Potentiating glymphatic drainage minimizes post-traumatic cerebral oedema.

Cerebral oedema is associated with morbidity and mortality after traumatic brain injury (TBI)1. Noradrenaline levels are increased after TBI2-4, and the amplitude of the increase in noradrenaline predicts both the extent of injury5 and the likelihood of mortality6. Glymphatic impairment is both a feature of and a contributor to brain injury7,8, but its relationship with the injury-associated surge in noradrenaline is unclear. Here we report that acute post-traumatic oedema results from a suppression of glymphatic and lymphatic fluid flow that occurs in response to excessive systemic release of noradrenaline. This post-TBI adrenergic storm was associated with reduced contractility of cervical lymphatic vessels, consistent with diminished return of glymphatic and lymphatic fluid to the systemic circulation. Accordingly, pan-adrenergic receptor inhibition normalized central venous pressure and partly restored glymphatic and cervical lymphatic flow in a mouse model of TBI, and these actions led to substantially reduced brain oedema and improved functional outcomes. Furthermore, post-traumatic inhibition of adrenergic signalling boosted lymphatic export of cellular debris from the traumatic lesion, substantially reducing secondary inflammation and accumulation of phosphorylated tau. These observations suggest that targeting the noradrenergic control of central glymphatic flow may offer a therapeutic approach for treating acute TBI.

Age- and glaucoma-induced changes to the ocular glymphatic system.

The ocular glymphatic system supports bidirectional fluid transport along the optic nerve, thereby removes metabolic wastes including amyloid-ß. To better understand this biological process, we examined the distributions of intravitreally and intracisternally infused tracers in full-length optic nerves from different age groups of mice. Aging was linked to globally impaired ocular glymphatic fluid transport, similar to what has seen previously in the brain. Aging also reduced the pupillary responsiveness to light stimulation and abolished light-induced facilitation in anterograde ocular glymphatic flow. In contrast to normal aging, in the DBA/2 J model of glaucoma, we found a pathological increase of glymphatic fluid transport to the anterior optic nerve that was associated with dilation of the perivascular spaces. Thus, aging and glaucoma have fundamentally different effects on ocular glymphatic fluid transport. Manipulation of glymphatic fluid transport might therefore present a new target for the treatment of glaucoma.

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.

Biomarkers of traumatic injury are transported from brain to blood via the glymphatic system.

The nonspecific and variable presentation of traumatic brain injury (TBI) has motivated an intense search for blood-based biomarkers that can objectively predict the severity of injury. However, it is not known how cytosolic proteins released from traumatized brain tissue reach the peripheral blood. Here we show in a murine TBI model that CSF movement through the recently characterized glymphatic pathway transports biomarkers to blood via the cervical lymphatics. Clinically relevant manipulation of glymphatic activity, including sleep deprivation and cisternotomy, suppressed or eliminated TBI-induced increases in serum S100ß, GFAP, and neuron specific enolase. We conclude that routine TBI patient management may limit the clinical utility of blood-based biomarkers because their brain-to-blood transport depends on glymphatic activity.

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

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 Aß in Alzheimer's disease (AD). In wild type mice, we show that Aß40 (fluorescently labeled Aß40 or unlabeled Aß40), was distributed from CSF to brain, via the peri-arterial space, and associated with neurons. In contrast, Aß42 was mostly restricted to the peri-arterial space due mainly to its greater propensity to oligomerize when compared to Aß40. Interestingly, pretreatment with Aß40 in the CSF, but not Aß42, reduced CSF transport into brain. In APP/PS1 mice, a model of AD, with and without extensive amyloid-ß deposits, glymphatic transport was reduced, due to the accumulation of toxic Aß species, such as soluble oligomers. CSF-derived Aß40 co-localizes with existing endogenous vascular and parenchymal amyloid-ß plaques, and thus, may contribute to the progression of both cerebral amyloid angiopathy and parenchymal Aß accumulation. Importantly, glymphatic failure preceded significant amyloid-ß 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.

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.

Photolysis of caged Ca2+ but not receptor-mediated Ca2+ signaling triggers astrocytic glutamate release.

Astrocytes in hippocampal slices can dynamically regulate synaptic transmission in a process mediated by increases in intracellular Ca(2+). However, it is debated whether astrocytic Ca(2+) signals result in release of glutamate. We here compared astrocytic Ca(2+) signaling triggered by agonist exposure versus photolysis side by side. Using transgenic mice in which astrocytes selectively express the MrgA1 receptor, we found that receptor-mediated astrocytic Ca(2+) signaling consistently triggered neuronal hyperpolarization and decreased the frequency of miniature excitatory postsynaptic currents (EPSCs). In contrast, photolysis of caged Ca(2+) (o-nitrophenyl-EGTA) in astrocytes led to neuronal depolarization and increased the frequency of mEPSCs through a metabotropic glutamate receptor-mediated pathway. Analysis of transgenic mice in which astrocytic vesicular release is suppressed (dominant-negative SNARE mice) and pharmacological manipulations suggested that glutamate is primarily released by opening of anion channels rather than exocytosis. Combined, these studies show that photolysis but not by agonists induced astrocytic Ca(2+) signaling triggers glutamate release.

Critical role of aquaporin-4 (AQP4) in astrocytic Ca2+ signaling events elicited by cerebral edema.

Aquaporin-4 (AQP4) is a primary influx route for water during brain edema formation. Here, we provide evidence that brain swelling triggers Ca(2+) signaling in astrocytes and that deletion of the Aqp4 gene markedly interferes with these events. Using in vivo two-photon imaging, we show that hypoosmotic stress (20% reduction in osmolarity) initiates astrocytic Ca(2+) spikes and that deletion of Aqp4 reduces these signals. The Ca(2+) signals are partly dependent on activation of P2 purinergic receptors, which was judged from the effects of appropriate antagonists applied to cortical slices. Supporting the involvement of purinergic signaling, osmotic stress was found to induce ATP release from cultured astrocytes in an AQP4-dependent manner. Our results suggest that AQP4 not only serves as an influx route for water but also is critical for initiating downstream signaling events that may affect and potentially exacerbate the pathological outcome in clinical conditions associated with brain edema.

Transient but not chronic hyperglycemia accelerates ocular glymphatic transport.

Glymphatic transport is vital for the physiological homeostasis of the retina and optic nerve. Pathological alterations of ocular glymphatic fluid transport and enlarged perivascular spaces have been described in glaucomatous mice. It remains to be established how diabetic retinopathy, which impairs vision in about 50% of diabetes patients, impacts ocular glymphatic fluid transport. Here, we examined ocular glymphatic transport in chronic hyperglycemic diabetic mice as well as in healthy mice experiencing a daily transient increase in blood glucose. Mice suffering from severe diabetes for two and four months, induced by streptozotocin, exhibited no alterations in ocular glymphatic fluid transport in the optic nerve compared to age-matched, non-diabetic controls. In contrast, transient increases in blood glucose induced by repeated daily glucose injections in healthy, awake, non-diabetic mice accelerated antero- and retrograde ocular glymphatic transport. Structural analysis showed enlarged perivascular spaces in the optic nerves of glucose-treated mice, which were absent in diabetic mice. Thus, transient repeated hyperglycemic events, but not constant hyperglycemia, ultimately enlarge perivascular spaces in the murine optic nerve. These findings indicate that fluid transport in the mouse eye is vulnerable to fluctuating glycemic levels rather than constant hyperglycemia, suggesting that poor glycemic control drives glymphatic malfunction and perivascular enlargement in the optic nerve.

Critical role of connexin 43 in secondary expansion of traumatic spinal cord injury.

Spinal cord injury (SCI) is often complicated by secondary injury as a result of the innate inflammatory response to tissue trauma and swelling. Previous studies have shown that excessive ATP release from peritraumatic regions contributes to the inflammatory response to SCI by activation of low-affinity P2X7 receptors. Because connexin hemichannels constitute an important route for astrocytic ATP release, we here evaluated the impact on post-traumatic ATP release of deletion of connexins (Cx30/Cx43) in astrocytes. In vivo bioluminescence imaging showed a significant reduction in ATP release after weight-drop injury in mice with deletion of Cx43 compared with Cx43-expressing littermates, both on a Cx30 knockout background. Moreover, astrogliosis and microglia activation were reduced in peritraumatic areas of those mice lacking Cx43; motor recovery was also significantly improved, and the traumatic lesion was smaller. Combined, these observations are consistent with a contribution by astrocytic hemichannels to post-traumatic ATP release that aggravates secondary injury and restrains functional recovery after experimental spinal cord injury. Connexins may thereby constitute a new therapeutic target in spinal cord injury.

Loss of aquaporin-4 results in glymphatic system dysfunction via brain-wide interstitial fluid stagnation.

The glymphatic system is a fluid transport network of cerebrospinal fluid (CSF) entering the brain along arterial perivascular spaces, exchanging with interstitial fluid (ISF), ultimately establishing directional clearance of interstitial solutes. CSF transport is facilitated by the expression of aquaporin-4 (AQP4) water channels on the perivascular endfeet of astrocytes. Mice with genetic deletion of AQP4 (AQP4 KO) exhibit abnormalities in the brain structure and molecular water transport. Yet, no studies have systematically examined how these abnormalities in structure and water transport correlate with glymphatic function. Here, we used high-resolution 3D magnetic resonance (MR) non-contrast cisternography, diffusion-weighted MR imaging (MR-DWI) along with intravoxel-incoherent motion (IVIM) DWI, while evaluating glymphatic function using a standard dynamic contrast-enhanced MR imaging to better understand how water transport and glymphatic function is disrupted after genetic deletion of AQP4. AQP4 KO mice had larger interstitial spaces and total brain volumes resulting in higher water content and reduced CSF space volumes, despite similar CSF production rates and vascular density compared to wildtype mice. The larger interstitial fluid volume likely resulted in increased slow but not fast MR diffusion measures and coincided with reduced glymphatic influx. This markedly altered brain fluid transport in AQP4 KO mice may result from a reduction in glymphatic clearance, leading to enlargement and stagnation of fluid in the interstitial space. Overall, diffusion MR is a useful tool to evaluate glymphatic function and may serve as valuable translational biomarker to study glymphatics in human disease.

Distinct astrocytic modulatory roles in sensory transmission during sleep, wakefulness, and arousal states in freely moving mice.

Despite extensive research on astrocytic Ca2+ in synaptic transmission, its contribution to the modulation of sensory transmission during different brain states remains largely unknown. Here, by using two-photon microscopy and whole-cell recordings, we show two distinct astrocytic Ca2+ signals in the murine barrel cortex: a small, long-lasting Ca2+ increase during sleep and a large, widespread but short-lasting Ca2+ spike when aroused. The large Ca2+ wave in aroused mice was inositol trisphosphate (IP3)-dependent, evoked by the locus coeruleus-norepinephrine system, and enhanced sensory input, contributing to reliable sensory transmission. However, the small Ca2+ transient was IP3-independent and contributed to decreased extracellular K+, hyperpolarization of the neurons, and suppression of sensory transmission. These events respond to different pharmacological inputs and contribute to distinct sleep and arousal functions by modulating the efficacy of sensory transmission. Together, our data demonstrate an important function for astrocytes in sleep and arousal states via astrocytic Ca2+ waves.

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.

An astrocytic basis of epilepsy.

Hypersynchronous neuronal firing is a hallmark of epilepsy, but the mechanisms underlying simultaneous activation of multiple neurons remains unknown. Epileptic discharges are in part initiated by a local depolarization shift that drives groups of neurons into synchronous bursting. In an attempt to define the cellular basis for hypersynchronous bursting activity, we studied the occurrence of paroxysmal depolarization shifts after suppressing synaptic activity using tetrodotoxin (TTX) and voltage-gated Ca(2+) channel blockers. Here we report that paroxysmal depolarization shifts can be initiated by release of glutamate from extrasynaptic sources or by photolysis of caged Ca(2+) in astrocytes. Two-photon imaging of live exposed cortex showed that several antiepileptic agents, including valproate, gabapentin and phenytoin, reduced the ability of astrocytes to transmit Ca(2+) signaling. Our results show an unanticipated key role for astrocytes in seizure activity. As such, these findings identify astrocytes as a proximal target for the treatment of epileptic disorders.

Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury.

Traumatic spinal cord injury is characterized by an immediate, irreversible loss of tissue at the lesion site, as well as a secondary expansion of tissue damage over time. Although secondary injury should, in principle, be preventable, no effective treatment options currently exist for patients with acute spinal cord injury (SCI). Excessive release of ATP by the traumatized tissue, followed by activation of high-affinity P2X7 receptors, has previously been implicated in secondary injury, but no clinically relevant strategy by which to antagonize P2X7 receptors has yet, to the best of our knowledge, been reported. Here we have tested the neuroprotective effects of a systemically administered P2X7R antagonist, Brilliant blue G (BBG), in a weight-drop model of thoracic SCI in rats. Administration of BBG 15 min after injury reduced spinal cord anatomic damage and improved motor recovery without evident toxicity. Moreover, BBG treatment directly reduced local activation of astrocytes and microglia, as well as neutrophil infiltration. These observations suggest that BBG not only protected spinal cord neurons from purinergic excitotoxicity, but also reduced local inflammatory responses. Importantly, BBG is a derivative of a commonly used blue food color (FD&C blue No. 1), which crosses the blood-brain barrier. Systemic administration of BBG may thus comprise a readily feasible approach by which to treat traumatic SCI in humans.

Cortical spreading depression causes and coincides with tissue hypoxia.

Cortical spreading depression (CSD) is a self-propagating wave of cellular depolarization that has been implicated in migraine and in progressive neuronal injury after stroke and head trauma. Using two-photon microscopic NADH imaging and oxygen sensor microelectrodes in live mouse cortex, we find that CSD is linked to severe hypoxia and marked neuronal swelling that can last up to several minutes. Changes in dendritic structures and loss of spines during CSD are comparable to those during anoxic depolarization. Increasing O2 availability shortens the duration of CSD and improves local redox state. Our results indicate that tissue hypoxia associated with CSD is caused by a transient increase in O2 demand exceeding vascular O2 supply.

Astrocyte-mediated control of cerebral blood flow.

Local increase in blood flow during neural activity forms the basis for functional brain imaging, but its mechanism remains poorly defined. Here we show that cortical astrocytes in vivo possess a powerful mechanism for rapid vasodilation. We imaged the activity of astrocytes labeled with the calcium (Ca(2+))-sensitive indicator rhod-2 in somatosensory cortex of adult mice. Photolysis of caged Ca(2+) in astrocytic endfeet ensheathing the vessel wall was associated with an 18% increase in arterial cross-section area that corresponded to a 37% increase in blood flow. Vasodilation occurred with a latency of only 1-2 s, and both indomethacin and the cyclooxygenase-1 inhibitor SC-560 blocked the photolysis-induced hyperemia. These observations implicate astrocytes in the control of local microcirculation and suggest that one of their physiological roles is to mediate vasodilation in response to increased neural activity.

[Evaluation of the value of ultrasound-guided core needle biopsy in the diagnosis of breast lesions].

OBJECTIVE: To investigate the accuracy, consistency and related affecting factors in pathological results of breast lesions diagnosed by ultrasound-guided core needle biopsy (CNB) and conventional excision histopathology. METHODS: The clinical data of 177 consecutive cases of breast lesions examined by ultrasound-guided CNB and subsequently excised were reviewed from Jan. 2003 to Nov. 2009. The agreement of pathological diagnosis between the CNB and subsequent excision pathology was analyzed. RESULTS: There were 136 cancers in the final diagnosis after surgical excision among 386 breast lesions and 129 of them were diagnosed by CNB. The sensitivity (true positive) of CNB was 94.9%, false negative rate was 5.1%, specificity (true negative) was 100%, false positive rate 0, Youden's index was 0.949, and positive predictive value and negative predictive value were 100% and 85.4%, respectively. Condensation rate was 96.0% and Kappa value was 0.895. CONCLUSION: Ultrasound-guided CNB with histopathological assessment is accurate in diagnosis of breast lesions and has a great consistency with conventional excision pathology. It is a reliable method for the diagnosis of breast lesions to avoid an over-reliance on excision pathological examination.

Cerebrospinal fluid influx drives acute ischemic tissue swelling.

Stroke affects millions each year. Poststroke brain edema predicts the severity of eventual stroke damage, yet our concept of how edema develops is incomplete and treatment options remain limited. In early stages, fluid accumulation occurs owing to a net gain of ions, widely thought to enter from the vascular compartment. Here, we used magnetic resonance imaging, radiolabeled tracers, and multiphoton imaging in rodents to show instead that cerebrospinal fluid surrounding the brain enters the tissue within minutes of an ischemic insult along perivascular flow channels. This process was initiated by ischemic spreading depolarizations along with subsequent vasoconstriction, which in turn enlarged the perivascular spaces and doubled glymphatic inflow speeds. Thus, our understanding of poststroke edema needs to be revised, and these findings could provide a conceptual basis for development of alternative treatment strategies.

Loss of astrocytic domain organization in the epileptic brain.

Gliosis is a pathological hallmark of posttraumatic epileptic foci, but little is known about these reactive astrocytes beyond their high glial fibrillary acidic protein (GFAP) expression. Using diolistic labeling, we show that cortical astrocytes lost their nonoverlapping domain organization in three mouse models of epilepsy: posttraumatic injury, genetic susceptibility, and systemic kainate exposure. Neighboring astrocytes in epileptic mice showed a 10-fold increase in overlap of processes. Concurrently, spine density was increased on dendrites of excitatory neurons. Suppression of seizures by the common antiepileptic, valproate, reduced the overlap of astrocytic processes. Astrocytic domain organization was also preserved in APP transgenic mice expressing a mutant variant of human amyloid precursor protein despite a marked upregulation of GFAP. Our data suggest that loss of astrocytic domains was not universally associated with gliosis, but restricted to seizure pathologies. Reorganization of astrocytes may, in concert with dendritic sprouting and new synapse formation, form the structural basis for recurrent excitation in the epileptic brain.