Small Interference RNA Targeting Connexin-43 Improves Motor Function and Limits Astrogliosis After Juvenile Traumatic Brain Injury

International audienceJuvenile 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

Changements de l’unité neurovasculaire après un traumatisme crânien juvénile léger

Traumatic brain injury (TBI) is the first cause for emergency department visits in the pediatric population. Regardless of the severity of TBI, pediatric patients suffer long-term cognitive and emotional impairments but the underlying cellular and molecular mechanisms are still poorly understood and there are no effective treatments available. The neurovascular unit is composed by blood vessels, neurons and astrocytes. Astrocytes are crucial for various physiological functions of this unit such as brain homeostasis and neurovascular coupling. In injuries astrocytes become “reactive”, and this “astrocytopathy” can impact their physiological roles and worsen the outcome after injury.We investigated astrocytopathy in juvenile TBI and hypothesized that: (1) reactive astrocytes contribute to spread of edema through connexin gap junctions after juvenile moderate TBI; and that (2) astrocytopathy also develops after juvenile mild TBI with calcium changes that could contribute to (3) impaired vascular reactivity, all of which impacts the behavioral outcome after injury.We have shown that:(1)Reducing astrocytopathy by downregulating the gap junction protein connexin 43 improved the behavioral outcome after juvenile moderate TBI, but did not impact the spread of edema.(2)Astrocytes became reactive and underwent morphological changes after juvenile mild TBI with disturbances in purinergic-calcium signaling related to expression changes of the water channel aquaporin 4 (AQP4).(3)Major vascular dysfunction developed after juvenile mild TBI with functional and morphological changes of the intraparenchymal vessels that paralleled behavioral impairments and preceded axonal damage after injury.This work brings new insights in the pathophysiology of juvenile TBI and opens prospects for developing therapeutics targeting astrocytopathy after injury.Le traumatisme crânien (TC) est la première cause de visite aux urgences pour la population pédiatrique. Indépendamment du niveau de sévérité du TC, les patients pédiatriques souffrent sur le long-terme de troubles cognitifs et émotionnels, cependant les mécanismes moléculaires et cellulaires sous-jacents sont encore peu connus, et il n’existe pas de traitement efficace disponible à ce jour. L’unité neurovasculaire est composée de vaisseaux sanguins, neurones et astrocytes. Les astrocytes sont essentiels à une variété de fonctions physiologiques assurées par cette unité tels que l’homéostasie cérébrale et le couplage neurovasculaire. Suite à une lésion, les astrocytes deviennent « réactifs », et cette « astrocytopatie » peut impacter leur rôle physiologique et empirer les conséquences de la lésion.Nous avons étudié le rôle de l’astrocytopatie dans le TC juvénile et fait l’hypothèse que : (1) les astrocytes réactifs contribuent à la propagation de l’œdème via les jonctions serrées connexines après un TC juvénile modéré ; (2) l’astrocytopatie se développe également après un TC juvénile léger avec des changements calciques qui pourraient contribuer à (3) une altération de la réactivité vasculaire, tout cela impactant sur les conséquences comportementales qui font suite à la lésion.Nous avons montré que :(1)Réduire l’astrocythopatie en sous-régulant la connexine 43 permettait d’améliorer les conséquences comportementales après un TC modéré juvénile, mais n’impactait pas la propagation de l’œdème.(2)Les astrocytes devenaient réactifs et subissaient des changements morphologiques après un TC juvénile léger avec des perturbations dans les signaux purinergiques-calciques liés à des changements dans l’expression du canal aqueux aquaporine 4 (AQP4).(3)Une dysfonction vasculaire majeure s’était développée après le TC juvénile léger avec des changements fonctionnels et morphologiques des vaisseaux intraparenchymaux parallèles aux altérations comportementales et précédant les dommages axonaux après la lésion.Ce travail apporte un nouvel aperçu de la pathophysiologie du TC juvénile et ouvre des possibilités pour développer des thérapies ciblant l’astrocytopatie après une lésion

Neurovascular unit changes after juvenile traumatic brain injury

Le traumatisme crânien (TC) est la première cause de visite aux urgences pour la population pédiatrique. Indépendamment du niveau de sévérité du TC, les patients pédiatriques souffrent sur le long-terme de troubles cognitifs et émotionnels, cependant les mécanismes moléculaires et cellulaires sous-jacents sont encore peu connus, et il n’existe pas de traitement efficace disponible à ce jour. L’unité neurovasculaire est composée de vaisseaux sanguins, neurones et astrocytes. Les astrocytes sont essentiels à une variété de fonctions physiologiques assurées par cette unité tels que l’homéostasie cérébrale et le couplage neurovasculaire. Suite à une lésion, les astrocytes deviennent « réactifs », et cette « astrocytopatie » peut impacter leur rôle physiologique et empirer les conséquences de la lésion.Nous avons étudié le rôle de l’astrocytopatie dans le TC juvénile et fait l’hypothèse que : (1) les astrocytes réactifs contribuent à la propagation de l’œdème via les jonctions serrées connexines après un TC juvénile modéré ; (2) l’astrocytopatie se développe également après un TC juvénile léger avec des changements calciques qui pourraient contribuer à (3) une altération de la réactivité vasculaire, tout cela impactant sur les conséquences comportementales qui font suite à la lésion.Nous avons montré que :(1) Réduire l’astrocythopatie en sous-régulant la connexine 43 permettait d’améliorer les conséquences comportementales après un TC modéré juvénile, mais n’impactait pas la propagation de l’œdème.(2) Les astrocytes devenaient réactifs et subissaient des changements morphologiques après un TC juvénile léger avec des perturbations dans les signaux purinergiques-calciques liés à des changements dans l’expression du canal aqueux aquaporine 4 (AQP4).(3) Une dysfonction vasculaire majeure s’était développée après le TC juvénile léger avec des changements fonctionnels et morphologiques des vaisseaux intraparenchymaux parallèles aux altérations comportementales et précédant les dommages axonaux après la lésion.Ce travail apporte un nouvel aperçu de la pathophysiologie du TC juvénile et ouvre des possibilités pour développer des thérapies ciblant l’astrocytopatie après une lésion.Traumatic brain injury (TBI) is the first cause for emergency department visits in the pediatric population. Regardless of the severity of TBI, pediatric patients suffer long-term cognitive and emotional impairments but the underlying cellular and molecular mechanisms are still poorly understood and there are no effective treatments available. The neurovascular unit is composed by blood vessels, neurons and astrocytes. Astrocytes are crucial for various physiological functions of this unit such as brain homeostasis and neurovascular coupling. In injuries astrocytes become “reactive”, and this “astrocytopathy” can impact their physiological roles and worsen the outcome after injury.We investigated astrocytopathy in juvenile TBI and hypothesized that: (1) reactive astrocytes contribute to spread of edema through connexin gap junctions after juvenile moderate TBI; and that (2) astrocytopathy also develops after juvenile mild TBI with calcium changes that could contribute to (3) impaired vascular reactivity, all of which impacts the behavioral outcome after injury.We have shown that:(1) Reducing astrocytopathy by downregulating the gap junction protein connexin 43 improved the behavioral outcome after juvenile moderate TBI, but did not impact the spread of edema.(2) Astrocytes became reactive and underwent morphological changes after juvenile mild TBI with disturbances in purinergic-calcium signaling related to expression changes of the water channel aquaporin 4 (AQP4).(3) Major vascular dysfunction developed after juvenile mild TBI with functional and morphological changes of the intraparenchymal vessels that paralleled behavioral impairments and preceded axonal damage after injury.This work brings new insights in the pathophysiology of juvenile TBI and opens prospects for developing therapeutics targeting astrocytopathy after injury

Small Interference RNA Targeting Connexin-43 Improves Motor Function and Limits Astrogliosis After Juvenile Traumatic Brain Injury.

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

Small Interference RNA Targeting Connexin-43 Improves Motor Function and Limits Astrogliosis After Juvenile Traumatic Brain Injury.

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

Chronic cerebrovascular dysfunction after traumatic brain injury

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

Modulating the water channel AQP4 alters miRNA expression, astrocyte connectivity and water diffusion in the rodent brain.

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

Early cerebrovascular and long-term neurological modifications ensue following juvenile mild traumatic brain injury in male mice

International audienceClinical evidence suggests that a mild traumatic brain injury occurring at a juvenile age (jmTBI) may be sufficient to elicit pathophysiological modifications. However, clinical reports are not adequately integrated with experimental studies examining brain changes occurring post-jmTBI. We monitored the cerebrovascular modifications and assessed the long-term behavioral and electrographic changes resulting from experimental jmTBI.In vivo photoacoustic imaging demonstrated a decrease of cerebrovascular oxygen saturation levels in the impacted area hours post-jmTBI. Three days post-jmTBI oxygenation returned to pre-jmTBI levels, stabilizing at 7 and 30 days after the injury. At the functional level, cortical arterioles displayed no NMDA vasodilation response, while vasoconstriction induced by thromboxane receptor agonist was enhanced at 1 day post-jmTBI. Arterioles showed abnormal NMDA vasodilation at 3 days post-jmTBI, returning to normality at 7 days post injury. Histology showed changes in vessel diameters from 1 to 30 days post-jmTBI. Neurological evaluation indicated signs of anxiety-like behavior up to 30 days post-jmTBI. EEG recordings performed at the cortical site of impact 30 days post-jmTBI did not indicate seizures activity, although it revealed a reduction of gamma waves as compared to age matched sham. Histology showed decrease of neuronal filament staining. In conclusion, experimental jmTBI triggers an early cerebrovascular hypo‑oxygenation in vivo and faulty vascular reactivity. The exact topographical coherence and the direct casualty between early cerebrovascular changes and the observed long-term neurological modifications remain to be investigated. A potential translational value for cerebro-vascular oxygen monitoring in jmTBI is discussed