Multiscale Imaging Approaches to Decipher Blood-Brain Barrier Disruption in Parkinson’s Disease: A Bridge to Novel Therapeutic Targets

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Data may be preliminary. 16 May 2025 V1 Latest version Share on Multiscale Imaging Approaches to Decipher Blood-Brain Barrier Disruption in Parkinson’s Disease: A Bridge to Novel Therapeutic Targets Authors : Zihao Lu , Haolin Yin , Pan Xiang , Xuan Yi , Xiaohe Tian , and Qiyong Gong 0000-0002-5912-4871 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174736644.44597516/v1 514 views 246 downloads Contents Abstract Abstract Introduction Composition and Function of the BBB Functional and Structural Changes of the BBB in PD Patients Targeted Therapy and Monitoring of the BBB Multiscale Imaging Techniques for BBB Assessment Discussion and Conclusion Abbreviations Acknowledgements Author contributions Declaration of competing interest Reference Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The blood-brain barrier (BBB) plays a crucial role in the onset and progression of neurodegenerative diseases, particularly in the pathophysiology of Parkinson’s disease (PD). Under normal physiological conditions, the BBB maintains the barrier function between the central nervous system (CNS) and the peripheral circulation, precisely regulating molecular transport to prevent the infiltration of pathogens and harmful substances into the brain. However, BBB disruption may lead to the accumulation of neurotoxic substances, ultimately resulting in irreversible neuronal damage. In PD, BBB impairment has been implicated as a contributing factor to α-synuclein (α-syn) aggregation, oxidative stress, and chronic neuroinflammation, collectively accelerating neurodegeneration. This review focuses on BBB alterations associated with PD, integrating multimodal imaging techniques and clinical studies to systematically analyze its pathological characteristics. By thoroughly examining BBB changes in PD patients, this study aims to identify potential therapeutic targets and provide novel insights for optimizing diagnostic and intervention strategies. A comprehensive understanding of the structural and functional changes of the BBB in PD will facilitate the development of more precise diagnostic approaches and innovative therapeutic strategies. Multiscale Imaging Approaches to Decipher Blood-Brain Barrier Disruption in Parkinson’s Disease: A Bridge to Novel Therapeutic Targets Zihao Lu a1 , Haolin Yin a1 , Pan Xiang 1 , Xuan Yi 4 , Xiaohe Tian 1,2 *, Qiyong Gong 1,2,3 * 1 Department of Radiology, Huaxi MR Research Center (HMRRC), Institution of Radiology and Medical Imaging, West China Hospital of Sichuan University, Sichuan University, Chengdu, Sichuan, China 2 The Xiamen Key Laboratory of Psychoradiology and Neuromodulation, West China Xiamen Hospital of Sichuan University, Xiamen, Fujian, China 3 Department of Radiology, West China Xiamen Hospital of Sichuan University, Xiamen, Fujian, China 4 Department of Machine Design & Manufacturing and Automation, School of Mechanical Engineering, Sichuan University, Chengdu, China *Corresponding author: Xiaohe Tian, email: [email protected] ; Qiyong Gong, email: [email protected] . a ZHL and HLY contributed equally to this work. Abstract The blood-brain barrier (BBB) plays a crucial role in the onset and progression of neurodegenerative diseases, particularly in the pathophysiology of Parkinson’s disease (PD). Under normal physiological conditions, the BBB maintains the barrier function between the central nervous system (CNS) and the peripheral circulation, precisely regulating molecular transport to prevent the infiltration of pathogens and harmful substances into the brain. However, BBB disruption may lead to the accumulation of neurotoxic substances, ultimately resulting in irreversible neuronal damage. In PD, BBB impairment has been implicated as a contributing factor to α-synuclein (α-syn) aggregation, oxidative stress, and chronic neuroinflammation, collectively accelerating neurodegeneration. This review focuses on BBB alterations associated with PD, integrating multimodal imaging techniques and clinical studies to systematically analyze its pathological characteristics. By thoroughly examining BBB changes in PD patients, this study aims to identify potential therapeutic targets and provide novel insights for optimizing diagnostic and intervention strategies. A comprehensive understanding of the structural and functional changes of the BBB in PD will facilitate the development of more precise diagnostic approaches and innovative therapeutic strategies. Introduction PD is the second most common neurodegenerative disorder after Alzheimer’s disease (AD), with its incidence and prevalence having risen significantly over the past two decades. Due to global population aging, the number of PD patients is projected to exceed 12 million by 2040. The disease course of PD typically spans several decades, gradually progressing to severe disability, imposing substantial physical and psychological burdens on both patients and caregivers 1 . Currently, there are no effective treatments capable of halting or slowing PD progression. In recent years, studies have revealed the multifactorial etiology of PD, encompassing genetic and environmental factors, as well as their interactions; While genetic factors have been extensively studied, etiological research on environmental exposures remains challenging due to their heterogeneity and long-term effects 2,3 . Among these, increasing attention has been directed toward the role of the neurovascular unit (NVU). The concept of the NVU was first introduced in 2001, highlighting the critical role of BBB disruption and cerebral blood flow dysregulation in the pathogenesis of neurodegenerative diseases. This understanding has laid the foundation for exploring novel therapeutic strategies targeting the NVU 4,5 . The NVU is the fundamental structural and functional unit of the BBB, comprising endothelial cells, pericytes, glial cells (such as microglia, oligodendrocytes, and astrocytic endfeet), and neurons 4 . This intricate network regulates BBB permeability and cerebral blood flow, ensuring the maintenance of the microenvironment essential for neuronal circuits. As a selective barrier, the BBB prevents the entry of pathogens, toxins, and other bloodborne substances into the central nervous system (CNS). Therefore, BBB impairment may lead to the accumulation of neurotoxic substances, ultimately resulting in irreversible neuronal damage 6 . In the progression of Parkinson’s disease (PD), the disruption of the blood-brain barrier (BBB) is closely associated with α-synuclein (α-Syn) aggregation, oxidative stress, and chronic neuroinflammation. These factors interact with each other, ultimately driving neurodegenerative changes 7,8 . Advancements in neuroimaging techniques and computational data analysis have enabled in vivo detection of early BBB microstructural changes in PD, facilitating the development of therapeutic strategies targeting transport systems or metabolic abnormalities. This review comprehensively examines the intrinsic relationship between BBB disruption and pathological mechanisms in PD patients and provides an in-depth discussion of imaging techniques used to assess BBB alterations across different scales, from laboratory to clinical settings, including fluorescence imaging, magnetic resonance imaging (MRI), and nuclear imaging. This review is the first to systematically integrate BBB alterations in PD with imaging methodologies, offering new insights into potential therapeutic targets. Composition and Function of the BBB The BBB is a complex vascular interface that permeates the brain and spinal cord 9 , where endothelial cells form a continuous tubular structure, restricting paracellular solute diffusion through tight junctions (TJs) and maintaining selective substance exchange via specialized transport systems. Pericytes envelop endothelial cells, regulating capillary diameter and enhancing barrier stability through paracrine signaling. The basement membrane, composed of extracellular matrix proteins such as type IV collagen and laminin, provides structural support to the BBB and mediates cellular signal transduction. Astrocytic endfeet enwrap blood vessels, regulating water homeostasis, ion balance, and neurovascular coupling while maintaining BBB integrity through the secretion of growth factors. The coordination of these cellular components and functional interfaces ensures the barrier properties and homeostasis of the CNS. Endothelial Cells (ECs) are the core component of the BBB, exhibiting highly specialized anatomical and functional characteristics. Compared to peripheral microvascular ECs, BBB microvascular ECs are more flattened, with a thickness reduced by approximately 39% and a luminal-to-brain surface distance of less than 0.25 μm 10 . This structure facilitates efficient material exchange between blood vessels and neurons while ensuring the physical stability of the barrier. Furthermore, BBB ECs form a continuous barrier via TJs, primarily composed of transmembrane proteins such as Claudins (e.g., Claudin-5), Occludins, and Zonula Occludens-1 (ZO-1), along with their cytoskeletal adaptor proteins. This highly compact junctional architecture greatly restricts solute movement through intercellular spaces, making the BBB structurally distinct from peripheral vasculature and conferring superior barrier function 11,12 . Functionally, BBB ECs exhibit extremely low levels of transcytosis compared to peripheral microvascular ECs. This property effectively prevents large molecules in the bloodstream from entering the CNS indiscriminately in the absence of a selective mechanism. Instead, transmembrane exchange is primarily mediated by specific transport mechanisms, including glucose transporter 1 (GLUT1), which supplies energy substrates to neurons 13 ; large neutral amino acid transporters (LAT1/2), which mediate the uptake of essential amino acids and levodopa (L-Dopa); and receptor-mediated transcytosis systems such as transferrin receptor (TfR) 14 , which regulates brain iron homeostasis 15 . These transport mechanisms ensure the selective permeability of the BBB, allowing the brain to precisely regulate the uptake and clearance of specific substances to meet neuronal metabolic demands. Pericytes, located within the basement membrane, are in close contact with ECs but are typically separated by their respective basement membranes 16,17 . This structure facilitates selective adhesion while promoting intercellular signaling and metabolic exchange 18 . Pericyte projections extend along the luminal surface of ECs, are rich in contractile proteins, and dynamically regulate capillary diameter, thereby influencing local cerebral blood flow 19 . Pericyte loss has been associated with abnormal hemodynamic responses and metabolic stress. Recent studies suggest that pericyte dysfunction may serve as an early indicator of neurodegenerative manifestations such as cognitive decline. In the early stages of metabolic stress responses, pericytes may contribute to BBB leakage 20 , whereas in later disease stages, platelet-derived growth factor receptor-β (PDGFR-β) expressed by pericytes mediates platelet-derived growth factor-BB (PDGF-BB) signaling, promoting the release of basic fibroblast growth factor (bFGF), which enhances BBB structural integrity 21,22 . This mechanism underscores the role of pericytes in stabilizing the BBB and providing neuroprotection. Astrocytes serve as a crucial bridge in neurovascular coupling 23,24 . Morphologically, their extensive cytoplasmic projections give them a stellate shape, with endfeet closely enwrapping brain capillaries 25 . These endfeet are enriched with aquaporins, particularly aquaporin-4 (AQP4) 26 , which plays a vital role in CNS water homeostasis and potassium (K⁺) regulation 27 . AQP4 is localized near the basement membrane and anchored to the perivascular region via heparan sulfate proteoglycans such as agrin. K⁺ distribution is modulated by AQP4, wherein local increases in K⁺ concentration stimulate astrocytic endfeet to absorb K⁺ and promote distal efflux 28,29 . Interestingly, AQP4 levels are downregulated in both PD and AD patients 30–32 . Additionally, astrocytes secrete multiple factors such as bFGF and angiopoietin, which promote TJ formation and enhance barrier stability 33 . Functional and Structural Changes of the BBB in PD Patients This section systematically explores the structural and functional changes of the BBB during the progression of PD, with a focus on α-synuclein-related transport dysfunction, BBB integrity impairment, and inflammatory responses (Figure 1). Additionally, relevant clinical studies are summarized (Table 1). The analysis of these pathological mechanisms not only contributes to a deeper understanding of BBB abnormalities in PD but also lays the theoretical foundation for subsequent investigations using imaging techniques at different scales. [insert Table 1.] [insert Figure 1.] Abnormal deposition of Lewy bodies in the substantia nigra and striatum is a hallmark pathological feature of PD, with aggregated α-Syn being the core component 49 . Under physiological conditions, α-Syn is widely distributed in brain tissue, cerebrospinal fluid, and blood, primarily participating in synaptic vesicle cycling and neurotransmitter release 50,51 . However, during the pathological progression of PD, misfolding and aggregation of α-Syn can trigger endoplasmic reticulum stress, mitochondrial dysfunction, and reactive oxygen species (ROS) accumulation, ultimately leading to neuronal damage 52,53 . Studies have demonstrated a gradient distribution of α-Syn within the vascular system. Strong positive staining is observed in the endothelial cells and smooth muscle cells of the leptomeningeal vessel walls, whereas weaker staining is detected in intracerebral blood vessels, with no α-Syn found in capillary walls 54 . Although the precise regulatory mechanisms between α-Syn and vascular endothelial cells remain unclear, research has confirmed that α-Syn can be transported across the BBB through multiple mechanisms, including clathrin-mediated endocytic transport, extracellular vesicle pathways 55,56 , and and low-density lipoprotein receptor-related protein 1 (LRP-1)-mediated transmembrane transport 57 . Low-Density Lipoprotein Receptor-Related Protein 1 (LRP-1), a member of the low-density lipoprotein receptor (LDL receptor) superfamily, is widely expressed in vascular endothelial cells, astrocytes, pericytes, and neurons, playing a crucial role in transmembrane transport and cellular signal regulation 58 . As a receptor-mediated efflux pathway, LRP-1 serves as the primary receptor for α-Syn uptake and propagation within neurons in the brain parenchyma 57 . In the exploration of the relationship between apolipoprotein E expression, α-Syn aggregation, and neurodegeneration, Hoozemans found that LRP-1 levels increase in the substantia nigra during the early stages of PD 34 . Further studies by Chen et al. demonstrated that knockdown of LRP-1 or the use of LRP-1 antagonists effectively inhibited the propagation of α-Syn in neurons 57,59 .The role of LRP-1 in the clearance of α-Syn at the BBB is particularly intriguing. The clearance function of LRP-1 at the BBB for amyloid-β has already been well established 60 . Sui et al. discovered that this function could be inhibited by α-Syn and further demonstrated that α-Syn utilizes LRP-1 for bidirectional transport across the BBB, facilitating movement between the brain parenchyma and the peripheral system 61 . This suggests that differences in LRP-1 distribution across the brain parenchyma, the abluminal (brain-facing) side of endothelial cells, and the luminal (blood-facing) side could influence PD pathogenesis. Modifying the spatiotemporal expression pattern of LRP-1 may therefore represent a promising therapeutic target for PD. The receptor for advanced glycation end products (RAGE) is another receptor-mediated transport system, located on the luminal side of the BBB. RAGE functions as an influx transporter for amyloid-β, and its increased expression in AD patients contributes to toxic protein accumulation 62,63 . Although RAGE does not directly transport α-Syn, potential interactions between RAGE and α-Syn have been suggested. Early studies reported elevated cortical RAGE expression in early-stage PD patients, accompanied by lipid peroxidation damage 64 . More recently, Long et al. confirmed that RAGE interacts with α-Syn fibrils, mediating microglial inflammatory responses 35 . During PD progression, the dynamic balance between LRP-1 and RAGE may determine whether α-Syn accumulates or is cleared from the brain. Upregulation of LRP-1 in the cerebrovascular system of PD patients may represent a compensatory response to pathological protein aggregation, whereas overexpression of RAGE may amplify α-Syn-related inflammatory signaling, creating a vicious cycle 65 . P-glycoprotein (P-gp) is an ATP-dependent transmembrane transporter widely expressed on the luminal side of endothelial cells in the BBB 66 . It plays a crucial role as the primary drug efflux pump of the BBB, transporting a broad range of xenobiotics and pharmaceuticals 67,68 . Although P-gp does not directly participate in α-Syn transport, its role in neurodegenerative diseases such as PD has been extensively studied 69 . Early studies used ¹¹C-verapamil positron emission tomography (PET) imaging to assess P-gp uptake function in PD patients and found an 18% increase in ¹¹C-verapamil uptake in the brains of PD patients compared to healthy controls, suggesting potential alterations in P-gp function 36 . However, subsequent studies failed to detect significant differences in distribution volume (DV)—defined as the ratio of the total amount of a drug in the body to its plasma (or blood) concentration, reflecting the efflux capacity of P-gp at the BBB—between early-stage PD patients and healthy controls 38 . Further investigations revealed that among PD, progressive supranuclear palsy (PSP), and multiple system atrophy (MSA) patients, late-stage PD and PSP patients exhibited increased ¹¹C-verapamil uptake in the frontal white matter, indicating P-gp dysfunction. In contrast, newly diagnosed PD patients showed lower uptake in the midbrain and frontal regions, possibly reflecting an upregulation of P-gp function 37 . Whether P-gp dysfunction is a causative factor in PD or a consequence of disease progression and long-term treatment remains controversial. Recent research on the vitamin D receptor (VDR) signaling pathway has provided insights into this issue 70,71 . Studies have shown that 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) can restore VDR and P-gp expression in PD mouse models. Furthermore, α-Syn aggregation has been found to downregulate VDR and P-gp expression 72 , whereas 1,25(OH)₂D₃ treatment can reverse this effect.These findings suggest that dysregulation of the VDR-P-gp signaling pathway may play a key role in the pathological progression of PD, and activation of VDR could represent a potential As previously mentioned, the physical stability of the BBB’s highly selective function is primarily maintained by the tight junctions of vascular endothelial cells. Changes in BBB permeability are closely associated with the disruption of tight junctions, basement membrane damage, and the loss of adhesion factors 6 . Recent studies by Kuan et al. found a reduction in tight junction proteins in postmortem brain tissue from PD patients. In vitro models further confirmed that misfolded α-Syn interferes with the normal function of occludins and claudins 73 . Early studies on postmortem PD striatal tissue detected extravasation of red blood cells, white blood cells, and fibrinogen, indicating BBB integrity loss and increased permeability 40,43,74 . Using DCE-MRI, Al-Bachari et al. assessed BBB integrity in PD, cerebrovascular disease (CP), and healthy controls (HC), finding increased \(K_{\text{trans}}\) values in the PD group, suggesting BBB leakage, particularly in the substantia nigra, white matter, and posterior cortex 42 . Janelidze et al. used cerebrospinal fluid (CSF) angiogenic markers and the CSF albumin (mg/L) to plasma albumin (g/L) ratio, a marker of BBB function, to show that PD patients exhibited greater BBB dysfunction compared to controls. Moreover, CSF angiogenic biomarkers (such as VEGF) were associated with gait disturbances and orthostatic hypotension in PD patients 44 . Vascular degeneration and abnormal angiogenesis further affect BBB integrity. In postmortem PD brain tissue, multiple brain regions exhibited vascular loss and reduced capillary branching, similar to AD 75 . Additionally, PD patients displayed an increase in string vessels 74 , characterized by degenerated, fragmented, non-luminal capillary remnants composed of basement membrane and cellular debris but lacking functional endothelial cells 76 . Some studies also observed abnormal angiogenesis in PD, with newly formed blood vessel basement membranes being discontinuous, intercellular junctions widening, and fenestration numbers increasing 46,77 . Excessive angiogenesis is expected to impair the selective barrier function of the BBB. Oxidative stress is closely linked to increased vascular growth 78,79 . Faucheux et al. found a 2.5-fold increase in endothelial cell nuclei in the SNpc of PD patients 45 . Additionally, a study by Bradaric et al. on postmortem brain tissue from PD, incidental Lewy body disease (iLBD), PSP, and HC groups found elevated αvβ3 expression in the LC and SNpc regions in PD, PSP, and iLBD groups, with additional increased αvβ3 expression in the globus pallidus of PD and PSP patients. Furthermore, SNpc microglial activation was increased in PD and PSP patients, suggesting that angiogenesis and neuroinflammation may play key roles in PD and related diseases 46 . Since newly formed blood vessels may lack a mature BBB protective function, regulating the VEGF signaling pathway to balance angiogenesis, stabilizing BBB structure to reduce string vessel formation, and inhibiting oxidative stress and neuroinflammation to improve microvascular function may serve as effective strategies for mitigating PD-related vascular pathology 80 . Interestingly, the striatum exhibits high sensitivity to BBB damage. During ischemic stress, the permeability of the striatum increases earlier than in other brain regions 40,81 . As the core hub of information integration and regulation within the cortico-basal ganglia circuit, the striatum receives cortical input and facilitates movement execution through the direct pathway 82 . The pathophysiology of PD is primarily centered on the selective and progressive loss of dopaminergic neurons in the SNpc and the nigrostriatal pathway 83 . The substantia nigra exhibits high microglial activation and elevated dopamine levels, creating a neurotoxic environment. This suggests that basal ganglia-related brain regions may represent the primary sites of BBB integrity loss in PD patients 84–86 . However, it is important to note that BBB disruption is age-related. In normal aging, increased BBB leakage has been observed in the dentate gyrus and CA1 region of the hippocampus 87,88 . Additionally, hemorrhagic events following traumatic brain injury may increase the risk of developing PD, further supporting the potential link between BBB dysfunction and PD pathogenesis 89,90 . Currently, both ex vivo tissue sample studies and in vivo imaging research must address the following critical questions: Is increased BBB permeability in PD a primary initiating factor or a secondary consequence of disease progression. Do PD patients exhibit spatiotemporal-specific patterns of BBB permeability changes, or are these alterations simply attributable to cerebrovascular diseases commonly coexisting in the elderly population? The discussion of neuroinflammation in PD is a broad and complex topic. As previously mentioned, BBB-associated neuroinflammation is closely related to abnormal α-Syn accumulation, the release of inflammatory mediators by perivascular cells, pro-inflammatory factors in systemic circulation, oxidative stress, and mitochondrial dysfunction. Vascular endothelial cells respond rapidly to α-Syn-induced inflammatory activation by undergoing contraction and remodeling, leading to the formation of intercellular gaps and disruption of endothelial layer continuity 91 . Miklossy et al. found that ICAM-1 was overexpressed in the SN of both PD patients and MPTP-treated monkeys, accompanied by astrocyte and microglial activation and leukocyte infiltration. Leukocyte counts in the PD group were five times higher than those in the HC group 92 . Endothelial activation induces the upregulation of VCAM-1 and ICAM-1, enhancing the invasiveness of peripheral immune cells 93 . The subsequent increase in local blood flow and inflammatory factor infiltration further promotes neuroinflammation, ultimately leading to BBB dysfunction. Astrocytes play a key role in antioxidant defense, removing reactive oxygen species (ROS) and reactive nitrogen species (RNS). Cellular stress and inflammation can lead to reactive astrogliosis, increasing ROS/RNS production in astrocytes, thereby promoting oxidative/nitrosative stress-induced neurodegeneration in PD 94,95 . In familial PD patients, mutations in LRRK2, GBA, SNCA, and PARK7 are also expressed in astrocytes. These mutations affect mitochondrial function, inflammatory responses, lipid transport, and lysosomal function 96 . De Rus Jacquet et al. found that iPSC-derived astrocytes from LRRK2 G2019S mutation carriers exhibited pro-inflammatory characteristics and failed to support capillary formation, resulting in BBB dysfunction. MEK1/2 signaling inhibition was shown to reduce astrocytic inflammation and improve BBB integrity 97 . Pericyte activation induced by abnormal aggregation of α-Syn may lead to the release of MMP-9 and pro-inflammatory cytokines, such as IL-1β, IL-6, and monocyte chemoattractant protein-1 (MCP-1). These pro-inflammatory factors can activate MMPs, resulting in the degradation of the endothelial glycocalyx and damage to tight junction proteins, histopathological analysis has revealed plasma fibrinogen and serum protein leakage, indicating BBB disruption 8,98 . Thus, targeting pericyte function may help stabilize BBB structure and mitigate neurodegenerative damage. Inhibiting MMP-9 activity could serve as a potential therapeutic strategy to reduce BBB impairment and neuroinflammation. Additionally, pericytes may exert neuroprotective effects. For example, platelet-derived growth factor receptor β (PDGFR-β) is highly expressed in brain pericytes and mediates the release of basic fibroblast growth factor (bFGF) and platelet-derived growth factor BB (PDGF-BB), both of which have demonstrated neurorestorative effects in PD animal models 22 . Therefore, activation of the PDGFR-β pathway could be a promising therapeutic intervention for PD-associated vascular pathology. Targeted Therapy and Monitoring of the BBB Therapeutic strategies targeting the BBB in PD can be categorized into two core concepts: ”protection” and ”crossing”. Given the high selectivity of the BBB, drug-based interventions focused on α-Syn transport regulation, BBB stabilization, and neuroinflammation modulation all face significant drug delivery challenges. To efficiently cross the BBB in PD patients for drug delivery, multiple strategies have been proposed 99 . These approaches include receptor-mediated transcytosis, carrier cell-assisted delivery, physical BBB opening techniques, and non-invasive delivery routes (e.g., intranasal and peripheral nerve delivery) 100,101 . Each method has its unique mechanisms and limitations. Receptor-mediated transcytosis (RMT) utilizes endogenous transport systems, such as the transferrin receptor (TfR), low-density lipoprotein receptor (LDL-R), and glucose transporter (GLUT1), to facilitate the entry of functionalized nanoparticles or biomolecules into the CNS 102 . However, BBB alterations in PD may lead to dynamic changes in receptor expression, causing significant individual variations in targeting efficiency 103 . Carrier cell-assisted delivery exploits the natural targeting ability and biocompatibility of biological cells to transport drugs or nanoparticles across the BBB into the CNS. In recent years, exosomes have emerged as highly promising delivery vectors for PD treatment, owing to their excellent biocompatibility, low immunogenicity, and superior BBB-crossing ability 104 . Notably, macrophage-derived exosomes exhibit a five-fold increase in BBB permeability under inflammatory conditions compared to healthy states and can be further engineered for enhanced targeting 105 . However, this strategy still faces technical bottlenecks, such as scalability, drug-loading efficiency, and targeting specificity. Additionally, cell-mediated drug delivery involves complex in vivo metabolic pathways, and further research is needed to achieve controlled delivery kinetics and retention time 106 . Physical methods for BBB opening provide a temporary increase in BBB permeability. Among these, focused ultrasound (FUS) + microbubble technology is considered one of the most clinically translatable approaches for PD treatment 107,108 . This technique applies low-intensity ultrasound stimulation to induce microbubble oscillation within cerebral capillaries, leading to localized BBB opening and facilitating the entry of large-molecule drugs or gene therapies into brain tissue. FUS has demonstrated good controllability in PD animal models and clinical studies, enabling region-specific drug delivery and enhancing intracerebral accumulation of therapeutic agents 109–111 . However, this strategy still faces several technical challenges, including limited spatial resolution, potential vascular damage due to localized heating effects, and long-term structural changes in the BBB with repeated use 112 . Additionally, hyperosmotic agents (e.g., mannitol) have been used to induce BBB opening, significantly enhancing drug penetration. However, their non-specific effects may lead to brain edema and other adverse reactions, restricting their clinical applicability 113,114 . In the diagnosis and treatment of PD, a comprehensive assessment of the BBB serves as the foundation for therapeutic strategy design and optimization 115 . The necessity of this evaluation arises from the multidimensional interactions between the dynamic pathological changes of the BBB and drug delivery mechanisms 6 . First, the spatiotemporal heterogeneity of BBB receptor expression directly determines targeted delivery efficiency. For example, the local upregulation of the transferrin receptor (TfR) in the substantia nigra 116,117 , combined with the systemic downregulation of the low-density lipoprotein receptor (LDL-R), creates a paradoxical pathological pattern 118,119 . Without precise assessment of receptor abundance through TfR-PET or cerebrospinal fluid (CSF) biomarkers, receptor-dependent nanoparticles may fail due to target depletion 120 . Additionally, neuroinflammation-driven fluctuations in BBB permeability (e.g., tight junction protein disruption by pro-inflammatory cytokines) necessitate that drug delivery strategies dynamically adapt to BBB permeability changes 121 . Monitoring BBB permeability levels enables precise identification of the optimal time window for exosome delivery and the appropriate phase for receptor-targeted strategies. Second, BBB structural integrity is a key threshold for the safety of physical interventions. While focused ultrasound (FUS) technology can selectively open the BBB, patients with microvascular fragility or tight junction protein loss (observed in late-stage PD patients) require baseline permeability quantification to prevent vascular rupture or pathogen infiltration. Similarly, hyperosmotic agents like mannitol, which significantly increase BBB permeability, may induce brain edema in patients with pre-existing α-Syn-mediated BBB leakage 111 . Therefore, non-invasive imaging techniques are essential for pre-selecting suitable patients. Furthermore, the pathological state of the BBB influences the fate of delivery vectors. Altered P-glycoprotein (P-gp) efflux pump activity may accelerate nanoparticle clearance 122 , necessitating ¹¹C-verapamil PET tracking to predict vector retention time. Most importantly, as PD progresses, the BBB undergoes dynamic changes. Early-stage GLUT1 downregulation may impair glucose carrier-mediated delivery 123 , whereas late-stage P-gp overexpression may require a combination of ultrasound and exosome-based approaches to bypass the BBB. This necessitates the use of multi-scale imaging assessment tools (e.g., nanoparticle-tracing PET combined with DCE-MRI) to achieve individualized therapeutic window matching. Fundamentally, the BBB functions as a ”dynamic cipher lock” of the CNS, where receptor distribution, structural integrity, and metabolic characteristics collectively define the three key dimensions of therapeutic strategy design. Only through a systematic evaluation using multi-scale imaging tools can we successfully decode its pathological patterns, achieving precise therapeutic adaptation, risk mitigation, and treatment outcome prediction. Multiscale Imaging Techniques for BBB Assessment This section focuses on the fundamental principles, advantages, and limitations of two-photon fluorescence microscopy (TPFM), MRI, and PET, as well as potential future improvements in BBB imaging research. As shown in Figure. 2, these techniques span spatial scales ranging from the nanometer to the millimeter level. Super-resolution microscopy enables the visualization of cellular ultrastructure with nanometer-scale resolution, while optical microscopy techniques provide insights into cell-cell interactions within the BBB. In contrast, MRI and nuclear imaging techniques are used to assess macroscopic brain structure and function, aiding in the identification of potential links between BBB dysfunction and neurological disorders. [insert Figure 2.] Before introducing SRM, it is essential to briefly review the principles of the laser confocal microscopy. In simple terms, this technique utilizes a precisely focused laser beam to illuminate the sample and employs pinholes in front of both the excitation light source and the detector to exclude out-of-focus light, thereby achieving high-resolution imaging of a specific focal plane 124 . This method provides higher contrast and clearer three-dimensional structural images while reducing background noise, thus extending the diffraction-limited resolution to smaller values, approximately 0.1–0.2 micrometers 125 . However, it still faces limitations in visualizing the precise distribution of proteins within neurons or cells of the blood-brain barrier. Stimulated Emission Depletion (STED) microscopy, a form of SRM, builds upon the principles of confocal microscopy. Stimulated Emission Depletion (STED) microscopy, a form of super-resolution microscopy (SRM), enhances confocal imaging by using a doughnut-shaped STED laser to selectively deplete fluorescence around the focal point, reducing spot size and surpassing the diffraction limit 126,127 . As a purely optical technique, it supports various dyes without requiring complex post-processing. Other widely used SRM techniques include Structured Illumination Microscopy (SIM) and Single Molecule Localization Microscopy (SMLM). SMLM precisely localizes individual molecules by controlling fluorescence activation cycles, while SIM employs structured illumination patterns to reconstruct high-resolution images. For further details, refer to additional literature 128,129 . SRM is particularly valuable for studying the interactions of α-syn with neurons and the BBB during PD progression. As previously discussed, the abnormal aggregation of α-syn within cells leads to neuronal dysfunction and eventually neurodegeneration. Recent studies employing STED microscopy have enhanced our understanding of the mechanisms by which α-syn is transported across the BBB. These investigations focused on the uptake and transport of α-syn monomers and oligomers through an in vitro BBB model system. Nielsen and et al. discovered that both α-syn monomers and oligomers can be internalized by primary brain endothelial cells, though the transport of oligomers is more restricted than that of monomers. STED microscopy further demonstrated that Rab7 directly participates in the polarized transport of α-syn monomers across BBB endothelial cells, suggesting a new potential therapeutic target for PD and related synucleinopathies 55 . One plausible mechanism for the intercellular transmission of α-syn involves the templated-seeding process, wherein exogenous α-syn fibrils promote the self-assembly of endogenous α-syn, leading to the formation of new aggregates 130–132 . A study led by Jason and his team, utilizing SMLM with DNA-PAINT imaging, examined the uptake and seeding of unlabeled exogenous α-syn fibrils in SH-SY5Y cells. The research revealed that exogenous fibrils exhibit low efficiency in inducing the self-assembly of endogenous α-syn. However, this process is accelerated by proteasomal activity, ultimately causing cells to respond to protein aggregation by increasing the secretion of nanoscale aggregates. These findings suggest that the secretion of α-syn aggregates could serve as a mechanism to maintain protein homeostasis. The subcellular distribution of α-syn in the brains of PD patients has also been a focus of research 133 . Wilma’s team used 3D multicolor confocal microscopy and STED to examine different post-translationally modified forms of α-syn in postmortem brain tissue from PD patients. Their observations indicated that mature Lewy bodies possess an ”onion skin-like” structure. Moreover, they identified that Ser129-phosphorylated α-synuclein forms a distinctive network within neurons before the formation of Lewy bodies. The study also highlighted that the accumulation of Lewy bodies, alongside a substantial buildup of mitochondria and lysosomes within neurons, may cause abnormal aggregation of organelles and proteins 134 . This buildup has the potential to impair axonal transport, contributing to neuronal dysfunction and the progression of PD. In addition to exploring the mechanisms underlying PD pathology, SRM provides superior resolution and a more detailed observation of molecular dynamics at a scale of 50 nm, surpassing the capabilities of traditional microscopy 135,136 . This advanced resolution offers significant advantages for monitoring the progression of PD, drug development, and therapeutic assessments, allowing for more precise visualization of protein aggregates and enhancing our understanding of molecular processes involved in disease progression. For imaging the BBB, STED microscopy offers significant advantages due to its exceptional optical sectioning capability and high resolution, allowing for precise three-dimensional and time-lapse imaging in live tissues 137 . Additionally, it supports multicolor imaging, enabling simultaneous analysis of the relative positions and interactions of multiple molecules or structures 138 . However, the limitations of STED primarily stem from its requirement for high light intensity; achieving ultra-high resolution necessitates the use of extremely intense lasers. For example, increasing the resolution from 100 nm to 30 nm requires a six-order-of-magnitude increase in laser intensity (from KW/cm² to GW/cm²). This can lead to phototoxic damage and fluorescence bleaching of the samples, which affects the quality and duration of live imaging. Organic far-red fluorescent dyes are suitable for live-cell imaging because they have less light scattering, lower autofluorescence, and reduced phototoxicity 127 . The future direction of STED microscopy lies in developing nanoprobes and optimizing imaging conditions to enhance long-term imaging quality and expand its applications in live biological BBB imaging research. Thanks to advancements in electrophysiology and integrated imaging technologies, researchers can now incorporate different cell types into three-dimensional culture systems, enabling the development of more complex in vitro BBB models 139,140 . However, these models still face challenges in fully replicating the anatomical and physiological context necessary for studying BBB dysfunction. High-resolution fluorescence microscopy, particularly two-photon laser scanning microscopy (TPLSM), allows for precise localization and evaluation of BBB cellular components in vivo, as well as the detection of BBB permeability marker leakage at the single-vessel level 141 . Due to its unique advantages, two-photon and multiphoton fluorescence microscopy have become essential tools in BBB research. Traditional fluorescence microscopy excites a fluorescent molecule using a single photon, whereas two-photon excitation requires two photons to arrive simultaneously at a fluorescent molecule to excite it. Due to the simultaneous arrival requirement, the probability of excitation is very low, meaning that fluorescence intensity is proportional to the square of light intensity—an effect known as ”nonlinear fluorescence.” This implies that significant fluorescence signals are only observable within a small region near the laser’s focal point. Because two-photon excitation occurs only at the focal point, the rest of the sample remains minimally affected, greatly reducing photobleaching and phototoxicity 142,143 . This selective excitation improves image clarity and depth, making two-photon microscopy highly advantageous for in vivo imaging. The infrared excitation light used in two-photon microscopy, ranging from 600 to 1,100 nm, is minimally scattered and absorbed by tissues, allowing for deep tissue imaging without the need for a pinhole to exclude out-of-focus light, thereby enhancing the signal-to-noise ratio. Two-photon microscopy achieves extremely high three-dimensional spatial resolution, with lateral resolution typically ranging from 300 to 500 nm and axial resolution approximately 800 to 1000 nm. For instance, using advanced techniques like adaptive optical two-photon multifocal structured illumination microscopy (AO 2P-MSIM), the lateral resolution can be enhanced to approximately 153 nm and the axial resolution to about 735 nm 144 . These improvements are particularly effective at depths up to 500 µm, making this technique ideal for observing neurons and their signals in all layers of the mouse cerebral cortex 145 . Imaging the BBB in living rodents with the miniature fluorescence microscope (two-Photon and multiphoton microscopy) can be achieved by injecting sodium fluorescein or fluorescein dextrose. Typically, smaller dextrose (< 3 kDa) can cross the compromised BBB, and the leakage through the barrier can be quantified by the fluorescence signals captured by the miniature fluorescence microscope. Larger dextran glycosides are retained within the cerebral vasculature, revealing the density and morphology of the blood vessels 146,147 . Barr, J. L et al. explored a method for assessing BBB permeability using microfluorescence microscopy in free-ranging rats 148 . First, the researchers implanted imaging tubes (cannula) into regions of interest in the brains of anesthetized rats. After the postoperative recovery and acclimatization period, the low molecular weight tracer sodium sulfate was injected intravenously. A microscope was utilized to observe the leakage of sodium sulfate from the cerebral microcirculation, and the average fluorescence intensity near the microvessels was recorded in real time to illustrate changes in BBB permeability. Similarly, the research team discovered that cocaine increased the permeability of the BBB in awake rats in a dose- and time-dependent manner 148 . Emily A. Gibson’s team used a head-mounted mini two-photon fiber-coupled microscope (2P-FCM) to observe the cerebral vasculature of mice for up to 17 consecutive days 149 . As previously mentioned, vascular endothelial cells, pericytes, basement membranes, and astrocytes constitute the primary barriers limiting the passage of molecules into and out of the brain. Analyzing the individual components of the BBB is challenging due to the limited resolution of two-photon microscopy (0.5 micron) 150 . In this context, Kucharz summarized two methods for visualization. The first method is the ”zoomed-in view,” which begins by recording an image of a large area (several hundred microns wide) containing multiple blood vessels (Figure. 3D). It distinguishes the brain parenchyma from the blood vessels either manually, automatically, or by combining different fluorescence channels 151–153 . However, this method is unable to differentiate the permeability of the BBB in various types of vessels (e.g., arterioles, capillaries, and venules) and can only assess the average BBB permeability across all vessels within the image area. The second approach involves ”narrowing the field of view” by imaging a small area and reducing the recording time to ensure that the fluorescence recorded is solely from this vessel 150 . This approach not only eliminates the need to model multiple vessels but also enables the modeling of transvascular transport by symmetrically distributing the fluorescence signal around the target vessel and calculating the permeability parameters of the BBB in the region of interest. In the second approach, it is considered that the glycocalyx, BEC, and the extravascular compartment, which includes the end-feet of astrocytes, together form a triple barrier called ”the tripartite BBB” (Figure. 3C). However, this model, as a simplistic description of the blood-brain barrier, fails to characterize the individual barrier properties of each component of the BBB. Through fluorescent labeling of specific cell or subcellular populations, high-resolution optical in vivo imaging plays a crucial role in investigating the specific mechanisms of normal function and impairment of the BBB. This is especially important in providing direct visualization of capillary blood flow and its modulation by neural activity154–156. This technique enables us to investigate the regulation of microvascular blood flow in the brain by smooth muscle cells and pericytes. It reveals the precise temporal pattern of smooth muscle cell and pericyte activation and demonstrates how this activation spreads upstream from the locus to the small, pliable meningeal arteries to regulate functional congestion157. In vivo microscopy requires craniotomy or cranial thinning of experimental animals before imaging (Fig. 3A/B). The resulting inflammation, edema, and proliferation of neuroglia may degrade the quality of acquired images, particularly in the case of rat models158–160. In this regard, future studies should be conducted under strict aseptic surgical conditions, optimizing the size of the cranial window and minimizing damage by precisely localizing the tissue. [insert Figure 3.] The future of two-photon microscopy will focus on expanding the imaging field of view, increasing depth and resolution, and miniaturizing equipment. For example, Cheng’s team developed a miniature two-photon microscope probe that combines MEMS scanners, electrowetting lenses, and miniature objectives, weighing only 2 to 3 grams, which can be fixed to the heads of experimental mice for continuous neuronal activity recording161. Moser’s team further developed the MINI2P system, weighing less than 3 grams, enabling rapid, multilayered imaging of up to 1,000 neurons in freely moving mice, uncovering spatial connections between neurons in different brain regions 162. Integrated with drug delivery techniques, two-photon microscopy provides an effective method to assess target engagement and predict therapeutic efficacy, showing immense potential in exploring BBB mechanisms like paracellular permeability and transcytosis, and paving the way for the next generation of therapeutic drugs 163. As a non-invasive test, Dynamic Contrast-Enhanced MRI performs rapid, consecutive, and repeated scans to obtain images before and after contrast injection. In normal circumstances, Gadolinium-Based Contrast Agents (GBCAs) administered intravenously have difficulty crossing the BBB to enter the central nervous system 164 . However, when inflammation or similar causes lead to a decrease in the expression of Tight Junction proteins on endothelial cells, GBCAs can penetrate the BBB. Their paramagnetic properties interact with nearby water molecules, primarily shortening the T1 relaxation time. This leads to higher signal intensity on T1-weighted images 165 . DCE-MRI fits a Time-Intensity-Curve (TIC) to all pixels within the ROI Region of Interest (ROI) and applies a hypothetical pharmacokinetic model to analyze the results. This process allows for the assessment of the structure and function of the microvasculature, obtaining a series of semiquantitative or quantitative parameters. Compared to traditional magnetic resonance enhancement scanning, which can only capture the enhancement characteristics of tissue at a specific time point, DCE can provide more comprehensive information about the pathophysiological characteristics of the displayed region 166 . This includes tissue vascular permeability, local blood perfusion, and other relevant factors. DCE-MRI has proven valuable in assessing BBB pathology in oncology, acute ischemic stroke, and multiple sclerosis 167–169 . Usually, the spatial resolution of the fast sequence in DCE-MRI can reach 1mm. However, sometimes to maintain high temporal resolution, the spatial resolution might be reduced from 1mm to 1.7mm. Currently, it is the most widely used and advanced imaging technique for quantitatively evaluating the integrity of the BBB. Semi-quantitative parameters are relatively easy to measure, do not involve the use of pharmacokinetic models, and provide pseudo-colour maps with good contrast. Commonly used parameters include curve morphology, onset time, peak time, peak signal intensity, and area under the curve. However, comparing the results between different patients and studies is challenging due to the influence of scanning equipment, scanning technology, and the individuals being examined 170,171 . In addition, semi-quantitative parameters make it difficult to distinguish the contribution of different factors, such as hemodynamics and vascular permeability, to signal changes 172 . Quantitative analyses of DCE-MRI use various pharmacokinetic models, commonly including the conventional Tofts model, the modified Tofts model, the Patlak model, and the measurement of the arterial input function (AIF) is required 173 (Figure. 4). Quantitative parameters include the \(K_{\text{trans}}\), transfer constant from the blood plasma to the extravascular extracellular space;\(V_{P}\), the fractional plasma volume. \(K_{\text{trans}}\) is the primary index recommended by the National Cancer Institute for DCE-MRI studies to reflect vascular permeability. [insert Figure 4.] Relative to the disruption of the BBB in acute inflammatory conditions, tumors, and cerebral infarction, the BBB leakage in neurodegenerative diseases such as PD is more subtle and challenging to detect. The research by Barnes et al. indicates that the Patlak model is especially well-suited for measuring low-level BBB leakage 174 . Olmedo-Díaz et al. used the 6-hydroxydopamine (6-OHDA) rat model of PD to explore the potential connection between brain iron accumulation and BBB impairment, neuroinflammation, and abnormal ferritin expression 175 . Within the first week after the injection of 6-OHDA, disruption of the BBB occurred, preceding the major phase of dopamine neurodegeneration. After one week, the permeability to gadolinium decreased, indicating a partial restoration of the BBB. However, even after five weeks post-injection, disturbances in the BBB were still evident, along with an increased number of GFAP-positive astrocytes. This suggests that after dopamine neuronal degeneration, the BBB is unlikely to fully recover. Even a small amount of iron leaking through the compromised BBB would be sufficient to catalyze the production of reactive oxygen species (ROS) and induce oxidative stress caused by 6-OHDA. Moreover, changes in astrogliosis may also contribute to neurovascular dysfunction. As previously mentioned, Dynamic Contrast-Enhanced (DCE) technology uses gadolinium as a contrast agent for imaging. The principle involves shortening the spin-lattice relaxation time (T1 constant) to enhance the signal on T1-weighted images. Similarly, iron oxide particle contrast agents do not generate magnetic resonance signals themselves. Instead, they reduce the signal on T2 and T2*-weighted images by shortening the spin-spin relaxation time (T2 constant) and causing phase shifts in adjacent protons, thereby achieving image contrast. Iron, a natural component of hemoglobin, endows iron oxide particle contrast agents with greater biocompatibility in comparison to gadolinium-based agents 176 . Iron oxide particles are categorized based on their diameter into ultra-small superparamagnetic iron oxide particles (10-50 nm), superparamagnetic iron oxide particles (50-300 nm), and micrometer-sized iron oxide particles (1-5 μm) 177 . Iron oxide particles offer a key advantage in their capacity to conduct non-invasive, targeted in vivo molecular imaging of the BBB. Imaging methods include coupling with specific endothelial cells such as E-selectin, P-selectin, VCAM-1, and ICAM-1, or crossing the BBB using ultra-small superparamagnetic iron oxide particles 178,179 . These particles are internalized by reactive astrocytes, microglia, and dendritic cells in the central nervous system, tracking the migration of phagocytic cell migration and reflecting inflammatory responses due to BBB disruption 180 . Superparamagnetic iron oxide (SPIO) nanoparticles are used as markers to track the presence and migration of phagocytic cells within the CNS, serving as an indicator of BBB disruption. Early research suggests that diets high in antioxidants, when given to animals with damage to the substantia nigra striatal dopaminergic system, promote dopaminergic regeneration and prolong the reduction of inflammation after neuroinflammation induction. However, in the early stages, animals fed an antioxidant-rich diet show a greater accumulation of immune cells compared to those not fed antioxidants 181,182 . To determine whether macrophages in the CNS originate from resident microglia or circulating blood cells, Ana Virel and et al. used superparamagnetic iron oxide (SPIO) nanoparticles to label phagocytic cells in the blood 183 . They combined this with T2*-weighted MRI and DCE-MRI techniques to investigate the distribution of immune cells. DCE imaging comparisons between the bilberry diet and control groups after 6-OHDA treatment showed no significant difference in the extent of BBB disruption. However, in T2*-weighted imaging, the group that consumed a bilberry diet showed larger low-signal areas, indicating increased infiltration of immune cells into the brain. Notably, within the damaged striatum, blood-derived cells carrying SPIO were found attached to capillary walls, indicating that these cells might enter the brain via capillaries, particularly in the bilberry diet group. The researchers suggest that the migration of macrophages, which is not solely reliant on BBB disruption, could be a driving factor in neuroinflammation within the PD rat model. In recent years, there has been growing interest in ultra-small superparamagnetic iron oxide (USPIO) nanoparticles as MRI contrast agents. USPIO nanoparticles have the ability to decrease magnetization intensity and shorten the T1 relaxation time of water protons, due to their spin-off effect, making them suitable for enhancing T1-weighted MRI by improving image contrast and sensitivity 184 . The ultra-small size of these nanoparticles helps them avoid non-specific uptake by mononuclear phagocytes, leading to prolonged circulation. This is beneficial for high-resolution targeted imaging and steady-state imaging. Additionally, USPIO nanoparticles can be cleared by the kidneys, reducing the risk of iron overload, and demonstrating good biocompatibility and biosafety 185 . In addition to these applications, USPIO nanoparticles are also used to track the movement of white blood cells across the BBB. This is because white blood cells can be loaded with USPIO ex vivo before reintroduction into the vascular system, allowing sufficient time for them to reach sites of inflammation and the central nervous system. Although intravenous injection of USPIO can be utilized to label circulating white blood cells, its specificity is limited to passive diffusion through the BBB 141 . Furthermore, while USPIOs have lower sensitivity to antibody binding, they exhibit better safety compared to MPIOs that lack a biodegradable coating. Hence, FDA-approved USPIO agents, such as ferumoxytol and ferumoxtran-10, are available for diagnosing various diseases. Despite the limitations of (U)SPIO-MRI technology in differentiating macrophages with pro-inflammatory and anti-inflammatory characteristics, it still shows significant potential in tracking neuroinflammatory phenomena 176 . These phenomena include the disruption of the BBB and the migration of macrophages. The research using (U)SPIO-MRI technology in PD animal models or in patients with PD is still in its early stages. To date, there have been relatively few studies using this technique in Patients with PD. Given the potential application of this technology in neuroinflammation related to the BBB, further research in this field is considered valuable. The use of radioactive isotopes (such as FDG-PET) as tracers involves the risk of radiation exposure and often has less-than-ideal spatial resolution. 186 . Recently, imaging techniques using D-glucose (Glc) or other glucose analogs, such as 3-O-methyl-D-glucose (3OMG), as magnetic resonance contrast agents, have shown significant potential for non-invasive research in areas such as BBB transport and amino acid metabolism 187 . This technique, known as Gluco-CEST (glucose Chemical Exchange Saturation Transfer), primarily measures the exchange of protons between the hydroxyl groups in glucose molecules and water molecules in biological tissues. Before introducing Gluco-CEST, it is essential to briefly explain the principles of CEST imaging. CEST imaging relies on two key conditions: 1. A pool containing target protons that will exchange with water, and 2. A frequency-specific Radio Frequency pulse to saturate these protons. In simple terms, this process involves using a selective saturation pulse to pre-saturate the hydrogen protons in specific large molecules within a solute pool. These fully saturated hydrogen protons undergo chemical exchange with the lower-energy hydrogen protons in the surrounding free water, partially saturating them and consequently reducing the magnetic resonance signal of the free water protons 188,189 . By detecting the reduced signal of free water, one can indirectly determine the distribution and concentration of the exchangeable large molecules. Based on the properties of the solute, the contrast agents used in CEST can be divided into diamagnetic CEST (dia-CEST) and paramagnetic CEST (para-CEST). During the progression of neurodegenerative diseases, significant changes occur in the brain’s energy metabolism system. These changes can be evaluated using the glucoCEST technique, especially in detecting minute leakages in the BBB. The transportation of glucose in the brain primarily relies on glucose transporter proteins (GLUTs) located on the end-feet of vascular endothelial cells and astrocytes, which are tightly regulated. Notably, D-glucose not only passively crosses the BBB through GLUT1 proteins but can also enter the brain via tight junctions on a compromised BBB 186 . Compared to gadolinium contrast agents, D-glucose has a smaller molecular weight and may demonstrate greater sensitivity to leakages in the BBB 190,191 . Currently, several factors influence the CEST effect, including the uniformity of the ultra-high magnetic field B0. Even slight non-uniformities in the magnetic field can result in substantial measurement errors in CEST asymmetry 192 . Therefore, it is essential to develop Gluco-CEST technology that is compatible with low-field devices. However, as the field strength decreases, the difference between the saturation frequency of hydroxyl protons and the resonance frequency of free water becomes smaller. This decrease in chemical shift results in an increase in background signals, which in turn reduces the sensitivity of the technique 193 . Furthermore, CEST acquisition requires multiple saturation frequencies with extended repetition times (TR), and the advancement of faster imaging sequences would facilitate the clinical application of CEST.A critical challenge with Gluco-CEST technology is the requirement to inject high concentrations of glucose (approximately 1 g/kg), which could potentially modify the distribution of transport proteins or increase paracellular permeability at the BBB 194 . Clinical translation must also take into account the risk of venous thrombosis and the tolerance of diabetic patients. Nuclear Imaging Techniques Compared to MRI, which uses easily manageable endogenous tracers, PET employs exogenous radiolabeled tracers. During the scanning process, a radiotracer is injected into the body and accumulates in metabolically active target tissues. As the radioactive isotope decays, it emits positrons. These positrons travel a short distance within the body before encountering electrons, resulting in an annihilation reaction. During this process, the positrons and electrons are annihilated, producing two gamma photons with an energy of 511 keV each, emitted in nearly opposite directions. The PET scanner is equipped with a ring of highly sensitive detectors that record the time and position of these photons. By measuring the time difference and position, the computer reconstructs the origin of the gamma photons. This technique, known as coincidence detection, allows for the creation of detailed three-dimensional images of the tracer distribution within the body, providing comprehensive information on the physiological and metabolic activities of the tissues 195 . Although PET sacrifices some spatial resolution, it offers high sensitivity, particularly for the molecular imaging of active transport proteins. Clinical PET scanners typically have a spatial resolution of 3-5 mm, while small animal scanners can achieve resolutions of up to 1 mm. High-performance PET scanners using large matrix technology can even attain sub-millimeter resolution (<1 mm) 196,197 . As an absolute quantitative technique, PET can measure the concentration and kinetics of tracers directly related to the expression and activity of BBB transport proteins. The most commonly used parameter described by PET for assessing radioactivity in tissue and plasma is the brain-to-plasma partition coefficient. According to the expert consensus published by Summerfield et al, the brain-to-plasma partition coefficient, also known as the volume of distribution (\(V_{D}\)), describes the ratio of the total concentration of a drug in the tissue to its total concentration in the plasma at steady state 198 .The parameter used to measure the net rate of drug transfer across the BBB into the brain is\(K_{1}\), Similar to \(K_{\text{trans}}\) mentioned earlier,\(K_{1}\)also relies on dynamic imaging data and uses compartmental models and Patlak plots to describe the process of drug passage through the BBB into brain tissue. Similarly, \(K_{2}\)represents the rate of transfer from brain tissue back to the blood, while \(K_{3}\) represents the rate of drug transfer within brain tissue from the rapidly equilibrating compartment to the slowly equilibrating compartment 199,200 . Beyond the previously discussed P-gp studies, recent years have seen the development of various PET tracers designed to investigate different BBB components and functions. Aquaporin (AQP) tracers, such as [¹¹C] TGN-020, have been utilized to study water exchange across the BBB. However, their binding specificity between AQP1 and AQP4 is suboptimal, potentially leading to quantification errors 201–203 . RAGE-specific tracers, including [ 18 F] RAGER and [ 18 F] FlnRAGER, target both the intracellular and extracellular domains of RAGE, demonstrating good brain uptake. However, these tracers may also bind to other targets, such as the melatonin receptor, which could affect specificity. Matrix metalloproteinase (MMP) PET imaging is another promising approach for assessing BBB damage and distinguishing leukocyte infiltration status. MMP PET can be combined with MRI to evaluate lesion-induced alterations in water exchange and cerebral blood flow dynamics. In addition, nanobody-based PET tracers have been developed to target BBB inflammation markers such as VCAM-1 and ICAM-1. These tracers are not only useful for BBB research but can also be applied to imaging peripheral immune interactions.α-Syn PET tracers, such as [ 18 F]ACI-12589, exhibit high in vitro affinity and specificity, enabling the detection of α-Syn aggregation pathology and distinguishing PD from other synucleinopathies, including MSA. Discussion and Conclusion The onset and progression of PD is a multiscale and multi-level pathological process, encompassing α-Syn aggregation, mitochondrial dysfunction, and neuroinflammatory activation at the molecular level, vascular endothelial dysfunction and synaptic damage at the cellular level, and dopaminergic pathway degeneration and global brain network reorganization at the tissue level 204 . Imaging technologies play a crucial role in deciphering this complex pathological process. They are not only essential for drug delivery research but also hold great potential in elucidating PD pathophysiology, optimizing individualized treatment strategies, and facilitating the translation of laboratory findings into clinical applications. By leveraging multimodal and multiscale imaging techniques, researchers can bridge different levels of analysis, from molecular and cellular changes to whole-brain network alterations, ultimately constructing a comprehensive framework that integrates fundamental PD research with clinical practice. At the molecular and cellular scales, SRM enables the visualization of α-Syn aggregation within BBB cells and its transmembrane transport, revealing how abnormal protein accumulation—through interactions with endothelial cells and astrocytes—impacts BBB permeability and structural integrity 205 . With its nanometer-scale resolution, SRM captures intracellular molecular changes, providing crucial evidence for early BBB pathology in PD. This insight helps elucidate the initial mechanisms of BBB damage and facilitates the development of targeted therapies against protein aggregation. However, since SRM is primarily applicable to fixed samples, it lacks the ability to monitor dynamic pathological processes. To address this limitation, two-photon microscopy (TPM) is required for real-time in vivo imaging. TPM allows for the assessment of functional BBB changes, including hemodynamic alterations, permeability shifts, and cell-cell interactions, thereby elucidating the dynamic mechanisms of BBB disruption during PD progression. In PD models, TPM enables the observation of BBB leakage, impaired cerebral blood flow, and the cascading effects of neuroinflammatory responses. These real-time in vivo insights provide essential evidence for developing targeted therapeutic strategies aimed at restoring BBB integrity 163,206 . At the tissue and whole-brain levels, MRI provides a non-invasive and repeatable approach for assessing global BBB structure and function. DCE-MRI allows for the quantification of BBB permeability changes, evaluation of vascular integrity, and assessment of cerebral perfusion. In PD patients, it can be used to monitor BBB disruption, as well as evaluate microvascular pathology, brain atrophy, and alterations in blood flow perfusion 141,207 . These MRI-based findings complement the insights from SRM and TPM, while also serving as valuable clinical tools for tracking disease progression and evaluating therapeutic efficacy. Additionally, PET offers unique advantages in BBB function and metabolic assessments, particularly in detecting transporter activity (e.g., P-gp) and brain metabolic abnormalities. When combined with novel PET tracers targeting various BBB components, PET imaging provides functional evidence that enhances the understanding of BBB dysfunction in PD pathology and informs the development of targeted therapies for transport system and metabolic abnormalities. The integration of multimodal imaging techniques enables researchers to bridge multiple scales, from nanometer-level molecular insights to macroscopic assessments, providing a systematic understanding of BBB disruption in PD and its impact on disease progression. From the molecular, cellular, tissue, and system levels, SRM reveals the fine molecular and cellular structures of the BBB, TPM provides dynamic functional data at the tissue level, MRI offers non-invasive global structural and functional assessments, and PET contributes metabolic and functional imaging insights 208,209 . This multimodal approach not only provides comprehensive imaging evidence for investigating BBB damage mechanisms in PD but also facilitates the discovery of new therapeutic targets and validation of treatment efficacy. More importantly, the synergistic application of multimodal imaging accelerates the translation of laboratory research into clinical practice, strengthening the scientific foundation for precise PD diagnosis, individualized treatment strategies, and therapy evaluation. Future research should focus on optimizing the integration capacity of multimodal imaging, enhancing the sensitivity and specificity of these techniques in detecting pathological changes. Key directions include the development of more efficient imaging probes and contrast agents, such as high-sensitivity MRI and PET tracers, as well as multifunctional dyes compatible with multiple imaging modalities. Additionally, the incorporation of artificial intelligence (AI) and advanced high-dimensional imaging analysis will facilitate the automated processing of complex multimodal data, ensuring a seamless transition from fundamental research to clinical applications. Large-scale, multicenter clinical studies will be essential to validate the applicability of these imaging technologies in early PD diagnosis, disease monitoring, and therapeutic evaluation, ultimately driving their widespread clinical adoption. Imaging is not only a crucial tool in PD research but also a core driving force behind precision medicine, accelerating the translation of laboratory discoveries into clinical applications. Abbreviations 6-OHDA: 6-Hydroxydopamine AQP1: Aquaporin-1 AQP4: Aquaporin-4 BBB: Blood-Brain Barrier BEC: Brain Endothelial Cells CEST: Chemical Exchange Saturation Transfer CSF: Cerebrospinal Fluid DCE: Dynamic Contrast-Enhanced FDG: Fluorodeoxyglucose FUS: Focused Ultrasound GFAP: Glial Fibrillary Acidic Protein GLUT1: Glucose Transporter 1 ICAM-1: Intercellular Adhesion Molecule-1 IL: Interleukin LRRK2: Leucine-Rich Repeat Kinase 2 MMP: Matrix Metalloproteinase MRI: Magnetic Resonance Imaging MSA: Multiple System Atrophy NVU: Neurovascular Unit P-gp: P-Glycoprotein PD: Parkinson’s Disease PET: Positron Emission Tomography PSP: Progressive Supranuclear Palsy RAGE: Receptor for Advanced Glycation End Products ROI: Region of Interest ROS: Reactive Oxygen Species SN: Substantia Nigra SNCA: Alpha-Synuclein SPIO: Superparamagnetic Iron Oxide SRM: Super-Resolution Microscopy STED: Stimulated Emission Depletion Microscopy TIC: Time-Intensity Curve TJ: Tight Junction TNF: Tumor Necrosis Factor TPM: Two-Photon Microscopy USPIO: Ultra-Small Superparamagnetic Iron Oxide VCAM-1: Vascular Cell Adhesion Molecule-1 VDR: Vitamin D Receptor Acknowledgements This study was supported by National Key R&D Program of China (2022YFC2009900) and the National Natural Science Foundation of China (Grant Nos. 81621003, 81761128023, 81820108018 and 82027808) and NIH/NIMH R01MH112189-01. Author contributions All authors have read and approved this paper. Zihao Lu drafted, developed, and revised the manuscript, and conceived the figures. Zihao Lu and Haolin Yin created the figures and organized the tables. Pan Xiang and Xuan Yi contributed to the literature search, review, and manuscript refinement. Qiyong Gong and Xiaohe Tian revised this paper. Declaration of competing interest All the authors declare that they have no competing interests in this paper. Reference 1. Dorsey ER, Sherer T, Okun MS, et al. The Emerging Evidence of the Parkinson Pandemic. J Park Dis 2018; 8: S3–S8.2. Bloem BR, Okun MS, Klein C. Parkinson’s disease. Lancet Lond Engl 2021; 397: 2284–2303.3. Pan G, Jiang Y, Zhang W, et al. Identification of Parkinson’s disease subtypes with distinct brain atrophy progression and its association with clinical progression. Psychoradiology ; 4. Epub ahead of print 7 February 2024. DOI: 10.1093/psyrad/kkae002.4. Iadecola C. 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Keywords alpha-synuclein blood brain barrier mri parkinson's disease Authors Affiliations Zihao Lu Sichuan University West China Hospital Department of Radiology View all articles by this author Haolin Yin Sichuan University West China Hospital Department of Radiology View all articles by this author Pan Xiang Sichuan University West China Hospital Department of Radiology View all articles by this author Xuan Yi Sichuan University School of Mechanical Engineering View all articles by this author Xiaohe Tian Sichuan University West China Hospital Department of Radiology View all articles by this author Qiyong Gong 0000-0002-5912-4871 [email protected] Sichuan University West China Hospital Department of Radiology View all articles by this author Metrics & Citations Metrics Article Usage 514 views 246 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Zihao Lu, Haolin Yin, Pan Xiang, et al. Multiscale Imaging Approaches to Decipher Blood-Brain Barrier Disruption in Parkinson’s Disease: A Bridge to Novel Therapeutic Targets. Authorea . 16 May 2025. DOI: https://doi.org/10.22541/au.174736644.44597516/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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