Cerebrospinal fluid is a significant fluid source for anoxic cerebral oedema.

Cerebral oedema develops after anoxic brain injury. In two models of asphyxial and asystolic cardiac arrest without resuscitation, we found that oedema develops shortly after anoxia secondary to terminal depolarizations and the abnormal entry of CSF. Oedema severity correlated with the availability of CSF with the age-dependent increase in CSF volume worsening the severity of oedema. Oedema was identified primarily in brain regions bordering CSF compartments in mice and humans. The degree of ex vivo tissue swelling was predicted by an osmotic model suggesting that anoxic brain tissue possesses a high intrinsic osmotic potential. This osmotic process was temperature-dependent, proposing an additional mechanism for the beneficial effect of therapeutic hypothermia. These observations show that CSF is a primary source of oedema fluid in anoxic brain. This novel insight offers a mechanistic basis for the future development of alternative strategies to prevent cerebral oedema formation after cardiac arrest.

Biological sex does not predict glymphatic influx in healthy young, middle aged or old mice.

Sexual dimorphism is evident in brain structure, size, and function throughout multiple species. Here, we tested whether cerebrospinal fluid entry into the glymphatic system, a network of perivascular fluid transport that clears metabolic waste from the brain, was altered between male and female mice. We analyze glymphatic influx in 244 young reproductive age (2-4 months) C57BL/6 mice. We found no male/female differences in total influx under anesthesia, or across the anterior/posterior axis of the brain. Circadian-dependent changes in glymphatic influx under ketamine/xylazine anesthesia were not altered by sex. This was not true for diurnal rhythms under pentobarbital and avertin, but both still showed daily oscillations independent of biological sex. Finally, although glymphatic influx decreases with age there was no sex difference in total influx or subregion-dependent tracer distribution in 17 middle aged (9-10 months) and 36 old (22-24 months) mice. Overall, in healthy adult C57BL/6 mice we could not detect male/female differences in glymphatic influx. This finding contrasts the gender differences in common neurodegenerative diseases. We propose that additional sex-dependent co-morbidities, such as chronic stress, protein misfolding, traumatic brain injury or other pathological mechanisms may explain the increased risk for developing proteinopathies rather than pre-existing suppression of glymphatic influx.

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 magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-ß). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

PDGF-B Is Required for Development of the Glymphatic System.

The glymphatic system is a highly polarized cerebrospinal fluid (CSF) transport system that facilitates the clearance of neurotoxic molecules through a brain-wide network of perivascular pathways. Herein we have mapped the development of the glymphatic system in mice. Perivascular CSF transport first emerges in hippocampus in newborn mice, and a mature glymphatic system is established in the cortex at 2 weeks of age. Formation of astrocytic endfeet and polarized expression of aquaporin 4 (AQP4) consistently coincided with the appearance of perivascular CSF transport. Deficiency of platelet-derived growth factor B (PDGF-B) function in the PDGF retention motif knockout mouse line Pdgfbret/ret suppressed the development of the glymphatic system, whose functions remained suppressed in adulthood compared with wild-type mice. These experiments map the natural development of the glymphatic system in mice and define a critical role of PDGF-B in the development of perivascular CSF transport.

Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections.

Analysis of entire transparent rodent bodies after clearing could provide holistic biological information in health and disease, but reliable imaging and quantification of fluorescent protein signals deep inside the tissues has remained a challenge. Here, we developed vDISCO, a pressure-driven, nanobody-based whole-body immunolabeling technology to enhance the signal of fluorescent proteins by up to two orders of magnitude. This allowed us to image and quantify subcellular details through bones, skin and highly autofluorescent tissues of intact transparent mice. For the first time, we visualized whole-body neuronal projections in adult mice. We assessed CNS trauma effects in the whole body and found degeneration of peripheral nerve terminals in the torso. Furthermore, vDISCO revealed short vascular connections between skull marrow and brain meninges, which were filled with immune cells upon stroke. Thus, our new approach enables unbiased comprehensive studies of the interactions between the nervous system and the rest of the body.

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

The glymphatic system is a brain-wide clearance pathway; its impairment contributes to the accumulation of amyloid-ß. 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 Aqp4 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 Aqp4 KO lines and in one line that lacks perivascular AQP4 (Snta1 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.

Fluorescent Ca2+ indicators directly inhibit the Na,K-ATPase and disrupt cellular functions.

Fluorescent Ca2+ indicators have been essential for the analysis of Ca2+ signaling events in various cell types. We showed that chemical Ca2+ indicators, but not a genetically encoded Ca2+ indicator, potently suppressed the activity of Na+- and K+-dependent adenosine triphosphatase (Na,K-ATPase), independently of their Ca2+ chelating activity. Loading of commonly used Ca2+ indicators, including Fluo-4 acetoxymethyl (AM), Rhod-2 AM, and Fura-2 AM, and of the Ca2+ chelator BAPTA AM into cultured mouse or human neurons, astrocytes, cardiomyocytes, or kidney proximal tubule epithelial cells suppressed Na,K-ATPase activity by 30 to 80%. Ca2+ indicators also suppressed the agonist-induced activation of the Na,K-ATPase, altered metabolic status, and caused a dose-dependent loss of cell viability. Loading of Ca2+ indicators into mice, which is carried out for two-photon imaging, markedly altered brain extracellular concentrations of K+ and ATP. These results suggest that a critical review of data obtained with chemical Ca2+ indicators may be necessary.

A Distinct Population of Microglia Supports Adult Neurogenesis in the Subventricular Zone.

Microglia are involved in synaptic pruning both in development and in the mature CNS. In this study, we investigated whether microglia might further contribute to circuit plasticity by modulating neuronal recruitment from the neurogenic subventricular zone (SVZ) of the adult mouse striatum. We found that microglia residing in the SVZ and adjacent rostral migratory stream (RMS) comprise a morphologically and antigenically distinct phenotype of immune effectors. Whereas exhibiting characteristics of alternatively activated microglia, the SVZ/RMS microglia were clearly distinguished by their low expression of purinoceptors and lack of ATP-elicitable chemotaxis. Furthermore, the in vivo depletion of these microglia hampered the survival and migration of newly generated neuroblasts through the RMS to the olfactory bulb. SVZ and RMS microglia thus appear to comprise a functionally distinct class that is selectively adapted to the support and direction of neuronal integration into the olfactory circuitry. Therefore, this unique microglial subpopulation may serve as a novel target with which to modulate cellular addition from endogenous neural stem and progenitor cells of the adult brain. SIGNIFICANCE STATEMENT: Microglial cells are a specialized population of macrophages in the CNS, playing key roles as immune mediators. As integral components in the CNS, the microglia stand out for using the same mechanisms, phagocytosis and cytochemokine release, to promote homeostasis, synaptic pruning, and neural circuitry sculpture. Here, we addressed microglial functions in the subventricular zone (SVZ), the major postnatal neurogenic niche. Our results depict microglia as a conspicuous component of SVZ and its anterior extension, the rostral migratory stream, a pathway used by neuroblasts during their transit toward olfactory bulb layers. In addition to other unique populations residing in the SVZ niche, microglia display distinct morphofunctional properties that boost neuronal progenitor survival and migration in the mammalian brain.

Screening for neuropathic characteristics in failed back surgery syndromes: challenges for guiding treatment.

OBJECTIVE: Neuropathic pain screening tools have shown promise in identifying common neuropathic pain characteristics that derive from diverse etiologies (e.g., diabetic peripheral neuropathy, postherpetic neuralgia). However, no prior studies have specifically assessed whether these tools are capable of discerning the underlying pain mechanisms in the vast, heterogeneous group of patients diagnosed with failed back surgery syndrome (FBSS). DESIGN: In this clinical observational study, two tests for neuropathic pain characteristics, the Douleur Neuropathique en 4 (DN4) and Leeds Assessment of Neuropathic Symptoms and Signs (LANSS) questionnaires, were performed on 43 subjects with FBSS. Subjects underwent physical or neurosensory exam components of the DN4 and LANSS in the region of most severe pain (e.g., axial low back or lower extremities). DN4 and LANSS scores were correlated with clinical history and neurologic exam, pain-related quality of life questionnaires, and compared to an independent assessment of pain distribution. RESULTS: The presence of neuropathic characteristics, determined by the DN4 (62% sensitivity, 44% specificity), LANSS (38% sensitivity, 75% specificity; cut-offs of 4 and 12, respectively), or their combination (20% sensitivity, 58% specificity) was associated with higher pain intensity as measured by the visual analog scale (DN4 > 4, P = 0.001; LANSS ≥ 12, P = 0.042), modified Brief Pain Inventory-Short Form (DN4 > 4, P = 0.001; LANSS ≥ 12, P = 0.082), and Neuropathic Pain Symptom Inventory (DN4 > 4, P = 0.001; LANSS ≥ 12, P = 0.001), and greater pain-related functional impairment as measured by the Roland-Morris Disability Questionnaire (DN4 > 4, P = 0.006; LANSS ≥ 12, P = 0.018). The percentage of subjects characterized as neuropathic by the DN4 and LANSS lacked concordance (67.4 vs. 25.6), and the distribution of most severe symptoms (i.e., axial vs radicular) did not correlate with subjects determined to have neuropathic pain. CONCLUSIONS: Unlike other neuropathic syndromes, the neuropathic component of FBSS is less reliably identified by the LANSS and DN4.

Neuronal transgene expression in dominant-negative SNARE mice.

Experimental advances in the study of neuroglia signaling have been greatly accelerated by the generation of transgenic mouse models. In particular, an elegant manipulation that interferes with astrocyte vesicular release of gliotransmitters via overexpression of a dominant-negative domain of vesicular SNARE (dnSNARE) has led to documented astrocytic involvement in processes that were traditionally considered strictly neuronal, including the sleep-wake cycle, LTP, cognition, cortical slow waves, depression, and pain. A key premise leading to these conclusions was that expression of the dnSNARE was specific to astrocytes. Inconsistent with this premise, we report here widespread expression of the dnSNARE transgene in cortical neurons. We further demonstrate that the activity of cortical neurons is reversibly suppressed in dnSNARE mice. These findings highlight the need for independent validation of astrocytic functions identified in dnSNARE mice and thus question critical evidence that astrocytes contribute to neurotransmission through SNARE-dependent vesicular release of gliotransmitters.

Impairment of paravascular clearance pathways in the aging brain.

OBJECTIVE: In the brain, protein waste removal is partly performed by paravascular pathways that facilitate convective exchange of water and soluble contents between cerebrospinal fluid (CSF) and interstitial fluid (ISF). Several lines of evidence suggest that bulk flow drainage via the glymphatic system is driven by cerebrovascular pulsation, and is dependent on astroglial water channels that line paravascular CSF pathways. The objective of this study was to evaluate whether the efficiency of CSF-ISF exchange and interstitial solute clearance is impaired in the aging brain. METHODS: CSF-ISF exchange was evaluated by in vivo and ex vivo fluorescence microscopy and interstitial solute clearance was evaluated by radiotracer clearance assays in young (2-3 months), middle-aged (10-12 months), and old (18-20 months) wild-type mice. The relationship between age-related changes in the expression of the astrocytic water channel aquaporin-4 (AQP4) and changes in glymphatic pathway function was evaluated by immunofluorescence. RESULTS: Advancing age was associated with a dramatic decline in the efficiency of exchange between the subarachnoid CSF and the brain parenchyma. Relative to the young, clearance of intraparenchymally injected amyloid-ß was impaired by 40% in the old mice. A 27% reduction in the vessel wall pulsatility of intracortical arterioles and widespread loss of perivascular AQP4 polarization along the penetrating arteries accompanied the decline in CSF-ISF exchange. INTERPRETATION: We propose that impaired glymphatic clearance contributes to cognitive decline among the elderly and may represent a novel therapeutic target for the treatment of neurodegenerative diseases associated with accumulation of misfolded protein aggregates.

Evaluating glymphatic pathway function utilizing clinically relevant intrathecal infusion of CSF tracer.

BACKGROUND: Neurodegenerative diseases such as Alzheimer's are associated with the aggregation of endogenous peptides and proteins that contribute to neuronal dysfunction and loss. The glymphatic system, a brain-wide perivascular pathway along which cerebrospinal fluid (CSF) and interstitial fluid (ISF) rapidly exchange, has recently been identified as a key contributor to the clearance of interstitial solutes from the brain, including amyloid ß. These findings suggest that measuring changes in glymphatic pathway function may be an important prognostic for evaluating neurodegenerative disease susceptibility or progression. However, no clinically acceptable approach to evaluate glymphatic pathway function in humans has yet been developed. METHODS: Time-sequenced ex vivo fluorescence imaging of coronal rat and mouse brain slices was performed at 30-180 min following intrathecal infusion of CSF tracer (Texas Red- dextran-3, MW 3 kD; FITC- dextran-500, MW 500 kD) into the cisterna magna or lumbar spine. Tracer influx into different brain regions (cortex, white matter, subcortical structures, and hippocampus) in rat was quantified to map the movement of CSF tracer following infusion along both routes, and to determine whether glymphatic pathway function could be evaluated after lumbar intrathecal infusion. RESULTS: Following lumbar intrathecal infusions, small molecular weight TR-d3 entered the brain along perivascular pathways and exchanged broadly with the brain ISF, consistent with the initial characterization of the glymphatic pathway in mice. Large molecular weight FITC-d500 remained confined to the perivascular spaces. Lumbar intrathecal infusions exhibited a reduced and delayed peak parenchymal fluorescence intensity compared to intracisternal infusions. CONCLUSION: Lumbar intrathecal contrast delivery is a clinically useful approach that could be used in conjunction with dynamic contrast enhanced MRI nuclear imaging to assess glymphatic pathway function in humans.

Astrocytic CX43 hemichannels and gap junctions play a crucial role in development of chronic neuropathic pain following spinal cord injury.

Chronic neuropathic pain is a frequent consequence of spinal cord injury (SCI). Yet despite recent advances, upstream releasing mechanisms and effective therapeutic options remain elusive. Previous studies have demonstrated that SCI results in excessive ATP release to the peritraumatic regions and that purinergic signaling, among glial cells, likely plays an essential role in facilitating inflammatory responses and nociceptive sensitization. We sought to assess the role of connexin 43 (Cx43) as a mediator of CNS inflammation and chronic pain. To determine the extent of Cx43 involvement in chronic pain, a weight-drop SCI was performed on transgenic mice with Cx43/Cx30 deletions. SCI induced robust and persistent neuropathic pain including heat hyperalgesia and mechanical allodynia in wild-type control mice, which developed after 4 weeks and was maintained after 8 weeks. Notably, SCI-induced heat hyperalgesia and mechanical allodynia were prevented in transgenic mice with Cx43/Cx30 deletions, but fully developed in transgenic mice with only Cx30 deletion. SCI-induced gliosis, detected as upregulation of glial fibrillary acidic protein in the spinal cord astrocytes at different stages of the injury, was also reduced in the knockout mice with Cx43/Cx30 deletions, when compared with littermate controls. In comparison, a standard regimen of post-SCI treatment of minocycline attenuated neuropathic pain to a significantly lesser degree than Cx43 deletion. These findings suggest Cx43 is critically linked to the development of central neuropathic pain following acute SCI. Since Cx43/Cx30 is expressed by astrocytes, these findings also support an important role of astrocytes in the development of chronic pain.