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In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...

University of Rochester Medical Center

Glymphatic transport, defined as cerebrospinal fluid (CSF) peri-arterial inflow into brain, and interstitial fluid (ISF) clearance, is reduced in the aging brain. However, it is unclear whether glymphatic transport affects the distribution of soluble Aβ in Alzheimer's disease (AD). In wild type mice, we show that Aβ40 (fluorescently labeled Aβ40 or unlabeled Aβ40), was distributed from CSF to brain, via the peri-arterial space, and associated with neurons. In contrast, Aβ42 was mostly restricted to the peri-arterial space due mainly to its greater propensity to oligomerize when compared to Aβ40. Interestingly, pretreatment with Aβ40 in the CSF, but not Aβ42, reduced CSF transport into brain. In APP/PS1 mice, a model of AD, with and without extensive amyloid-β deposits, glymphatic transport was reduced, due to the accumulation of toxic Aβ species, such as soluble oligomers. CSF-derived Aβ40 co-localizes with existing endogenous vascular and parenchymal amyloid-β plaques, and thus, may contribute to the progression of both cerebral amyloid angiopathy and parenchymal Aβ accumulation. Importantly, glymphatic failure preceded significant amyloid-β deposits, and thus, may be an early biomarker of AD. By extension, restoring glymphatic inflow and ISF clearance are potential therapeutic targets to slow the onset and progression of AD.

Suppression of glymphatic fluid transport in a mouse model of Alzheimer's disease.

Cerebral small vessel diseases (SVDs) range broadly in etiology but share remarkably overlapping pathology. Features of SVD including enlarged perivascular spaces (EPVS) and formation of abluminal protein deposits cannot be completely explained by the putative pathophysiology. The recently discovered glymphatic system provides a new perspective to potentially address these gaps. This work provides a comprehensive review of the known factors that regulate glymphatic function and the disease mechanisms underlying glymphatic impairment emphasizing the role that aquaporin-4 (AQP4)-lined perivascular spaces (PVSs), cerebrovascular pulsatility, and metabolite clearance play in normal CNS physiology. This review also discusses the implications that glymphatic impairment may have on SVD inception and progression with the aim of exploring novel therapeutic targets and highlighting the key questions that remain to be answered.

Perivascular spaces, glymphatic dysfunction, and small vessel disease.

Despite the initial promise of immunotherapy for CNS disease, multiple recent clinical trials have failed. This may be due in part to characteristically low penetration of antibodies to cerebrospinal fluid (CSF) and brain parenchyma, resulting in poor target engagement. We here utilized transcranial macroscopic imaging to noninvasively evaluate in vivo delivery pathways of CSF fluorescent tracers. Tracers in CSF proved to be distributed through a brain-wide network of periarterial spaces, previously denoted as the glymphatic system. CSF tracer entry was enhanced approximately 3-fold by increasing plasma osmolality without disruption of the blood-brain barrier. Further, plasma hyperosmolality overrode the inhibition of glymphatic transport that characterizes the awake state and reversed glymphatic suppression in a mouse model of Alzheimer's disease. Plasma hyperosmolality enhanced the delivery of an amyloid-β (Aβ) antibody, obtaining a 5-fold increase in antibody binding to Aβ plaques. Thus, manipulation of glymphatic activity may represent a novel strategy for improving penetration of therapeutic antibodies to the CNS.

Transcranial optical imaging reveals a pathway for optimizing the delivery of immunotherapeutics to the brain.

The glymphatic (glial-lymphatic) pathway is a fluid-clearance pathway identified in the rodent brain in 2012. This pathway subserves the flow of CSF into the brain along arterial perivascular spaces and subsequently into the brain interstitium, facilitated by aquaporin 4 (AQP4) water channels. The pathway then directs flow towards the venous perivascular and perineuronal spaces, ultimately clearing solutes from the neuropil into meningeal and cervical lymphatic drainage vessels. In rodents, the glymphatic pathway is predominantly active during sleep, when the clearance of harmful metabolites such as amyloid β (Aβ) increases two-fold relative to the waking state. Glymphatic dysfunction, probably related to perturbed AQP4 expression, has been shown in animal models of traumatic brain injury, Alzheimer's disease, and stroke. The recent characterisations of the glymphatic and meningeal lymphatic systems in rodents and in humans call for revaluation of the anatomical routes for CSF-interstitial fluid flow and the physiological role that these pathways play in CNS health.

The glymphatic pathway in neurological disorders.

Flow of cerebrospinal fluid (CSF) through perivascular spaces (PVSs) in the brain is important for clearance of metabolic waste. Arterial pulsations are thought to drive flow, but this has never been quantitatively shown. We used particle tracking to quantify CSF flow velocities in PVSs of live mice. CSF flow is pulsatile and driven primarily by the cardiac cycle. The speed of the arterial wall matches that of the CSF, suggesting arterial wall motion is the principal driving mechanism, via a process known as perivascular pumping. Increasing blood pressure leaves the artery diameter unchanged but changes the pulsations of the arterial wall, increasing backflow and thereby reducing net flow in the PVS. Perfusion-fixation alters the normal flow direction and causes a 10-fold reduction in PVS size. We conclude that particle tracking velocimetry enables the study of CSF flow in unprecedented detail and that studying the PVS in vivo avoids fixation artifacts.

Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension.

The glymphatic system is a brain-wide clearance pathway; its impairment contributes to the accumulation of amyloid-β. Influx of cerebrospinal fluid (CSF) depends upon the expression and perivascular localization of the astroglial water channel aquaporin-4 (AQP4). Prompted by a recent failure to find an effect of  knock-out (KO) on CSF and interstitial fluid (ISF) tracer transport, five groups re-examined the importance of AQP4 in glymphatic transport. We concur that CSF influx is higher in wild-type mice than in four different  KO lines and in one line that lacks perivascular AQP4 ( KO). Meta-analysis of all studies demonstrated a significant decrease in tracer transport in KO mice and rats compared to controls. Meta-regression indicated that anesthesia, age, and tracer delivery explain the opposing results. We also report that intrastriatal injections suppress glymphatic function. This validates the role of AQP4 and shows that glymphatic studies must avoid the use of invasive procedures.

Aquaporin-4-dependent glymphatic solute transport in the rodent brain.

Spontaneous waves of cortical spreading depolarization (CSD) are induced in the setting of acute focal ischemia. CSD is linked to a sharp increase of extracellular K that induces a long-lasting suppression of neural activity. Furthermore, CSD induces secondary irreversible damage in the ischemic brain, suggesting that K homeostasis might constitute a therapeutic strategy in ischemic stroke. Here we report that adrenergic receptor (AdR) antagonism accelerates normalization of extracellular K, resulting in faster recovery of neural activity after photothrombotic stroke. Remarkably, systemic adrenergic blockade before or after stroke facilitated functional motor recovery and reduced infarct volume, paralleling the preservation of the water channel aquaporin-4 in astrocytes. Our observations suggest that AdR blockers promote cerebrospinal fluid exchange and rapid extracellular K clearance, representing a potent potential intervention for acute stroke.

Adrenergic receptor antagonism induces neuroprotection and facilitates recovery from acute ischemic stroke.

The glymphatic system is a brain-wide cerebrospinal fluid (CSF) transport system that utilizes the perivascular space for fast inflow of CSF. Arterial pulsations are a major driver of glymphatic CSF inflow, and hypertension that causes vascular pathologies, such as arterial stiffening and perivascular alterations, may impede the inflow. We used dynamic contrast-enhanced MRI (DCE-MRI) to assess the effect of hypertension on glymphatic transport kinetics in male young and adult spontaneously hypertensive (SHR) rats compared with age-matched normotensive Wistar-Kyoto rats (WKY). We anesthetized the rats with dexmedetomidine/isoflurane and infused paramagnetic contrast (Gd-DOTA) into the cisterna magna (CM) during DCE-MRI to quantify glymphatic transport kinetics. Structural MRI analysis showed that cerebroventricular volumes are larger and brain volumes significantly smaller in SHR compared to WKY rats, irrespective of age. We observed ventricular reflux of Gd-DOTA in SHR rats only, indicating abnormal CSF flow dynamics secondary to innate hydrocephalus. One-tissue compartment analysis revealed impeded glymphatic transport of Gd-DOTA in SHR compared with WKY rats in both age groups, implying that glymphatic transport, including solute clearance from brain parenchyma, is impaired during evolving hypertension in young SHR, an effect that worsens in states of chronic hypertension. The study demonstrates the suppression of glymphatic clearance in SHR rats and thus offers new insight into the co-existence of hypertension and concomitant vascular pathologies in Alzheimer's disease. The study further highlights the importance of considering the distribution of tracers in the CSF compartment in the analysis of the glymphatic system. The glymphatic system contributes to the removal of amyloid beta from the brain and is disrupted in Alzheimer's disease and aging. Using a rat model of hypertension, we measured gross CSF flow and tracked glymphatic influx and efflux rates with dynamic contrast-enhanced MRI, showing that glymphatic transport is compromised in both early and advanced stages of hypertension. The study provides a new perspective on the importance for brain metabolite and fluid homeostasis of maintaining healthy blood vessels, an increasingly pertinent issue in an aging population that in part may explain the link between vascular pathology and Alzheimer's disease.

Impaired Glymphatic Transport in Spontaneously Hypertensive Rats.

Periarterial spaces (PASs) are annular channels that surround arteries in the brain and contain cerebrospinal fluid (CSF): a flow of CSF in these channels is thought to be an important part of the brain's system for clearing metabolic wastes. In vivo observations reveal that they are not concentric, circular annuli, however: the outer boundaries are often oblate, and the arteries that form the inner boundaries are often offset from the central axis.

Humberto Mestre

Center for Translational Neuromedicine

University of Rochester Medical Center

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy