Endocannabinoid-mediated rescue of somatosensory cortex activity, plasticity and related behaviors following an early in life concussion.

Due to the assumed plasticity of immature brain, early in life brain alterations are thought to lead to better recoveries in comparison to the mature brain. Despite clinical needs, how neuronal networks and associated behaviors are affected by early in life brain stresses, such as pediatric concussions, have been overlooked. Here we provide first evidence in mice that a single early in life concussion durably increases neuronal activity in the somatosensory cortex into adulthood, disrupting neuronal integration while the animal is performing sensory-related tasks. This represents a previously unappreciated clinically relevant mechanism for the impairment of sensory-related behavior performance. Furthermore, we demonstrate that pharmacological modulation of the endocannabinoid system a year post-concussion is well-suited to rescue neuronal activity and plasticity, and to normalize sensory-related behavioral performance, addressing the fundamental question of whether a treatment is still possible once post-concussive symptoms have developed, a time-window compatible with clinical treatment.

Essential omega-3 fatty acids tune microglial phagocytosis of synaptic elements in the mouse developing brain.

Omega-3 fatty acids (n-3 PUFAs) are essential for the functional maturation of the brain. Westernization of dietary habits in both developed and developing countries is accompanied by a progressive reduction in dietary intake of n-3 PUFAs. Low maternal intake of n-3 PUFAs has been linked to neurodevelopmental diseases in Humans. However, the n-3 PUFAs deficiency-mediated mechanisms affecting the development of the central nervous system are poorly understood. Active microglial engulfment of synapses regulates brain development. Impaired synaptic pruning is associated with several neurodevelopmental disorders. Here, we identify a molecular mechanism for detrimental effects of low maternal n-3 PUFA intake on hippocampal development in mice. Our results show that maternal dietary n-3 PUFA deficiency increases microglia-mediated phagocytosis of synaptic elements in the rodent developing hippocampus, partly through the activation of 12/15-lipoxygenase (LOX)/12-HETE signaling, altering neuronal morphology and affecting cognitive performance of the offspring. These findings provide a mechanistic insight into neurodevelopmental defects caused by maternal n-3 PUFAs dietary deficiency.

Caveolin expression changes in the neurovascular unit after juvenile traumatic brain injury: signs of blood-brain barrier healing?

Traumatic brain injury (TBI) is one of the major causes of death and disability in pediatrics, and results in a complex cascade of events including the disruption of the blood-brain barrier (BBB). A controlled-cortical impact on post-natal 17-day-old rats induced BBB disruption by IgG extravasation from 1 to 3 days after injury and returned to normal at day 7. In parallel, we characterized the expression of three caveolin isoforms, caveolin 1 (cav-1), caveolin 2 (cav-2) and caveolin 3 (cav-3). While cav-1 and cav-2 are expressed on endothelial cells, both cav-1 and cav-3 were found to be present on reactive astrocytes, in vivo and in vitro. Following TBI, cav-1 expression was increased in blood vessels at 1 and 7 days in the perilesional cortex. An increase of vascular cav-2 expression was observed 7 days after TBI. In contrast, astrocytic cav-3 expression decreased 3 and 7 days after TBI. Activation of endothelial nitric oxide synthase (eNOS) (via its phosphorylation) was detected 1 day after TBI and phospho-eNOS was detected both in association with blood vessels and with astrocytes. The molecular changes involving caveolins occurring in endothelial cells following juvenile-TBI might participate, independently of eNOS activation, to a mechanism of BBB repair while, they might subserve other undefined roles in astrocytes.

Vascular neural network phenotypic transformation after traumatic injury: potential role in long-term sequelae.

The classical neurovascular unit (NVU), composed primarily of endothelium, astrocytes, and neurons, could be expanded to include smooth muscle and perivascular nerves present in both the up- and downstream feeding blood vessels (arteries and veins). The extended NVU, which can be defined as the vascular neural network (VNN), may represent a new physiological unit to consider for therapeutic development in stroke, traumatic brain injury, and other brain disorders (Zhang et al., Nat Rev Neurol 8(12):711-716, 2012). This review is focused on traumatic brain injury and resultant post-traumatic changes in cerebral blood flow, smooth muscle cells, matrix, blood-brain barrier structures and function, and the association of these changes with cognitive outcomes as described in clinical and experimental reports. We suggest that studies characterizing TBI outcomes should increase their focus on changes to the VNN, as this may yield meaningful therapeutic targets to resolve posttraumatic dysfunction.

Delayed increase of astrocytic aquaporin 4 after juvenile traumatic brain injury: possible role in edema resolution?

Traumatic brain injury (TBI) is one of the leading causes of death and disability in children and adolescents. The neuropathological sequelae that result from TBI are a complex cascade of events including edema formation, which occurs more frequently in the pediatric than the adult population. This developmental difference in the response to injury may be related to higher water content in the young brain and also to molecular mechanisms regulating water homeostasis. Aquaporins (AQPs) provide a unique opportunity to examine the mechanisms underlying water mobility, which remain poorly understood in the juvenile post-traumatic edema process. We examined the spatiotemporal expression pattern of principal brain AQPs (AQP1, AQP4, and AQP9) after juvenile TBI (jTBI) related to edema formation and resolution observed using magnetic resonance imaging (MRI). Using a controlled cortical impact in post-natal 17 day-old rats as a model of jTBI, neuroimaging analysis showed a global decrease in water mobility (apparent diffusion coefficient, ADC) and an increase in edema (T2-values) at 1 day post-injury, which normalized by 3 days. Immunohistochemical analysis of AQP4 in perivascular astrocyte endfeet was increased in the lesion at 3 and 7days post-injury as edema resolved. In contrast, AQP1 levels distant from the injury site were increased at 7, 30, and 60 days within septal neurons but did not correlate with changes in edema formation. Group differences were not observed for AQP9. Overall, our observations confirm that astrocyticAQP4 plays a more central role than AQP1 or AQP9 during the edema process in the young brain.

Aquaporins in cerebrovascular disease: a target for treatment of brain edema?

In cerebrovascular disease, edema formation is frequently observed within the first 7 days and is characterized by molecular and cellular changes in the neurovascular unit. The presence of water channels, aquaporins (AQPs), within the neurovascular unit has led to intensive research in understanding the underlying roles of each of the AQPs under normal conditions and in different diseases. In this review, we summarize some of the recent knowledge on AQPs, focusing on AQP4, the most abundant AQP in the central nervous system. Several experimental models illustrate that AQPs have dual, complex regulatory roles in edema formation and resolution. To date, no specific therapeutic agents have been developed to inhibit water flux through these channels. However, experimental results strongly suggest that this is an important area for future investigation. In fact, early inhibition of water channels may have positive effects in the prevention of edema formation. At later time points during the course of disease, AQP is important for the clearance of water from the brain into blood vessels. Thus, AQPs, and in particular AQP4, have important roles in the resolution of edema after brain injury. The function of these water channel proteins makes them an excellent therapeutic target.

A neurovascular perspective for long-term changes after brain trauma.

Traumatic brain injury (TBI) affects all age groups in a population and is an injury generating scientific interest not only as an acute event, but also as a complex brain disease with several underlying neurobehavioral and neuropathological characteristics. We review early and long-term alterations after juvenile and adult TBI with a focus on changes in the neurovascular unit (NVU), including neuronal interactions with glia and blood vessels at the blood-brain barrier (BBB). Post-traumatic changes in cerebral blood-flow, BBB structures and function, as well as mechanistic pathways associated with brain aging and neurodegeneration are presented from clinical and experimental reports. Based on the literature, increased attention on BBB changes should be integrated in studies characterizing TBI outcome and may provide a meaningful therapeutic target to resolve detrimental post-traumatic dysfunction.

Cell locations for AQP1, AQP4 and 9 in the non-human primate brain.

The presence of three water channels (aquaporins, AQP), AQP1, AQP4 and AQP9 were observed in normal brain and several rodent models of brain pathologies. Little is known about AQP distribution in the primate brain and its knowledge will be useful for future testing of drugs aimed at preventing brain edema formation. We studied the expression and cellular distribution of AQP1, 4 and 9 in the non-human primate brain. The distribution of AQP4 in the non-human primate brain was observed in perivascular astrocytes, comparable to the observation made in the rodent brain. In contrast with rodent, primate AQP1 is expressed in the processes and perivascular endfeet of a subtype of astrocytes mainly located in the white matter and the glia limitans, possibly involved in water homeostasis. AQP1 was also observed in neurons innervating the pial blood vessels, suggesting a possible role in cerebral blood flow regulation. As described in rodent, AQP9 mRNA and protein were detected in astrocytes and in catecholaminergic neurons. However additional locations were observed for AQP9 in populations of neurons located in several cortical areas of primate brains. This report describes a detailed study of AQP1, 4 and 9 distributions in the non-human primate brain, which adds to the data already published in rodent brains. This relevant species differences have to be considered carefully to assess potential drugs acting on AQPs non-human primate models before entering human clinical trials.

Aquaglyceroporin 9 in brain pathologies.

Aquaglyceroporins belong to the aquaporin family and are permeable to water and also to small solutes such as glycerol and urea. In this review, we will compare the expression of aquaporin 9 (AQP9), an aquaglyceroporin, with that of AQP4, a pure water channel, in pathological conditions. In astrocytes, AQP4 is mainly involved in water and ionic homeostasis. Its expression is highly modified in several brain disorders and it plays a key role in cerebral edema formation. AQP9 is expressed in astrocytes and in catecholaminergic neurons. The level of expression of brain AQP9 is under the control of blood insulin concentrations, and its expression is increased in diabetes, suggesting that AQP9 could be involved in brain energy metabolism. The induction of AQP9 in astrocytes is observed over time after stroke onset, suggesting participation in the clearance of excess lactate in the extracellular space. In some models, AQP9 is also induced in non-catecholaminergic neurons after global ischemia and in the periphery of gliomas, however functional roles are still unclear. The review of literature underlies that each AQP has several distinctive roles which depend on the AQP and cell types.

Induction of brain aquaporin 9 (AQP9) in catecholaminergic neurons in diabetic rats.

Aquaporin 9 facilitates the diffusion of water but also glycerol and monocarboxylates, known as brain energy substrates. AQP9 was recently observed in catecholaminergic neurons that are implicated in energy homeostasis and also possibly in neuroendocrine effects of diabetes. Recently it has been observed that the level of AQP9 expression in hepatocytes is sensitive to the blood concentration of insulin. Furthermore, insulin injection in the brain is known to be related to the energy homeostasis. Based on these observations, we investigated if the concentration of insulin affects the level of brain AQP9 expression and if so, in which cell types. This study has been carried out, in a model of the diabetic rat generated by streptozotocin injection and on brainstem slices. In diabetic rats showing a decrease in systemic insulin concentration, AQP9 is only increased in brain areas containing catecholaminergic neurons. In contrast, no significant change is detected in the cerebral cortex and the cerebellum. Using immunocytochemistry, we are able to show that the increase in AQP9 expression is specifically present in catecholaminergic neurons. In brainstem slice cultures, 2 microM insulin induces a significant decrease in AQP9 protein levels 6 h after application, suggesting that brain AQP9 is also regulated by the insulin. These results show that the level of expression of brain AQP9 is affected by variations of the concentration of insulin in a diabetic model and in vitro.

Distribution and possible roles of aquaporin 9 in the brain.

Aquaporin 9 (AQP9) is a member of the aquaporin channel family involved in water flux through plasma membranes and exhibits the distinct feature of being also permeable to monocarboxylates, such as lactate, and various solutes, including glycerol, carbamides, purines, pyrimidines, and urea. AQP9 is constitutively expressed at high levels in the liver. In the brain under physiological conditions, AQP9 was first observed in tanycytes, and then in astrocytes. Only recently, its expression was also shown in neurons. Neurons expressing AQP9 are catecholaminergic and glucose sensitive. The expression of neuronal AQP9 can be negatively regulated by insulin and in diabetic animals an increase in AQP9 expression is observed in the catecholaminergic nuclei of the hindbrain, similar to the regulation of AQP9 by insulin in the liver. Furthermore, after transient brain ischemia, AQP9 expression is increased in astrocytes and its regulation may implicate the MAP-kinase pathways stimulated in such pathological conditions. Despite these new data, the exact role of AQP9 in the brain is still unclear. However, we may hypothesize that AQP9 is implicated in brain energy metabolism, as a neutral solute channel. AQP9 could facilitate the diffusion of lactate from the astrocyte to the neuron. In glucose sensitive neurons, diffusion of lactate and glycerol could stimulate these neurons in a similar manner to glucose and could regulate the energy balance. In pathological conditions, induction of AQP9 in astrocytes could participate in the clearance of excess lactate in the extracellular space. These hypotheses concerning the function of brain AQP9 are still speculative and open new areas of investigation.

Distribution of Aquaporin 9 in the adult rat brain: preferential expression in catecholaminergic neurons and in glial cells.

Aquaporin 9 (AQP9) is a recently cloned water channel that is permeable to monocarboxylate, glycerol and urea. In rat, AQP9 has been found in testis and liver as well as in brain where its expression has been initially shown in glial cells in forebrain. However, the expression of AQP9 has not been investigated in the brainstem. The purpose of this study is to describe the distribution of AQP9-immunoreactive cells throughout the adult rat brain using reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry. We performed immunolabeling on brain from animals perfused with fixative and we show that AQP9 is expressed (i) in astrocytes in the glia limitans, in the white matter and in glial cells of the cerebellum, (ii) in the endothelial cells of pial vessels, and (iii) in specific groups of neurons. The neuronal AQP9 expression was almost exclusively observed in catecholaminergic cells including the adrenergic, noradrenergic and dopaminergic groups, but not in other monoaminergic neurons such as serotonergic or histaminergic cells. A slight labeling was also observed in non-catecholaminergic neurons localized in the paraventricular nucleus of the hypothalamus. These results indicate that AQP9 has a unique brain distribution with a preferential localization in catecholaminergic nuclei known to be involved in many cerebral functions. While the presence of AQP9 in glia limitans and in endothelial cells of the pial vessels could be related to water transport through the blood-brain barrier, its expression in neuronal cells, not directly involved in the osmoregulation, suggests that brain AQP9 could also be used as a metabolite channel since lactate and glycerol can be energy substrates for neurons.

Aquaporin 1 and aquaporin 4 expression in human brain after subarachnoid hemorrhage and in peritumoral tissue.

Aquaporins (AQPs) are a protein family of water channels which facilitate the water flux through the plasmatic membranes. The expression of AQPs has been described in rat brain by several studies. Despite recent reports that have shown an over-expression of AQP1 and 4 in human tumoral cells, little is known about AQP expression in human brain. The purpose of this study was to investigate the expression of AQP1 and AQP4 in human brain after subarachnoid hemorrhage (SAH) and in peritumoral tissue by western blot and immunohistochemistry. The results showed a marked increase of the expression of AQP1 and AQP4. This over-expression occurred on the astrocytic processes and polarization on astrocytic end-feet was lost. No expression was observed on neuronal cells. This study is the first demonstration of the induction of AQP1 and AQP4 on reactive astrocytes in an acute brain injury, such as SAH. These results reinforce the hypothesis that AQPs may be involved in the dynamics of brain edema formation or resolution. Further studies are needed to understand their functional role.

Astrocyte-specific expression of aquaporin-9 in mouse brain is increased after transient focal cerebral ischemia.

Aquaporin-9 (AQP9) is a new member of the aquaporin family of water-selective channels mainly expressed in liver and testis, presenting the characteristic of also being permeable to various solutes, particularly lactate. Recent data have shown the presence of AQP9 on tanycytes in the rat brain. In the current study, the authors show the expression of AQP9 in astrocytes in the mouse brain and changes in its expression after cerebral ischemia. Indeed, in control mouse, the AQP9 immunolabeling is present on astrocytic processes bordering the subarachnoid space and ventricles. The labeling also is observed on astrocytes in the white matter, hippocampus, hypothalamus, and lateral septum. After focal transient ischemia, an increase of the immunolabeling is detected on astrocytes in periinfarct areas. This AQP9 distribution study in mouse brain suggests a role of AQP9 in water homeostasis in the central nervous system. Furthermore, the overexpression of AQP9 on astrocytes surrounding an ischemic lesion suggests that AQP9 may also play a role in the regulation of postischemia edema and, in view of its permeability to monocarboxylates, in the clearance of lactate from the ischemic focus.

Presence of aquaporin-4 and muscarinic receptors in astrocytes and ependymal cells in rat brain: a clue to a common function?

Using combined double immunofluorescence and laser confocal microscopy, we studied the common cellular localization of cholinergic muscarinic receptors (mAChRs) and aquaporin-4 water channels (AQP4) in the cortex, the corpus callosum and in ependymal cells of the rat brain. In the cortex, AQP4 staining was restricted to the perivascular end-feet of astrocytes. It was more widely distributed on the astrocytes of the corpus callosum. On astrocytes, mAChRs were often present in regions immunoreactive to AQP4. Ependymal cells bordering the third ventricle were also stained by both antibodies. The double staining of mAChRs with AQP4 on two different cell-types might indicate that further interactions exist which may be important in the regulation of water and electrolyte movements in the brain.

Hypervascularization in the magnocellular nuclei of the rat hypothalamus: relationship with the distribution of aquaporin-4 and markers of energy metabolism.

In the magnocellular nuclei of the hypothalamus, there is a rich vascular network for which the function remains to be established. In the supraoptic nucleus, the high vascular density may be one element, which together with the water channel aquaporin-4 expressed in the astrocytes, is related to a role in osmoreception. We tested the osmoreception hypothesis by studying the correlation between vascular and cellular densities in the paraventricular nucleus and the supraoptic nucleus. Whether aquaporin-4 is likely to contribute to osmoreception was tested by studying the distribution in the magnocellular nuclei of the hypothalamus. The high vascular density may also reflect a high metabolic activity due to the synthesis of vasopressin and oxytocin. This metabolic hypothesis was tested by studying the regional cytochrome oxidase histochemistry, the local cerebral blood flow, and the density of glucose transporter type-1 in the supraoptic and paraventricular nuclei. All the magnocellular nuclei were characterized by an extended and intense aquaporin-4 labelling and a weak cytochrome oxidase histochemistry. The highest vascular density was found in the supraoptic nucleus and the magnocellular regions of the paraventricular nucleus. The local cerebral blood flow rates were surprisingly low in the paraventricular nucleus and the supraoptic nucleus in comparison to the cerebral cortex. Furthermore in these nuclei, the antibody for glucose transporter type-1 revealed two populations of vessels differing by their labelling intensity. The similarities observed between the different nuclei suggest that, in the hypothalamus, all magnocellular regions sense the plasma osmolarity. The low local cerebral blood flow, and the patterns of glucose transporter type-1 labelling and cytochrome oxidase histochemistry suggest that the high vascularization of these hypothalamic nuclei is not related to a high metabolic capacity in basal conditions.

Distribution of muscarinic receptors on the endothelium of cortical vessels in the rat brain.

Functional and pharmacological studies have suggested that there are muscarinic receptors (mAChRs) on the endothelial cells of major cerebral arteries, while recent immunological studies indicate that there are no mAChRs on the endothelium of brain capillaries. This difference may be because the distribution of mAChR on the endothelium varies with the type of vessel. This paper examines the distribution of mAChR on the vascular endothelium along intraparenchymal blood vessels in the rat brain by immunolabelling and laser confocal microscopy. Sections were immunostained by combinations of an anti-mAChR antibody (M35) with antibodies to endothelial (anti-GLUT1), or to smooth muscle markers (anti-actin). Antibody labellings were detected with fluorescent second antibodies. Most of the penetrating vessels bore mAChR immunolabelling which coincided over almost all the vessel surface with endothelial labelling. The mAChR immunolabelling was less widespread over the endothelium on the medium sized vessels (diameter < 50 microm) and only 50% of these vessels had mAChR staining on the endothelium. There was no mAChR immunostaining on the endothelium of the capillaries. In contrast with the basilar artery, there was no mAChR immunolabelling on the smooth muscle layer of the intracortical vessels. These data indicate that the intensity of mAChR immunolabelling decreases along the vascular tree from large conducting vessels to capillaries.

Regional study of the co-localization of neuronal nitric oxide synthase with muscarinic receptors in the rat cerebral cortex.

There is increasing evidence that nitric oxide is an important molecular messenger involved in a wide variety of biological processes including the regulation of the cerebral circulation. For instance, it has been implicated in the vascular response to nucleus basalis magnocellularis stimulation, a structure which is widely recognized as the predominant source of cholinergic fibres projecting to the neocortex. The present investigation was carried out to determine if muscarinic receptors are present on cortical neurons expressing neuronal nitric oxide synthase (nitric oxide-producing enzyme). To this aim, double labelling of both neuronal nitric oxide synthase/vessels and neuronal nitric oxide synthase/muscarinic receptors was performed on free-floating cryosections obtained from rat brain. The observations were made by confocal laser scanning microscopy. The double labelling of neuronal nitric oxide synthase with the arterioles demonstrated the presence of nitroxidergic fibres in the wall of intraparenchymal vessels. A rich network of nitroxidergic fibres independent of the vessels was also seen in the parenchyma. Since the maximal surface of a square of tissue without any nitroxidergic fibres corresponded to 1400 +/- 105 microns2, the distance separating any cortical point from its closest neuronal nitric oxide synthase-positive fibre was never higher than 25 microns (half diagonal of square). According to models of the diffusional spread of nitric oxide, it is likely that nitric oxide can reach the whole cortical volume. Our results on the regional study of neuronal nitric oxide synthase/muscarinic receptors showed a high density of neuronal nitric oxide synthase-positive neurons principally in the frontal and perirhinal cortices and a low density in the occipital cortex. These data fit well with the known pattern of cortical projections from the nucleus basalis magnocellularis as revealed by anterogradely transported markers. The double labelling showed that about 10% of neuronal nitric oxide synthase-positive neurons were co-localized with muscarinic receptors in the frontoparietal cortex. In agreement with previous papers, the vascular innervation by nitroxidergic neuronal processes was often found to lie near the branching points of arterioles. Such localization allows neuronal nitric oxide synthase-positive neurons an extensive control of the vascular tree without requiring a large number of neuronal commands. Therefore, despite the low level of neuronal nitric oxide synthase/muscarinic receptor co-localization, this neuronal subpopulation could represent a possible relay implicated in the vascular effects of the nucleus basalis magnocellularis.