Juvenile traumatic brain injury (jTBI) is the leading cause of death and disability for children and adolescents worldwide, but there are no pharmacological treatments available. Aquaporin 4 (AQP4), an astrocytic perivascular protein, is increased after jTBI, and inhibition of its expression with small interference RNA mitigates edema formation and reduces the number of reactive astrocytes after jTBI. Due to the physical proximity of AQP4 and gap junctions, coregulation of AQP4 and connexin 43 (Cx43) expressions, and the possibility of water diffusion via gap junctions, we decided to address the potential role of astrocytic gap junctions in jTBI pathophysiology. We evaluated the role of Cx43 in the spread of the secondary injuries via the astrocyte network, such as edema formation associated with blood–brain barrier dysfunctions, astrogliosis, and behavioral outcome. We observed that Cx43 was altered after jTBI with increased expression in the perilesional cortex and in the hippocampus at several days post injury. In a second set of experiments, cortical injection of small interference RNA against Cx43 decreased Cx43 protein expression, improved motor function recovery, and decreased astrogliosis but did not result in differences in edema formation as measured via T2-weighted imaging or diffusion-weighted imaging at 1 day or 3 days. Based on our findings, we can speculate that while decreasing Cx43 has beneficial roles, it likely does not contribute to the spread of edema early after jTBI.

Traumatic brain injuries (TBI) often involve vascular dysfunction that leads to long-term alterations in physiological and cognitive functions of the brain. Indeed, all the cells that form blood vessels and that are involved in maintaining their proper function can be altered by TBI. This Review focuses on the different types of cerebrovascular dysfunction that occur after TBI, including cerebral blood flow alterations, autoregulation impairments, subarachnoid hemorrhage, vasospasms, blood-brain barrier disruption, and edema formation. We also discuss the mechanisms that mediate these dysfunctions, focusing on the cellular components of cerebral blood vessels (endothelial cells, smooth muscle cells, astrocytes, pericytes, perivascular nerves) and their known and potential roles in the secondary injury cascade. © 2016 Wiley Periodicals, Inc.

Aquaporins (AQPs) facilitate water diffusion through the plasma membrane. Brain aquaporin-4 (AQP4) is present in astrocytes and has critical roles in normal and disease physiology. We previously showed that a 24.9% decrease in AQP4 expression after in vivo silencing resulted in a 45.8% decrease in tissue water mobility as interpreted from magnetic resonance imaging apparent diffusion coefficients (ADC). Similar to previous in vitro studies we show decreased expression of the gap junction protein connexin 43 (Cx43) in vivo after intracortical injection of siAQP4 in the rat. Moreover, siAQP4 induced a loss of dye-coupling between astrocytes in vitro, further demonstrating its effect on gap junctions. In contrast, silencing of Cx43 did not alter the level of AQP4 or water mobility (ADC) in the brain. We hypothesized that siAQP4 has off-target effects on Cx43 expression via modification of miRNA expression. The decreased expression of Cx43 in siAQP4-treated animals was associated with up-regulation of miR224, which is known to target AQP4 and Cx43 expression. This could be one potential molecular mechanism responsible for the effect of siAQP4 on Cx43 expression, and the resultant decrease in astrocyte connectivity and dramatic effects on ADC values and water mobility.

Traumatic brain injury (TBI) has the highest incidence amongst the pediatric population and its mild severity represents the most frequent cases. Moderate and severe injuries as well as repetitive mild TBI result in lasting morbidity. However, whether a single mild TBI sustained during childhood can produce long-lasting modifications within the brain is still debated. We aimed to assess the consequences of a single juvenile mild TBI (jmTBI) at 12 months post-injury in a mouse model. Non-invasive diffusion tensor imaging (DTI) revealed significant microstructural alterations in the hippocampus and the in the substantia innominata/nucleus basalis (SI/NB), structures known to be involved in spatial learning and memory. DTI changes paralled neuronal loss, increased astrocytic AQP4 and microglial activation in the hippocampus. In contrast, decreased astrocytic AQP4 expression and microglia activation were observed in SI/NB. Spatial learning and memory were impaired and correlated with alterations in DTI-derived derived fractional ansiotropy (FA) and axial diffusivity (AD). This study found that a single juvenile mild TBI leads to significant region-specific DTI microstructural alterations, distant from the site of impact, that correlated with cognitive discriminative novel object testing and spatial memory impairments at 12 months after a single concussive injury. Our findings suggest that exposure to jmTBI leads to a chronic abnormality, which confirms the need for continued monitoring of symptoms and the development of long-term treatment strategies to intervene in children with concussions.

A hallmark of stroke is water accumulation (edema) resulting from dysregulation of osmotic homeostasis. Brain edema contributes to tissue demise and may lead to increased intracranial pressure and lethal herniation. Currently, there are only limited treatments to prevent edema formation following stroke. Aquaporin 4 (AQP4), a brain water channel, has become a focus of interest for therapeutic approaches targeting edema. At present, there are no pharmacological tools to block AQP4. The role of AQP4 in edema after brain injury remains unclear with conflicting results from studies using AQP4^-/- mice and of AQP4 expression following stroke. Here, we studied AQP4 and its role in edema formation by testing AQP4^-/- mice in a model of middle cerebral artery occlusion using novel quantitative MRI water content measurements, histology and behavioral changes as outcome measures. Absence of AQP4 was associated with decreased mortality and increased motor recovery 3 to 14 days after stroke. Behavioral improvement was associated with decreased lesion volume, neuronal cell death and neuroinflammation in AQP4^-/- compared to wild type mice. Our data suggest that the lack of AQP4 confers an overall beneficial role at long term with improved neuronal survival and reduced neuroinflammation, but without a direct effect on edema formation.

In our juvenile traumatic brain injury (jTBI) model, emergence of cognitive dysfunctions was observed up to 6 months after trauma. Here we hypothesize that early brain injury induces changes in the neurovascular unit (NVU) that would be associated with amyloid-beta (Aβ) accumulation. We investigated NVU changes for up to 6 months in a rat jTBI model, with a focus on the efflux protein P-glycoprotein (P-gp) and on the basement membrane proteins perlecan and fibronectin, all known to be involved in Aβ clearance. Rodent-Aβ staining is present and increased after jTBI around cerebral blood microvessels, and the diameter of those is decreased by 25% and 34% at 2 and 6 months, respectively, without significant angiogenesis. P-glycoprotein staining in endothelium is decreased by 22% and parallels an increase of perlecan and fibronectin staining around cerebral blood vessels. Altogether, these results strongly suggest that the emergence of long-term behavioral dysfunctions observed in rodent jTBI may be related to endothelial remodeling at the blood-brain barrier alongside vascular dysfunction and altered Aβ trafficking. This study shows that it is important to consider jTBI as a vascular disorder with long-term consequences on cognitive functions.

Traumatic brain injury (TBI) is common in young children and adolescents and is associated with long-term disability and mortality. The neuropathologic sequelae that result from juvenile TBI are a complex cascade of events that include edema formation and brain swelling. Brain aquaporin-4 (AQP4) has a key role in edema formation. Thus, development of novel treatments targeting AQP4 to reduce edema could lessen the neuropathologic sequelae. We hypothesized that inhibiting AQP4 expression by injection of small-interfering RNA (siRNA) targeting AQP4 (siAQP4) after juvenile TBI would decrease edema formation, neuroinflammation, neuronal cell death, and improve neurologic outcomes. The siAQP4 or a RNA-induced silencing complex (RISC)-free control siRNA (siGLO) was injected lateral to the trauma site after controlled cortical impact in postnatal day 17 rats. Magnetic resonance imaging, neurologic testing, and immunohistochemistry were performed to assess outcomes. Pups treated with siAQP4 showed acute (3 days after injury) improvements in motor function and in spatial memory at long term (60 days after injury) compared with siGLO-treated animals. These improvements were associated with decreased edema formation, increased microglial activation, decreased blood-brain barrier disruption, reduced astrogliosis and neuronal cell death. The effectiveness of our treatment paradigm was associated with a 30% decrease in AQP4 expression at the injection site.

The past decade has brought tremendous progress in diagnostic and therapeutic options for cerebrovascular diseases as exemplified by the advent of thrombectomy in ischemic stroke, benefitting a steeply increasing number of stroke patients and potentially paving the way for a renaissance of neuroprotectants. Progress in basic science has been equally impressive. Based on a deeper understanding of pathomechanisms underlying cerebrovascular diseases, new therapeutic targets have been identified and novel treatment strategies such as pre- and post-conditioning methods were developed. Moreover, translationally relevant aspects are increasingly recognized in basic science studies, which is believed to increase their predictive value and the relevance of obtained findings for clinical application.This review reports key results from some of the most remarkable and encouraging achievements in neurovascular research that have been reported at the 10th International Symposium on Neuroprotection and Neurorepair. Basic science topics discussed herein focus on aspects such as neuroinflammation, extracellular vesicles, and the role of sex and age on stroke recovery. Translational reports highlighted endovascular techniques and targeted delivery methods, neurorehabilitation, advanced functional testing approaches for experimental studies, pre-and post-conditioning approaches as well as novel imaging and treatment strategies. Beyond ischemic stroke, particular emphasis was given on activities in the fields of traumatic brain injury and cerebral hemorrhage in which promising preclinical and clinical results have been reported. Although the number of neutral outcomes in clinical trials is still remarkably high when targeting cerebrovascular diseases, we begin to evidence stepwise but continuous progress towards novel treatment options. Advances in preclinical and translational research as reported herein are believed to have formed a solid foundation for this progress.