003c/h2\u003e \u003cp\u003eThe results of the current study showed the imaging and tracking of Lyve1 and CD34 in the mouse brain cortex. Analysis of coronal sections labeled for Lyve1 and CD34 confirmed the presence of Lyve1 vessels in the meninges. Our observations of the cortex area labeled for Lyve1 and CD34 revealed that Lyve1 vessels are located adjacent to the blood vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Coronal sections confirmed that Lyve1 vessels extend from the meninges to the parenchyma and that lymphatic vessels in the brain parenchyma are visible (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdditionally, the lymphatic vessels extend to the hippocampus and thalamus. Lyve1 was expressed in the choroid plexus adjacent to the blood vessels. Moreover, cortex, hippocampus, and thalamus areas labeled for both Lyve1 and CD31 markers showed that Lyve1 was expressed adjacent to CD31.\u003c/p\u003e \u003cp\u003eThe 3D reconstruction of the brain's functional lymphatic system will be a challenging task. To perform a 3D reconstruction of the vessel surfaces based on the signal thresholds of lymphatic vessels and blood vessels, the Surface plugin of Imaris software was used. Signals and their interactions and relationships detected in the selected areas for 3D reconstruction of blood and lymphatic vessels were analyzed using the Imaris Filament plugin. Moreover, Lyve1, CD34, and CD31 in the hippocampus region of the brain underwent 3D reconstruction. Tracking of Lyve1 and CD34 in the wild mouse brain cortex revealed that Lyve1 was adjacent to CD34, and lymphatic vessels turned over the blood vessels in the cortex area (Videos S1 and S2). Furthermore, CD31 and Lyve1 in the cortex area showed coiled 3D reconstruction of lymphatic vessels that extended to the hippocampus and thalamus areas.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Western blot analysis\u003c/h2\u003e \u003cp\u003eTo examine the expression of Lyve1, PROX1, VEGFC, CD31, CD34, and β-actin (control) in frozen mice hippocampus samples, we performed Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We identified the presence of Lyve1, PROX1, and VEGFC in the hippocampus, as well as CD31 and CD34. We also isolated total RNA from frozen hippocampus tissue and calculated the expression levels of each mRNA using relative quantification in the hippocampus of wild-type mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). We confirmed the expression of both CD31 and CD34 in the hippocampus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eWe have demonstrated lymphatic markers in the hippocampus and thalamus areas of the mouse brain which are entered to brain from meninges. In agreement with our current results, previous experiments have also reported the presence of lymphatic vessels in meninges (Louveau et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e; Chang et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Absinta et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The MLVs are known to play a crucial role in draining macromolecules and immune cells from the CSF within minutes (Louveau et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition, we have shown that both blood (CD34 and CD31) and lymphatic (Lyve1, Prox1, and VEGFC) markers are expressed in the hippocampus. The presence of three types of vessels in gliomas using immunohistochemical staining for Lyve1, Prox1, and CD34 has been shown (Meng et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). They have observed Lyve1\u0026thinsp;+\u0026thinsp;lymphatic vessels, CD34\u0026thinsp;+\u0026thinsp;blood vessels, and Lyve1\u0026thinsp;+\u0026thinsp;CD34\u0026thinsp;+\u0026thinsp;blood vessels (Meng et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). They proposed that the three types of vessels are related to the coiled reconstruction of lymphatic vessels. Notably, lymphatic markers were not observed in all sections that turned around a blood vessel in paraffin sections at a thickness of 4 \u0026micro;m.\u003c/p\u003e \u003cp\u003eVEGFC/vascular endothelial growth factor receptor 3 (VEGFR3) signaling is one of the main signaling pathways for lymphangiogenesis (Hogan et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and VEGFC mutants lack complete lymphatic vessels in the brain (van Lessen et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In contrast, transgenic zebrafish for prox1a, VEGFR4, and Lyve1 have displayed in vivo imaging of lymphangiogenic events in early embryos (van Impel et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Our experimental results show that VEGFC is expressed in the hippocampus. In this study, we have used a confocal microscope to detect blood vessels followed by lymphatic vessels in the mouse brain. Our results have revealed coiled 3D reconstruction of lymphatic vessels by Lyve1 labeling, indicating that lymphatic vessels turn over blood vessels. Recently, Lessen and colleagues have identified a unique population of cells (Brain lymphatic endothelial cells (BLECs)) in the embryonic and adult zebrafish brain. These cells had a loose connection with endothelial monolayers and absorbed macromolecules at a single-cell level. They also expressed lymphatic molecular markers, and Prox1a and Lyve1 markers were upregulated in these cells. BLECs covered different parts of the brain without forming endothelial tubular structures (van Lessen et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to current research, vessel pulsations, intracranial pressure, osmotic gradients, and various transporters are involved in the movement of lymphatic fluids, which are necessary for maintaining brain homeostasis. Recent studies have suggested that lymph is drained from the brain through the paravascular pathway into peripheral lymphatics, with exchange occurring between cerebrospinal fluid and cerebral ISF through astro-glial channels (van Lessen et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Ultimately, lymph passes through the blood-brain endothelial barrier via the glymphatic mechanism (Xie et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). MLVs have been shown to drain macromolecules from cerebrospinal and ISFs of the CNS into cervical lymph nodes in mice (Da Mesquita et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Using cellular, molecular, and neuroimaging techniques, anatomical features of vascular, glial, and lymphatic drainage have been demonstrated in rodents and other species (Matsumae et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDysfunction of the brain\u0026rsquo;s lymphatic system has been increasingly implicated in the pathogenesis of neurodegenerative diseases, such as Alzheimer\u0026rsquo;s disease (AD) and Parkinson\u0026rsquo;s disease (PD) (Buccellato et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In AD, the accumulation of toxic proteins, including amyloid-β (Aβ) and tau, is a hallmark of the disease (Bloom \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The glymphatic system and MLVs are critical for clearing these proteins from the brain (Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Impaired lymphatic drainage has been shown to exacerbate Aβ deposition and tau pathology, leading to neuronal damage and cognitive decline (Rego et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Similarly, in PD, the accumulation of α-synuclein aggregates is associated with neurodegeneration (Vidovic and Rikalovic \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Dysfunctional lymphatic clearance may contribute to the spread of α-synuclein pathology across brain regions, further exacerbating disease progression (Zou et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Chronic inflammation, a common feature of neurodegenerative diseases, is also linked to impaired lymphatic function (Cai et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Inefficient clearance of immune cells and inflammatory mediators by the lymphatic system can perpetuate neuroinflammation, creating a vicious cycle that accelerates neuronal loss (Beland et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These findings underscore the importance of maintaining a functional lymphatic system for brain health and highlight the potential of targeting lymphatic pathways to mitigate neurodegenerative disease progression.\u003c/p\u003e \u003cp\u003eThe identification of lymphatic vessels in the brain opens new avenues for therapeutic interventions. Enhancing lymphatic function could provide a novel strategy for treating neurodegenerative diseases. For example, upregulation of VEGFC, a key regulator of lymphangiogenesis, has been shown to improve lymphatic drainage and reduce Aβ accumulation in mouse models of AD (Wen et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similarly, pharmacological activation of the glymphatic system, through methods such as sleep modulation or aquaporin-4 (AQP4) channel enhancement, has demonstrated promise in improving waste clearance and reducing neuroinflammation (Verghese et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, targeting MLVs with gene therapy or small molecules could enhance their function and improve overall brain homeostasis. These approaches could be particularly beneficial in early-stage neurodegenerative diseases, where restoring lymphatic function might prevent or delay the onset of severe pathology. Furthermore, the development of non-invasive imaging techniques to monitor lymphatic function in patients could enable early diagnosis and personalized treatment strategies.\u003c/p\u003e \u003cp\u003eDysfunction of deep cervical lymph nodes has recently received significant attention as it relates to brain function (Thrane et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Previous studies have indicated that dysfunctional clearance of macromolecules through brain lymphatic pathways is the root cause of various brain disorders, including behavior and sleep disturbances, waste removal problems, AD, chronic stress (Le et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), brain tumors, and traumatic brain injury (van Lessen et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Brain edema, induced by brain infarction, tumors, and trauma, can cause high mortality rates, and current treatments are often ineffective (Cao et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The blood-brain barrier limits the movement of macromolecules into the CNS (Chen et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), but lymphatic drainage pathways can bypass these restrictions.\u003c/p\u003e \u003cp\u003eThe regulation of clearance mechanisms may provide a solution to neurodegenerative diseases (Iliff et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The development of techniques that allow for visualization of whole tissue has opened the possibility of analyzing entire pieces of tissue. Limitations of these techniques include unavailable transgenic animals, lack of antibody labeling, and high-tech microscopy, which are considered restricting factors for whole tissue imaging. Additionally, this study has a limitation related to a lower depth of imaging of a confocal microscope (7-micron thick paraffin-embedded sections). However, this issue can be addressed by analyzing more sections for imaging. We predict that data gathered from coiled 3D reconstructed lymphatic vessels can provide additional information from animal models of related functions. Furthermore, the development of new techniques and instrumentation could provide the framework for high-throughput analysis of brain lymphatic systems and drug discovery.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study provides groundbreaking insights into the intricate lymphatic drainage system of the mouse brain, highlighting its structural and functional complexities. Using advanced imaging techniques such as immunolabeling, confocal microscopy, and 3D reconstruction, we successfully identified the distribution and unique coiled architecture of lymphatic vessels in the hippocampus, cortex, and thalamus. The co-expression of lymphatic-specific markers Lyve1, Prox1, and VEGFC with endothelial markers CD31 and CD34 in these brain regions underscores the presence and potential role of lymphatic pathways in maintaining CNS homeostasis.\u003c/p\u003e \u003cp\u003eOur findings contribute to the evolving understanding of brain lymphatic networks by providing the first coiled 3D reconstructions of lymphatic vessels in paraffin-embedded brain sections, advancing the anatomical and functional mapping of the CNS lymphatic system. The results support the hypothesis that MLVs and deeper lymphatic structures collaborate in clearing macromolecules, immune cells, and metabolites, thereby sustaining neural health.\u003c/p\u003e \u003cp\u003eFurthermore, the robust expression patterns revealed by Western blotting and Real-Time PCR emphasize the critical involvement of lymphatic markers in brain physiology. These discoveries offer promising implications for future research and clinical applications, particularly in developing therapeutic strategies for neurological disorders that involve impaired lymphatic drainage or accumulation of toxic metabolites.\u003c/p\u003e \u003cp\u003eIn conclusion, this study paves the way for further exploration of the role of brain lymphatic systems in neurobiology and pathology. By enhancing our comprehension of the lymphatic mechanisms, these findings hold the potential to inform innovative treatments for human neurological diseases, establishing a vital link between anatomical discoveries and translational medical advancements.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, Amin Tamadon; Data curation, Alireza Afshar, Kulyash Zhilisbayeva and Akmaral Baspakova; Formal analysis, Amin Tamadon; Investigation, Amin Tamadon, Alireza Afshar, Nadiar Mussin, Madina Kurmanalina, Mehdi Mahdipour, Nader Tanideh , Arezoo Khoradmehr and Payam Taheri; Methodology, Amin Tamadon, Alireza Afshar, Nadiar Mussin, Madina Kurmanalina, Raisa Aringazina, Mehdi Mahdipour, Nader Tanideh , Arezoo Khoradmehr, Mostafa Najarasl and Payam Taheri; Project administration, Amin Tamadon; Resources, Nader Tanideh ; Software, Amin Tamadon, Alireza Afshar, Nadiar Mussin, Mostafa Najarasl and Payam Taheri; Validation, Akmaral Baspakova and Raisa Aringazina; Visualization, Kulyash Zhilisbayeva; Writing – original draft, Amin Tamadon and Alireza Afshar; Writing – review \u0026amp; editing, Nadiar Mussin, Kulyash Zhilisbayeva, Madina Kurmanalina, Akmaral Baspakova, Raisa Aringazina, Mehdi Mahdipour, Nader Tanideh , Arezoo Khoradmehr, Mostafa Najarasl and Payam Taheri.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThe present study adhered to established guidelines and regulations governing animal re-search. All experimental procedures received approval from the ethical committees of West Kazakhstan Marat Ospanov Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The original contributions presented in this study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author(s).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The authors would like to express their gratitude to Prof. Dr. Hossein Baharvand and Prof. Dr. Yaser Tahamtani for their support and for providing the necessary equipment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbsinta M, Ha SK, Nair G, Sati P, Luciano NJ, Palisoc M, Louveau A, Zaghloul KA, Pittaluga S, Kipnis J, Reich DS (2017) Human and nonhuman primate meninges harbor lymphatic vessels that can be visualized noninvasively by MRI. 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Transl Neurodegener 8:7. doi:10.1186/s40035-019-0147-y\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Lymphatic System, Brain Immunology, Lymphatic Vessels, Hippocampus, Neuroimmunology","lastPublishedDoi":"10.21203/rs.3.rs-6308497/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6308497/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe lymphatic system plays a vital role in immune function and waste removal, yet its involvement in the brain has remained largely unexplored. This study provides compelling evidence of lymphatic vessels in the mouse brain, specifically in the cortex, thalamus, and hippocampus. Using confocal microscopy, western blotting, and real-time PCR, lymphatic vessels were identified by the markers Lyve1, Prox1, and VEGFC. Paraffin-embedded brain slices from wild-type Balb/C mice, stained with antibodies against Lyve1, CD31, and CD34, revealed the presence of these vessels through detailed 2D and 3D imaging. Notably, a coiled 3D structure of lymphatic vessels was observed in the hippocampus, suggesting a complex lymphatic drainage network critical for brain homeostasis. Western blot analysis confirmed the expression of lymphatic markers Lyve1, Prox1, and VEGFC, along with endothelial markers CD31 and CD34. Real-time PCR demonstrated significant mRNA expression of these markers, further supporting their presence. This study uniquely integrates protein ex-pression analysis and gene expression profiling to characterize the brain's lymphatic system. The findings reveal a functional lymphatic system extending from the meninges into deeper brain regions, challenging the long-standing belief that the brain lacks lymphatic vessels. This finding marks a significant advancement in neuroimmunology, providing new insights into immune system involvement in the central nervous system. It highlights potential pathways for clearing macromolecules and immune cells, offering promising therapeutic strategies for brain diseases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Mouse brain lymphatic vessels","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 09:53:32","doi":"10.21203/rs.3.rs-6308497/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":"48df206b-4ca8-4b30-a2d7-602e89ac75de","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-22T14:54:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-21 09:53:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6308497","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6308497","identity":"rs-6308497","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}