The choroid plexus links innate immunity to CSF dysregulation in hydrocephalus.

The choroid plexus (ChP) is the blood-cerebrospinal fluid (CSF) barrier and the primary source of CSF. Acquired hydrocephalus, caused by brain infection or hemorrhage, lacks drug treatments due to obscure pathobiology. Our integrated, multi-omic investigation of post-infectious hydrocephalus (PIH) and post-hemorrhagic hydrocephalus (PHH) models revealed that lipopolysaccharide and blood breakdown products trigger highly similar TLR4-dependent immune responses at the ChP-CSF interface. The resulting CSF "cytokine storm", elicited from peripherally derived and border-associated ChP macrophages, causes increased CSF production from ChP epithelial cells via phospho-activation of the TNF-receptor-associated kinase SPAK, which serves as a regulatory scaffold of a multi-ion transporter protein complex. Genetic or pharmacological immunomodulation prevents PIH and PHH by antagonizing SPAK-dependent CSF hypersecretion. These results reveal the ChP as a dynamic, cellularly heterogeneous tissue with highly regulated immune-secretory capacity, expand our understanding of ChP immune-epithelial cell cross talk, and reframe PIH and PHH as related neuroimmune disorders vulnerable to small molecule pharmacotherapy.

Choroid plexus tissue perfusion and blood to CSF barrier function in rats measured with continuous arterial spin labeling.

The choroid plexus (ChP) of the cerebral ventricles is a source of cerebrospinal fluid (CSF) production and also plays a key role in immune surveillance at the level of blood-to-CSF-barrier (BCSFB). In this study, we quantify ChP blood perfusion and BCSFB mediated water exchange from arterial blood into ventricular CSF using non-invasive continuous arterial spin labelling magnetic resonance imaging (CASL-MRI). Systemic administration of anti-diuretic hormone (vasopressin) was used to validate BCSFB water flow as a metric of choroidal CSF secretory function. To further investigate the coupling between ChP blood perfusion and BCSFB water flow, we characterized the effects of two anesthetic regimens known to have large-scale differential effects on cerebral blood flow. For quantification of ChP blood perfusion a multi-compartment perfusion model was employed, and we discovered that partial volume correction improved measurement accuracy. Vasopressin significantly reduced both ChP blood perfusion and BCSFB water flow. ChP blood perfusion was significantly higher with pure isoflurane anesthesia (2-2.5%) when compared to a balanced anesthesia with dexmedetomidine and low-dose isoflurane (1.0 %), and significant correlation between ChP blood perfusion and BCSFB water flow was observed, however there was no significant difference in BCSFB water flow. In summary, here we introduce a non-invasive, robust, and spatially resolved in vivo imaging platform to quantify ChP blood perfusion as well as BCSFB water flow which can be applied to study coupling of these two key parameters in future clinical translational studies.

Studies on metabolic alterations due to hypobaric hypoxia in serum using NMR spectroscopy.

INTRODUCTION: The main physiological challenge in high altitude environment is hypoxia which affects the aerobic metabolism reducing the energy supply. These changes may further progress towards extreme environment related diseases. Rarely has the high-altitude biology been studied using system sciences and omics high-throughput technologies. OBJECTIVE: In the present study, 1H-NMR-based metabolomics, along with multivariate analysis, were employed in a preclinical rat model to characterise the serum metabolic changes under chronic hypobaric hypoxia (HH) stress. MATERIAL AND METHODS: Rats were exposed to simulated hypobaric hypoxia equivalent of 6700 m above the sea level. The serum samples were collected from control and HH-exposure (7, 14, and 21 days) of hypobaric hypoxia. RESULTS AND DISCUSSION: The 1H-NMR metabolomics of the serum showed alterations in the metabolism of membranes, amino-acids altered cellular bioenergetics and osmoregulation. Multivariate statistical analysis revealed alterations in acetoacetate, choline, glutamine, acetate, betaine, ketone bodies and branched amino acid metabolites. CONCLUSION: Present findings establishes the fingerprint biomarkers for chronic environmental hypoxia which will help in understanding extreme environment related health problems, early detection and developing strategies to clinically address high altitude hypoxia.

The Glymphatic System and Waste Clearance with Brain Aging: A Review.

The glymphatic system is a glial-dependent waste clearance pathway in the brain, in place of lymphatic vessels, dedicated to drain away soluble waste proteins and metabolic products. Specifically, the glymphatic network serves as a "front end" for waste clearance, and is connected downstream to an authentic lymphatic network, associated with dura covering the brain as well as cranial nerves and large vessels at the skull exits. The anatomical and functional interconnections between these two networks are not completely understood. Several key physiological processes have been identified that control glymphatic transport function and waste clearance from brain. In this review, we aim to provide an overview and discussion of the concept behind the glymphatic system, current evidence, and controversies, while specifically focusing on the consequences of aging and evidence of its existence in human brain. Discovering novel strategies for optimizing and maintaining efficient brain waste clearance across the lifespan may in the future prove to be important for preventing cognitive decline and sustaining healthy aging.

Altered metabolites of the rat hippocampus after mild and moderate traumatic brain injury - a combined in vivo and in vitro 1 H-MRS study.

Traumatic brain injury (TBI) has been shown to affect hippocampus-associated learning, memory and higher cognitive functions, which may be a consequence of metabolic alterations. Hippocampus-associated disorders may vary depending on the severity of injury [mild TBI (miTBI) and moderate TBI (moTBI)] and time since injury. The underlying hippocampal metabolic irregularities may provide an insight into the pathological process following TBI. In this study, in vivo and in vitro proton magnetic resonance spectroscopy (1 H-MRS) data were acquired from the hippocampus region of controls and TBI groups (miTBI and moTBI) at D0 (pre-injury), 4 h, Day 1 and Day 5 post-injury (PI). In vitro MRS results indicated trauma-induced changes in both miTBI and moTBI; however, in vivo MRS showed metabolic alterations in moTBI only. miTBI and moTBI showed elevated levels of osmolytes indicating injury-induced edema. Altered levels of citric acid cycle intermediates, glutamine/glutamate and amino acid metabolism indicated injury-induced aberrant bioenergetics, excitotoxicity and oxidative stress. An overall similar pattern of pathological process was observed in both miTBI and moTBI, with the distinction of depleted N-acetylaspartate levels (indicating neuronal loss) at 4 h and Day 1 and enhanced lactate production (indicating heightened energy depletion leading to the commencement of the anaerobic pathway) at Day 5 in moTBI. To the best of our knowledge, this is the first study to investigate the hippocampus metabolic profile in miTBI and moTBI simultaneously using in vivo and in vitro MRS.

Neurometabolic and structural alterations in rat brain due to acute hypobaric hypoxia: in vivo 1H MRS at 7 T.

In response to hypobaric hypoxia (HH), which occurs at high altitude, the brain undergoes deleterious changes at the structural and metabolite level. In vivo T2 weighted imaging (T2WI) and (1)H-MRS was performed to understand the structural and metabolic changes in the hippocampus region of rat brain. Data were acquired pre-exposure (baseline controls), immediately after exposure and subsequently at the first, fourth, seventh and 14th days post exposure at normoxia. T2 weighted images of rat brain showed hyperintensity in the CA2/CA3 region of the hippocampus 7 d after acute HH, which persisted till 14 d, probably indicating structural changes in the hippocampus. (1)H-MRS results showed no change in metabolite level immediately after acute HH exposure, but on the first day of normoxia the myo-inositol level was significantly decreased, possibly due to altered astrocyte metabolism. Metabolic alterations showing an increase in choline and decrease in glutamate on the fourth day of normoxia may be seen as a process of demyelination and loss of glutamate pool respectively. On the seventh and 14th days of normoxia, decreases in N-acetylaspartate, creatine and glutamine + glutamate were observed, which might be due to decreased viability of glutamatergic neurons. In vivo (1)H-MRS demonstrated early neurometabolic changes prior to probable structural changes post acute HH exposure. The extension of these studies will help in early risk assessment, developing intervention and strategies for combating HH related changes.

Continuous positive airway pressure increases CSF flow and glymphatic transport.

Respiration can positively influence cerebrospinal fluid (CSF) flow in the brain, yet its effects on central nervous system (CNS) fluid homeostasis, including waste clearance function via glymphatic and meningeal lymphatic systems, remain unclear. Here, we investigated the effect of supporting respiratory function via continuous positive airway pressure (CPAP) on glymphatic-lymphatic function in spontaneously breathing anesthetized rodents. To do this, we used a systems approach combining engineering, MRI, computational fluid dynamics analysis, and physiological testing. We first designed a nasal CPAP device for use in the rat and demonstrated that it functioned similarly to clinical devices, as evidenced by its ability to open the upper airway, augment end-expiratory lung volume, and improve arterial oxygenation. We further showed that CPAP increased CSF flow speed at the skull base and augmented glymphatic transport regionally. The CPAP-induced augmented CSF flow speed was associated with an increase in intracranial pressure (ICP), including the ICP waveform pulse amplitude. We suggest that the augmented pulse amplitude with CPAP underlies the increase in CSF bulk flow and glymphatic transport. Our results provide insights into the functional crosstalk at the pulmonary-CSF interface and suggest that CPAP might have therapeutic benefit for sustaining glymphatic-lymphatic function.

Characterization of perivascular space pathology in a rat model of cerebral small vessel disease by in vivo magnetic resonance imaging.

One of the most common causes of dementia is cerebral small vessel disease (SVD), which is associated with enlarged perivascular spaces (PVS). Clinically, PVS are visible as hyperintensities on T2-weighted (T2w) magnetic resonance images (MRI). While rodent SVD models exhibit arteriolosclerosis, PVS have not been robustly documented by MRI casting doubts on their clinical relevance. Here we established that the severity of SVD in spontaneously hypertensive stroke prone (SHRSP) rats correlated to 'moderate' SVD in human post-mortem tissue. We then developed two approaches for detecting PVS in SHRSP rats: 1) T2w imaging and 2) T1-weighted imaging with administration of gadoteric acid into cerebrospinal fluid. We applied the two protocols to six Wistar-Kyoto (WKY) control rats and thirteen SHRSP rats at ∼12 month of age. The primary endpoint was the number of hyperintense lesions. We found more hyperintensities on T2w MRI in the SHRSP compared to WKY rats (p-value = 0.023). CSF enhancement with gadoteric acid increased the visibility of PVS-like lesions in SHRSP rats. In some of the SHRSP rats, the MRI hyperintensities corresponded to enlarged PVS on histopathology. The finding of PVS-like hyperintensities on T2w MRI support the SHRSP rat's clinical relevance for studying the underlying pathophysiology of SVD.

Sustained glymphatic transport and impaired drainage to the nasal cavity observed in multiciliated cell ciliopathies with hydrocephalus.

BACKGROUND: Hydrocephalus (increased ventricular size due to CSF accumulation) is a common finding in human ciliopathies and in mouse models with genetic depletion of the multiciliated cell (MCC) cilia machinery. However, the contribution of MCC to CSF dynamics and, the mechanism by which impaired MCC function leads to hydrocephalus remains poorly understood. The aim of our study was to examine if defects in MCC ciliogenesis and cilia-generated CSF flow impact central nervous system (CNS) fluid homeostasis including glymphatic transport and solute waste drainage. METHODS: We used two distinct mouse models of MCC ciliopathy: MCC-specific CEP164 conditional knockout mice (FOXJ1-Cre;CEP164fl/fl (N = 10), 3-month-old) and p73 knock-out (p73-/- (N = 8), 5-month-old) mice. Age-matched, wild-type littermates for each of the mutants served as controls. Glymphatic transport and solute drainage was quantified using in vivo T1 mapping by magnetic resonance imaging (MRI) after CSF infusion of gadoteric acid. Brain morphometry and aquaporin 4 expression (AQP4) was also assessed. Intracranial pressure (ICP) was measured in separate cohorts. RESULTS: In both of the two models of MCC ciliopathy we found the ventriculomegaly to be associated with normal ICP. We showed that FOXJ1-Cre;CEP164fl/fl mice with hydrocephalus still demonstrated sustained glymphatic transport and normal AQP4 expression along capillaries. In p73-/- mice glymphatic transport was even increased, and this was paralleled by an increase in AQP4 polarization around capillaries. Further, solute drainage via the cribriform plate to the nasal cavity was severely impaired in both ciliopathy models and associated with chronic rhinitis and olfactory bulb hypoplasia. CONCLUSIONS: The combination of sustained glymphatic transport, impaired solute drainage via the cribriform plate to the nasal cavity and hydrocephalus has not previously been reported in models of MCC ciliopathy. Our data enhance our understanding of how different types of ciliopathies contribute to disruption of CNS fluid homeostasis, manifested in pathologies such as hydrocephalus.

Impaired neurogenesis alters brain biomechanics in a neuroprogenitor-based genetic subtype of congenital hydrocephalus.

Hydrocephalus, characterized by cerebral ventricular dilatation, is routinely attributed to primary defects in cerebrospinal fluid (CSF) homeostasis. This fosters CSF shunting as the leading reason for brain surgery in children despite considerable disease heterogeneity. In this study, by integrating human brain transcriptomics with whole-exome sequencing of 483 patients with congenital hydrocephalus (CH), we found convergence of CH risk genes in embryonic neuroepithelial stem cells. Of all CH risk genes, TRIM71/lin-41 harbors the most de novo mutations and is most specifically expressed in neuroepithelial cells. Mice harboring neuroepithelial cell-specific Trim71 deletion or CH-specific Trim71 mutation exhibit prenatal hydrocephalus. CH mutations disrupt TRIM71 binding to its RNA targets, causing premature neuroepithelial cell differentiation and reduced neurogenesis. Cortical hypoplasia leads to a hypercompliant cortex and secondary ventricular enlargement without primary defects in CSF circulation. These data highlight the importance of precisely regulated neuroepithelial cell fate for normal brain-CSF biomechanics and support a clinically relevant neuroprogenitor-based paradigm of CH.

Disparate volumetric fluid shifts across cerebral tissue compartments with two different anesthetics.

BACKGROUND: Large differences in glymphatic system transport-similar in magnitude to those of the sleep/wake cycle-have been observed during anesthesia with dexmedetomidine supplemented with low dose isoflurane (DEXM-I) in comparison to isoflurane (ISO). However, the biophysical and bioenergetic tissue status underlying glymphatic transport differences between anesthetics remains undefined. To further understand biophysical characteristics underlying these differences we investigated volume status across cerebral tissue compartments, water diffusivity, and T2* values in rats anesthetized with DEXM-I in comparison to ISO. METHODS: Using a crossover study design, a group of 12 Sprague Dawley female rats underwent repetitive magnetic resonance imaging (MRI) under ISO and DEXM-I. Physiological parameters were continuously measured. MRI included a proton density weighted (PDW) scan to investigate cerebrospinal fluid (CSF) and parenchymal volumetric changes, a multigradient echo scan (MGE) to calculate T2* maps as a measure of 'bioenergetics', and a diffusion scan to quantify the apparent diffusion coefficient (ADC). RESULTS: The heart rate was lower with DEXM-I in comparison to ISO, but all other physiological variables were similar across scans and groups. The PDW images revealed a 1% parenchymal volume increase with ISO compared to DEXM-I comprising multiple focal tissue areas scattered across the forebrain. In contrast, with DEXM-I the CSF compartment was enlarged by ~ 6% in comparison to ISO at the level of the basal cisterns and peri-arterial conduits which are main CSF influx routes for glymphatic transport. The T2* maps showed brain-wide increases in T2* in ISO compared to DEXM-I rats. Diffusion-weighted images yielded no significant differences in ADCs across the two anesthesia groups. CONCLUSIONS: We demonstrated CSF volume expansion with DEXM-I (in comparison to ISO) and parenchymal (GM) expansion with ISO (in comparison to DEXM-I), which may explain the differences in glymphatic transport. The T2* changes in ISO are suggestive of an increased bioenergetic state associated with excess cellular firing/bursting when compared to DEXM-I.

Glymphatic Cerebrospinal Fluid and Solute Transport Quantified by MRI and PET Imaging.

Over the past decade there has been an enormous progress in our understanding of fluid and solute transport in the central nervous system (CNS). This is due to a number of factors, including important developments in whole brain imaging technology and computational fluid dynamics analysis employed for the elucidation of glymphatic transport function in the live animal and human brain. In this paper, we review the technical aspects of dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) in combination with administration of Gd-based tracers into the cerebrospinal fluid (CSF) for tracking glymphatic solute and fluid transport in the CNS as well as lymphatic drainage. Used in conjunction with advanced computational processing methods including optimal mass transport analysis, one gains new insights into the biophysical forces governing solute transport in the CNS which leads to intriguing new research directions. Considering drainage pathways, we review the novel T1 mapping technique for quantifying glymphatic transport and cervical lymph node drainage concurrently in the same subject. We provide an overview of knowledge gleaned from DCE-MRI studies of glymphatic transport and meningeal lymphatic drainage. Finally, we introduce positron emission tomography (PET) and CSF administration of radiotracers as an alternative method to explore other pharmacokinetic aspects of CSF transport into brain parenchyma as well as efflux pathways.

Diffuse white matter loss in a transgenic rat model of cerebral amyloid angiopathy.

Diffuse white matter (WM) disease is highly prevalent in elderly with cerebral small vessel disease (cSVD). In humans, cSVD such as cerebral amyloid angiopathy (CAA) often coexists with Alzheimer's disease imposing a significant impediment for characterizing their distinct effects on WM. Here we studied the burden of age-related CAA pathology on WM disease in a novel transgenic rat model of CAA type 1 (rTg-DI). A cohort of rTg-DI and wild-type rats was scanned longitudinally using MRI for characterization of morphometry, cerebral microbleeds (CMB) and WM integrity. In rTg-DI rats, a distinct pattern of WM loss was observed at 9 M and 11 M. MRI also revealed manifestation of small CMB in thalamus at 6 M, which preceded WM loss and progressively enlarged until the moribund disease stage. Histology revealed myelin loss in the corpus callosum and thalamic CMB in all rTg-DI rats, the latter of which manifested in close proximity to occluded and calcified microvessels. The quantitation of CAA load in rTg-DI rats revealed that the most extensive microvascular Aß deposition occurred in the thalamus. For the first time using in vivo MRI, we show that CAA type 1 pathology alone is associated with a distinct pattern of WM loss.

In vivo T1 mapping for quantifying glymphatic system transport and cervical lymph node drainage.

Dynamic contrast-enhanced magnetic resonance imaging (MRI) for tracking glymphatic system transport with paramagnetic contrast such as gadoteric acid (Gd-DOTA) administration into cerebrospinal fluid (CSF) requires pre-contrast data for proper quantification. Here we introduce an alternative approach for glymphatic system quantification in the mouse brain via T1 mapping which also captures drainage of Gd-DOTA to the cervical lymph nodes. The Gd-DOTA injection into CSF was performed on the bench after which the mice underwent T1 mapping using a 3D spoiled gradient echo sequence on a 9.4 T MRI. In Ketamine/Xylazine (KX) anesthetized mice, glymphatic transport and drainage of Gd-DOTA to submandibular and deep cervical lymph nodes was demonstrated as 25-50% T1 reductions in comparison to control mice receiving CSF saline. To further validate the T1 mapping approach we also verified increased glymphatic transport of Gd-DOTA transport in mice anesthetized with KX in comparison with ISO. The novel T1 mapping method allows for quantification of glymphatic transport as well as drainage to the deep and superficial cervical lymph nodes. The ability to measure glymphatic transport and cervical lymph node drainage in the same animal longitudinally is advantageous and time efficient and the coupling between the two systems can be studied and translated to human studies.

The glymphatic system and its role in cerebral homeostasis.

The brain's high bioenergetic state is paralleled by high metabolic waste production. Authentic lymphatic vasculature is lacking in brain parenchyma. Cerebrospinal fluid (CSF) flow has long been thought to facilitate central nervous system detoxification in place of lymphatics, but the exact processes involved in toxic waste clearance from the brain remain incompletely understood. Over the past 8 yr, novel data in animals and humans have begun to shed new light on these processes in the form of the "glymphatic system," a brain-wide perivascular transit passageway dedicated to CSF transport and interstitial fluid exchange that facilitates metabolic waste drainage from the brain. Here we will discuss glymphatic system anatomy and methods to visualize and quantify glymphatic system (GS) transport in the brain and also discuss physiological drivers of its function in normal brain and in neurodegeneration.

Publisher Correction: Optimal Mass Transport with Lagrangian Workflow Reveals Advective and Diffusion Driven Solute Transport in the Glymphatic System.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Optimal Mass Transport with Lagrangian Workflow Reveals Advective and Diffusion Driven Solute Transport in the Glymphatic System.

The glymphatic system (GS) hypothesis states that advective driven cerebrospinal fluid (CSF) influx from the perivascular spaces into the interstitial fluid space rapidly transport solutes and clear waste from brain. However, the presence of advection in neuropil is contested and solutes are claimed to be transported by diffusion only. To address this controversy, we implemented a regularized version of the optimal mass transport (rOMT) problem, wherein the advection/diffusion equation is the only a priori assumption required. rOMT analysis with a Lagrangian perspective of GS transport revealed that solute speed was faster in CSF compared to grey and white matter. Further, rOMT analysis also demonstrated 2-fold differences in regional solute speed within the brain. Collectively, these results imply that advective transport dominates in CSF while diffusion and advection both contribute to GS transport in parenchyma. In a rat model of cerebral small vessel disease (cSVD), solute transport in the perivascular spaces (PVS) and PVS-to-tissue transfer was slower compared to normal rats. Thus, the analytical framework of rOMT provides novel insights in the local dynamics of GS transport that may have implications for neurodegenerative diseases. Future studies should apply the rOMT analysis approach to confirm GS transport reductions in humans with cSVD.

Brain Morphometry and Longitudinal Relaxation Time of Spontaneously Hypertensive Rats (SHRs) in Early and Intermediate Stages of Hypertension Investigated by 3D VFA-SPGR MRI.

Cerebral small vessel disease(s) (SVD) results from pathological changes of the small blood vessels in the brain and is common in older people. The diagnostic features by which SVD manifests in brain includes white matter hyperintensities, lacunes, dilated perivascular spaces, microbleeds, and atrophy. In the present study, we use in vivo magnetic resonance imaging (MRI) to characterize brain morphometry and longitudinal relaxation time (T1) of spontaneously hypertensive rats (SHRs) to study the contribution of chronic hypertension to SVD relevant pathology. Male SHR and Wistar-Kyoto (WKY) rats underwent 3D variable flip angle spoiled gradient echo brain MRI at 9.4 T at early (seven weeks old) and established (19 weeks old) stages of hypertension. The derived proton density weighted and T1 images were utilized for morphometry and to characterize T1 properties in gray matter (GM), white matter (WM) and cerebrospinal fluid (CSF). Custom tissue probability maps were constructed for accurate computerized whole brain tissue segmentations and voxel-wise analyses. Characteristic morphological differences between the two strains included enlarged ventricles, smaller corpus callosum (CC) volumes and general 'thinning' of CC in SHR compared to WKY rats at both age groups. While we did not observe parenchymal T1 differences, the T1 of CSF was elevated in SHR compared to controls. Collectively these findings indicate that SHRs develop WM atrophy which is a clinically robust MRI biomarker associated with WM degeneration.