Interplay between the glymphatic system and neurotoxic proteins in Parkinson’s disease and related disorders:current knowledge and future directions

Parkinson’s disease is a common neurodegenerative disorder that is associated with abnormal aggregation and accumulation of neurotoxic proteins,includingα-synuclein,amyloid-β,and tau,in addition to the impaired elimination of these neurotoxic protein.Atypical parkinsonism,which has the same clinical presentation and neuropathology as Parkinson’s disease,expands the disease landscape within the continuum of Parkinson’s disease and related disorders.The glymphatic system is a waste clearance system in the brain,which is responsible for eliminating the neurotoxic proteins from the interstitial fluid.Impairment of the glymphatic system has been proposed as a significant contributor to the development and progression of neurodegenerative disease,as it exacerbates the aggregation of neurotoxic proteins and deteriorates neuronal damage.Therefore,impairment of the glymphatic system could be considered as the final common pathway to neurodegeneration.Previous evidence has provided initial insights into the potential effect of the impaired glymphatic system on Parkinson’s disease and related disorders;however,many unanswered questions remain.This review aims to provide a comprehensive summary of the growing literature on the glymphatic system in Parkinson’s disease and related disorders.The focus of this review is on identifying the manifestations and mechanisms of interplay between the glymphatic system and neurotoxic proteins,including loss of polarization of aquaporin-4 in astrocytic endfeet,sleep and circadian rhythms,neuroinflammation,astrogliosis,and gliosis.This review further delves into the underlying pathophysiology of the glymphatic system in Parkinson’s disease and related disorders,and the potential implications of targeting the glymphatic system as a novel and promising therapeutic strategy.

Glymphatic system:a gateway for neuroinflammation

The glymphatic system is a relatively recently identified fluid exchange and transpo rt system in the brain.Accumulating evidence indicates thatglymphatic function is impaired not only in central nervous system disorders but also in systemic diseases.Systemic diseases can trigger the inflammatory responses in the central nervous system,occasionally leading to sustained inflammation and functional disturbance of the central nervous system.This review summarizes the current knowledge on the association between glymphatic dysfunction and central nervous system inflammation.In addition,we discuss the hypothesis that disease conditions initially associated with peripheral inflammation ove rwhelm the performance of the glymphatic system,thereby triggering central nervous system dysfun ction,chronic neuroinflammation,and neurodegeneration.Future research investigating the role of the glymphatic system in neuroinflammation may offer innovative therapeutic approaches for central nervous system disorders.

Peripheral origin exosomal micro RNAs aggravate glymphatic system dysfunction in diabetic cognitive impairment

Cognitive dysfunction is one of the common central nervous systems(CNS)complications of diabetes mellitus,which seriously affects the quality of life of patients and results in a huge economic burden.The glymphatic system dysfunction mediated by aquaporin-4(AQP4)loss or redistribution in perivascular astrocyte endfeet plays a crucial role in diabetes-induced cognitive impairment(DCI).However,the mechanism of AQP4 loss or redistribution in the diabetic states remains unclear.Accumulating evidence suggests that peripheral insulin resistance target tissues and CNS communication affect brain homeostasis and that exosomal miRNAs are key mediators.Glucose and lipid metabolism disorder is an important pathological feature of diabetes mellitus,and skeletal muscle,liver and adipose tissue are the key target insulin resistance organs.In this review,the changes in exosomal miRNAs induced by peripheral metabolism disorders in diabetes mellitus were systematically reviewed.We focused on exosomal miRNAs that could induce low AQP4 expression and redistribution in perivascular astrocyte endfeet,which could provide an interorgan communication pathway to illustrate the pathogenesis of DCI.Furthermore,the mechanisms of exosome secretion from peripheral insulin resistance target tissue and absorption to the CNS were summarized,which will be beneficial for proposing novel and feasible strategies to optimize DCI prevention and/or treatment in diabetic patients.

The lymphatic system:a therapeutic target for central nervous system disorders

The lymphatic vasculature forms an organized network that covers the whole body and is involved in fluid homeostasis,metabolite clearance,and immune surveillance.The recent identification of functional lymphatic vessels in the meninges of the brain and the spinal cord has provided novel insights into neurophysiology.They emerge as major pathways for fluid exchange.The abundance of immune cells in lymphatic vessels and meninges also suggests that lymphatic vessels are actively involved in neuroimmunity.The lymphatic system,through its role in the clearance of neurotoxic proteins,autoimmune cell infiltration,and the transmission of pro-inflammatory signals,participates in the pathogenesis of a variety of neurological disorders,including neurodegenerative and neuroinflammatory diseases and traumatic injury.Vascular endothelial growth factor C is the master regulator of lymphangiogenesis,a process that is critical for the maintenance of central nervous system homeostasis.In this review,we summarize current knowledge and recent advances relating to the anatomical features and immunological functions of the lymphatic system of the central nervous system and highlight its potential as a therapeutic target for neurological disorders and central nervous system repair.

From static to dynamic:live observation of the support system after ischemic stroke by two photon-excited fluorescence laser-scanning microscopy

Ischemic stroke is one of the most common causes of mortality and disability worldwide.However,treatment efficacy and the progress of research remain unsatisfactory.As the critical support system and essential components in neurovascular units,glial cells and blood vessels(including the bloodbrain barrier)together maintain an optimal microenvironment for neuronal function.They provide nutrients,regulate neuronal excitability,and prevent harmful substances from entering brain tissue.The highly dynamic networks of this support system play an essential role in ischemic stroke through processes including brain homeostasis,supporting neuronal function,and reacting to injuries.However,most studies have focused on postmortem animals,which inevitably lack critical information about the dynamic changes that occur after ischemic stroke.Therefore,a high-precision technique for research in living animals is urgently needed.Two-photon fluorescence laser-scanning microscopy is a powerful imaging technique that can facilitate live imaging at high spatiotemporal resolutions.Twophoton fluorescence laser-scanning microscopy can provide images of the whole-cortex vascular 3D structure,information on multicellular component interactions,and provide images of structure and function in the cranial window.This technique shifts the existing research paradigm from static to dynamic,from flat to stereoscopic,and from single-cell function to multicellular intercommunication,thus providing direct and reliable evidence to identify the pathophysiological mechanisms following ischemic stroke in an intact brain.In this review,we discuss exciting findings from research on the support system after ischemic stroke using two-photon fluorescence laser-scanning microscopy,highlighting the importance of dynamic observations of cellular behavior and interactions in the networks of the brain’s support systems.We show the excellent application prospects and advantages of two-photon fluorescence laser-scanning microscopy and predict future research developments and directions in the study of ischemic stroke.

The lymphatic drainage systems in the brain:a novel target for ischemic stroke?

Recent studies have proposed three lymphatic drainage systems in the brain,that is,the glymphatic system,the intramural periarterial drainage pathway,and meningeal lymphatic vessels,whose roles in various neurological diseases have been widely explored.The glymphatic system is a fluid drainage and waste clearance pathway that utilizes perivascular space and aquaporin-4 protein located in the astrocyte endfeet to provide a space for exchange of cerebrospinal fluid and interstitial fluid.The intramural periarterial drainage pathway drives the flow of interstitial fluid through the capillary basement membrane and the arterial tunica media.Meningeal lymphatic vessels within the dura mater are involved in the removal of cerebral macromolecules and immune responses.After ischemic stroke,impairment of these systems could lead to cerebral edema,accumulation of toxic factors,and activation of neuroinflammation,while restoration of their normal functions can improve neurological outcomes.In this review,we summarize the basic concepts of these drainage systems,including drainage routes,physiological functions,regulatory mechanisms,and detection technologies.We also focus on the roles of lymphatic drainage systems in brain injury after ischemic stroke,as well as recent advances in therapeutic strategies targeting these drainage systems.These findings provide information for potential novel strategies for treatment of stroke.

Propagation of tau and α-synuclein in the brain: therapeutic potential of the glymphatic system

Many neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease, are characterised by the accumulation of misfolded protein deposits in the brain, leading to a progressive destabilisation of the neuronal network and neuronal death. Among the proteins that can abnormally accumulate are tau and α-synuclein, which can propagate in a prion-like manner and which upon aggregation, represent the most common intracellular proteinaceous lesions associated with neurodegeneration. For years it was thought that these intracellular proteins and their accumulation had no immediate relationship with extracellular homeostasis pathways such as the glymphatic clearance system;however, mounting evidence has now suggested that this is not the case. The involvement of the glymphatic system in neurodegenerative disease is yet to be fully defined;however, it is becoming increasingly clear that this pathway contributes to parenchymal solute clearance. Importantly, recent data show that proteins prone to intracellular accumulation are subject to glymphatic clearance, suggesting that this system plays a key role in many neurological disorders. In this review, we provide a background on the biology of tau and α-synuclein and discuss the latest findings on the cell-to-cell propagation mechanisms of these proteins. Importantly, we discuss recent data demonstrating that manipulation of the glymphatic system may have the potential to alleviate and reduce pathogenic accumulation of propagation-prone intracellular cytotoxic proteins. Furthermore, we will allude to the latest potential therapeutic opportunities targeting the glymphatic system that might have an impact as disease modifiers in neurodegenerative diseases.

The Integrative Five-Fluid Circulation System in the Human Body

Water is the key medium to transport numerous constituents and to provide a platform for physiological processes to take place in the living organisms in general;it also participates actively in many of these processes. In humans, there are different vehicles to contain water and its constituents. Our objective is to find out whether there is an overall water-base circulation system in the human body by analyzing the updated findings of different research groups on the physiological functions of various seemingly isolated fluid systems. By 1963, there were five separate fluid systems discovered in mammalians: (i) The Primo Vasculature Fluid (PVF) with protein precursors and micro cells held in the Primo Vasculature System (PVS). (ii) Blood with its constituents held in the cardio vasculature. (iii) Extracranial interstitial fluid (ISF) whose vehicle had a very irregular structure—the interstitium all over the body. (iv) The cerebrospinal fluid had been considered to be within the brain ventricles and spinal canal. (v) The extra-cranial lymphatic system which drained ISF, and had been known to join the subclavian vein. Fluid (i) was first reported in 1963 and fluids (ii) to (v) have been known for many decades, but the failure to detect a lymphatic system inside the skull has also been a mystery for many decades. The intra-cranial ISF (which we name as BISF) has drawn little attention, apart from discussing the mechanism of the blood-brain-barrier. During the past decade, there has been direct evidence indicating that CSF and BISF are actually mixed. After that, the intracranial lymphatic system was discovered and confirmed in animal models only slightly over one year back, and we called such fluid as glymphatic-fluid. After reviewing the stated “classical” five fluid systems together with the new findings in Sections 2 - 7, we propose, for the first time, that the PVF, the blood, ISF, a mixture of CSF-BISF, and a mixture of glymphatic-fluid and lymph form an integrative circulation system in water base in the human and other mammalian bodies, as schematically represented in the last section. In this paper, we point out the positive correlation of chronic neuro degenerative diseases such as Alzheimer’s disease, Parkinson’s diseases and the insufficient brain wastes clearance by the glymphatic system. We also discuss the role played by the venous vessels as part of such clearance in upright posture. Moreover, simple non-invasive maneuver techniques are introduced here, as one example of enhancement of glymphatic fluid flow out of the skull to join the lymphatic system. A series of questions are raised in Section 8, the answers to which would help us to understand the transition from physio- to pathological states in the development of many diseases. Detailed analysis of this paper leads us to consider that research in understanding this integrative circulation system is only at the infancy stage, and fluid dynamics investigation seems to be the plausible modality of approach in the near future.

Glymphatic imaging and modulation of the optic nerve

Optic nerve health is essential for proper function of the visual system.However,the pathophysiology of certain neurodegenerative disease processes affecting the optic nerve,such as glaucoma,is not fully understood.Recently,it was hypothesized that a lack of proper clearance of neurotoxins contributes to neurodegenerative diseases.The ability to clear metabolic waste is essential for tissue homeostasis in mammals,including humans.While the brain lacks the traditional lymphatic drainage system identified in other anatomical regions,there is growing evidence of a glymphatic system in the central nervous system,which structurally includes the optic nerve.Named to acknowledge the supportive role of astroglial cells,this perivascular fluid drainage system is essential to remove toxic metabolites from the central nervous system.Herein,we review existing literature describing the physiology and dysfunction of the glymphatic system specifically as it relates to the optic nerve.We summarize key imaging studies demonstrating the existence of a glymphatic system in the optic nerves of wild-type rodents,aquaporin 4-null rodents,and humans;glymphatic imaging studies in diseases where the optic nerve is impaired;and current evidence regarding pharmacological and lifestyle interventions that may help promote glymphatic function to improve optic nerve health.We conclude by highlighting future research directions that could be applied to improve imaging detection and guide therapeutic interventions for diseases affecting the optic nerve.

Impact of apolipoprotein E isoforms on sporadic Alzheimer's disease:beyond the role of amyloid beta

The impact of apolipoprotein E(ApoE)isoforms on sporadic Alzheimer's disease has long been studied;however,the influences of apolipoprotein E gene(APOE)on healthy and pathological human brains are not fully understood.ApoE exists as three common isoforms(ApoE2,ApoE3,and ApoE4),which differ in two amino acid residues.Traditionally,ApoE binds cholesterol and phospholipids and ApoE isoforms display diffe rent affinities for their receptors,lipids transport and distribution in the brain and periphery.The role of ApoE in the human depends on ApoE isoforms,brain regions,aging,and neural injury.APOE E4 is the strongest genetic risk factor for sporadic Alzheimer's disease,considering its role in influencing amyloid-beta metabolism.The exact mechanisms by which APOE gene variants may increase or decrease Alzheimer's disease risk are not fully understood,but APOE was also known to affect directly and indirectly tau-mediated neurodegeneration,lipids metabolism,neurovascular unit,and microglial function.Consistent with the biological function of ApoE,ApoE4 isoform significantly alte red signaling pathways associated with cholesterol homeostasis,transport,and myelination.Also,the rare protective APOE variants confirm that ApoE plays an important role in Alzheimer's disease pathogenesis.The objectives of the present mini-review were to describe classical and new roles of various ApoE isoforms in Alzheimer's disease pathophysiology beyond the deposition of amyloid-beta and to establish a functional link between APOE,brain function,and memory,from a molecular to a clinical level.APOE genotype also exerted a heterogeneous effect on clinical Alzheimer's disease phenotype and its outcomes.Not only in learning and memory but also in neuro psychiatric symptoms that occur in a premorbid condition.Cla rifying the relationships between Alzheimer's disease-related pathology with neuropsychiatric symptoms,particularly suicidal ideation in Alzheimer's disease patients,may be useful for elucidating also the underlying pathophysiological process and its prognosis.Also,the effects of anti-amyloid-beta drugs,recently approved for the treatment of Alzheimer's disease,could be influenced by the APOE genotype.

The relationship between the lymphatic system and Alzheimer's disease wasdiscussed based on the tri-jiao theory

Type of the lymphatic system is a new concept put forward by foreign scholars in recent years.It is similar to the lymphatic system,is the brain of the internal fluid metabolism channel and its metabolites in the brain plays a major role in the process of removal,sleep disorders in alzheimer's disease brain stroke,traumatic brain injury and other central nervous system diseases.The Tri-jiao theory is a very important in traditional Chinese medicine plate structure,it is from the function to traffic from running water channel,but still no definite conclusion on structure.This paper attempts to explore the relationship between trioke and the lymphatic system and their influence on Alzheimer's disease through modern medical research on the lymphatic system,so as to provide new inspirations for the treatment of Alzheimer's disease.

Glymphatic系统与认知障碍相关疾病的研究进展

Glymphatic系统通过脑脊液和脑间质液之间的引流与物质交换使得脑内代谢废物被快速清除,影响因素包括AQP-4、脑动脉搏动、睡眠及体位等。Glymphatic系统功能障碍可导致多种认知障碍相关疾病发生,促使其功能恢复可能是防治认知障碍相关疾病的新思路。

Novel therapeutic strategies targeting mitochondria as a gateway in neurodegeneration

In recent years, multiple disciplines have focused on mitochondrial biology and contributed to understanding its relevance towards adult-onset neurodegenerative disorders. These are complex dynamic organelles that have a variety of functions in ensuring cellular health and homeostasis. The plethora of mitochondrial functionalities confers them an intrinsic susceptibility to internal and external stressors(such as mutation accumulation or environmental toxins), particularly so in long-lived postmitotic cells such as neurons. Thus, it is reasonable to postulate an involvement of mitochondria in aging-associated neurological disorders, notably neurodegenerative pathologies including Alzheimer’s disease and Parkinson’s disease. On the other hand, biological effects resulting from neurodegeneration can in turn affect mitochondrial health and function, promoting a feedback loop further contributing to the progression of neuronal dysfunction and cellular death. This review examines state-of-the-art knowledge, focus on current research exploring mitochondrial health as a contributing factor to neuroregeneration, and the development of therapeutic approaches aimed at restoring mitochondrial homeostasis in a pathological setting.

Impact of Manipulative Treatment on Professional Drivers

The experimental research, presented in the study, focuses on track tests with the aim of highlighting changes in lap times after manipulative treatment of drainage of the glymphatic system and stimulation of the sympathetic nervous system. Introduction: The experimental research, presented in this study, focuses on analyzing the potential effects of a manipulative treatment on the performance of a professional driver. The main objective is to evaluate the change in lap times after the application of the treatment, trying to understand whether it can actually positively influence the driver’s performance. The study stands an important opportunity to extend knowledge, regarding the use of manipulative therapies in the context of optimized driving skills. The results obtained could provide useful insights and contribute to improving the performance of professional drivers by offering new perspectives and strategies to improve their performance. Leveraging a rigorous scientific approach and a sample of highly skilled drivers, the research aims to provide concrete evidence on the effectiveness of manipulative treatment in driving skills. Monitoring lap times before and after the intervention also capture any temporary or long-term effects of the treatment, ensuring a thorough and reliable analysis of the results. Materials and methods: 15 professional drivers, aged 18 to 36 years, with at least 10 years of experience as drivers, participated in this study. The test consisted of analyzing lap times before and after treatment.

Functional aspects of the brain lymphatic drainage system in aging and neurodegenerative diseases

The phenomenon of an aging population is advancing at a precipitous rate.Alzheimer's disease(AD)and Parkinson's disease(PD)are two of the most common age-associated neurodegenerative diseases,both of which are primarily characterized by the accumulation of toxic proteins and the progressive demise of neuronal structures.Recent discoveries about the brain lymphatic drainage system have precipitated a growing body of investigations substantiating its novel roles,including the clearance of macromolecular waste and the trafficking of immune cells.Notably,aquaporin 4-mediated glymphatic transport,crucial for maintaining neural homeostasis,becomes disrupted during the aging process and is further compromised in the pathogenesis of AD and PD.Functional meningeal lymphatic vessels,which facilitate the drainage of cerebrospinal fluid into the deep cervical lymph nodes,are integral in bridging the central nervous system with peripheral immune responses.Dysfunction in these meningeal lymphatic vessels exacerbates pathological trajectory of the age-related neurodegenerative disease.This review explores modulatory influence of the glymphatic system and meningeal lymphatic vessels on the aging brain and its associated neurodegenerative disorders.It also encapsulates the insights of potential mechanisms and prospects of the targeted non-pharmacological interventions.

Aquaporin 4 deficiency eliminates the beneficial effects of voluntary exercise in a mouse model of Alzheimer’s disease

Regular exercise has been shown to reduce the risk of Alzheimer’s disease(AD).Our previous study showed that the protein aquaporin 4(AQP4),which is specifically expressed on the paravascular processes of astrocytes,is necessary for glymphatic clearance of extracellular amyloid beta(Aβ)from the brain,which can delay the progression of Alzheimer’s disease.However,it is not known whether AQP4-regulated glymphatic clearance of extracellular Aβis involved in beneficial effects of exercise in AD patients.Our results showed that after 2 months of voluntary wheel exercise,APP/PS1 mice that were 3 months old at the start of the intervention exhibited a decrease in Aβburden,glial activation,perivascular AQP4 mislocalization,impaired glymphatic transport,synapse protein loss,and learning and memory defects compared with mice not subjected to the exercise intervention.In contrast,APP/PS1 mice that were 7 months old at the start of the intervention exhibited impaired AQP4 polarity and reduced glymphatic clearance of extracellular Aβ,and the above-mentioned impairments were not alleviated after the 2-month exercise intervention.Compared with age-matched APP/PS1 mice,AQP4 knockout APP/PS1 mice had more serious defects in glymphatic function,Aβplaque deposition,and cognitive impairment,which could not be alleviated after the exercise intervention.These findings suggest that AQP4-dependent glymphatic transport is the neurobiological basis for the beneficial effects of voluntary exercises that protect against the onset of AD.

Updated Understanding of the Glial-Vascular Unit in Central Nervous System Disorders

The concept of the glial-vascular unit(GVU)was raised recently to emphasize the close associations between brain cells and cerebral vessels,and their coordinated reactions to diverse neurological insults from a“glio-centric”view.GVU is a multicellular structure composed of glial cells,perivascular cells,and perivascular space.Each component is closely linked,collectively forming the GVU.The central roles of glial and perivascular cells and their multi-level interconnections in the GVU under normal conditions and in central nervous system(CNS)disorders have not been elucidated in detail.Here,we comprehensively review the intensive interactions between glial cells and perivascular cells in the niche of perivascular space,which take part in the modulation of cerebral blood flow and angiogenesis,formation of the blood-brain barrier,and clearance of neurotoxic wastes.Next,we discuss dysfunctions of the GVU in various neurological diseases,including ischemic stroke,spinal cord injury,Alzheimer’s disease,and major depression disorder.In addition,we highlight the possible therapies targeting the GVU,which may have potential clinical applications.

Alzheimer’s Disease, Early Diagnosis

Aim: In this review paper we propose a method to make an early diagnosis of the Alzheimer’s Disease (AD), the most common form of neurodegenerative dementia. Background: Glymphatic System (GS) is the main means of eliminating waste substances in the central nervous system (CNS);if it does not work properly, waste substances accumulate in CNS until to cause AD. Basal Forebrain is the most important component of a much broader system of cholinergic cells distributed throughout the Central Nervous System (CNS). This structure regulates attention, learning and memory and its destruction is considered responsible for the cognitive AD alterations. The characteristics of AD patients, that interest us most, are the lack of Acetylcholine, and the Orexin excess;we think that the hypothalamus produces more Orexin to stimulate cholinergic cells, indispensable for a correct CNS functioning. We want to identify these patients by detecting the Orexin excess. Early Diagnosis Model. Of course we could take a cerebrospinal fluid sample and dose Orexin but this method is risky and painful for the patient’s health, therefore unsuitable for large numbers of patients. We propose a fairly simple method for the early diagnosis of AD: if we temporarily eliminate the Orexin excess, with Dual Orexin Receptor Antagonist (DORA), i.e. Suvorexant, we can intercept the Orexin increase and demonstrate the decrease in Acetylcholine with a Functional Magnetic Resonance or a Polysomnography, many years before the AD symptoms occur.

Digoxin Ameliorates Glymphatic Transport and Cognitive Impairment in a Mouse Model of Chronic Cerebral Hypoperfusion

The glymphatic system plays a pivotal role in maintaining cerebral homeostasis.Chronic cerebral hypoperfusion,arising from small vessel disease or carotid stenosis,results in cerebrometabolic disturbances ultimately manifesting in white matter injury and cognitive dysfunction.However,whether the glymphatic system serves as a potential therapeutic target for white matter injury and cognitive decline during hypoperfusion remains unknown.Here,we established a mouse model of chronic cerebral hypoperfusion via bilateral common carotid artery stenosis.We found that the hypoperfusion model was associated with significant white matter injury and initial cognitive impairment in conjunction with impaired glym・phatic system function.The glymphatic dysfunction was associated with altered cerebral perfusion and loss of aquaporin 4 polarization.Treatment of digoxin rescued changes in glymphatic transport,white matter structure,and cognitive function.Suppression of glymphatic functions by treatment with the AQP4 inhibitor TGN-020 abolished this protective effect of digoxin from hypoperfusion injury.Our research yields new insight into the relationship between hemodynamics,glymphatic transport,white matter injury,and cognitive changes after chronic cerebral hypoperfusion.

Quantitative Determination of Glymphatic Flow Using Spectrophotofluorometry

Following intrathecal injection of fluorescent tracers,ex vivo imaging of brain vibratome slices has been widely used to study the glymphatic system in the rodent brain.Tracer penetration into the brain is usually quantified by image-processing,even though this approach requires much time and manual operation.Here,we illustrate a simple protocol for the quantitative determination of glymphatic activity using spectrophotofluorometry.At specific time-points following intracisternal or intrastriatal injection of fluorescent tracers,certain brain regions and the spinal cord were harvested and tracers were extracted from the tissue.The intensity of tracers was analyzed spectrophotometrically and their concentrations were quantified from standard curves.Using this approach,the regional and dynamic delivery of subarachnoid CSF tracers into the brain parenchyma was assessed,and the clearance of tracers from the brain was also determined.Furthermore,the impairment of glymphatic influx in the brains of old mice was confirmed using our approach.Our method is more accurate and efficient than the imaging approach in terms of the quantitative determination of glymphatic activity,and this will be useful in preclinical studies.