Roles Of Aquanorin-4 Gene Deficiency In Different Types Of Brain Injury

BackgroundsThe aquaporins (AQPs) are a family of transmembrane water channel proteins that mediate water transmembrane movment. AQP4, the major subtype of AQPs in the CNS, is mainly expressed in pericapillary astrocyte foot processes, glial limiting membranes and ependyma, but lower in non-endfoot membranes. The expression pattern is showing consistence with its function in water homeostasis. It has been reported that the expression of AQP4 is up-regulated after brain injuries including traumatic brain injury, cerebral ischemia and brain tumours. The research on AQP4 gene knockout animals revealed that the expression and function of AQP4 are associated with both cellular and vasogenic brain edema. However, AQP4 plays a role in astrocyte growth and other functions, and is also associated with non-edema disorders, such as inflammation and excitatory toxicity.Post-ischemic inflammation is determinant for ischemic insults and recovery process, and is regulated by various pro-inflammatory cytokines and mediators. Cysteinyl leukotrienes (CysLTs) are potent inflammatory mediators, and play important roles in cerebral ischemia, traumatic brain injury and other diseases in the central nervous system. CysLTs act their specific receptors, the CysLT receptors including CysLT1 and CysLT2 receptor. Pharmacological studies have shown that CysLT1 receptor mediates acute in vivo neuronal injury and chronic astroglial proliferation after cerebral ischemia; it regulates post-ischemic inflammation by increasing the permeability of blood-brain barrier and mediates expression of intercellular adhesion molecule. CysLT2 receptor mediated microglial activation and phagocytosis in vivo and in vitro after ischemic injury.In AQP-deficient mice, brain edema after brain injury, such as brain ischemia and brain trauma, has been widely investigated; however, other injured changes, especially inflammation, remain to be investigated. Since AQP4 deficiency induces astrocyte dysfunction, non-edema alterations will occurs, which is our focus in the present study.AimsTo clarify the roles of AQP4 gene deficiency in different types of brain injury, in the present study we performed the following investigations:In AQP4-deficient (AQP4-/-) and wild-type (AQP4+/+) mice, we assessed the inflammatory responses after focal cerebral ischemia was induced by 30 min of middle cerebral arterial occlusion (MCAO). Ischemic neuronal injury and cellular inflammatory responses, as well as the expression and localization of cysteinyl leukotriene CysLT2 and CysLT1 receptors, were determined at 24 and 72 h after MCAO.2. In AQP4-/- and AQP4+/+ mice, we observed the chronic injury after focal cerebral ischemia was induced by 30 min of middle cerebral arterial occlusion (MCAO). Neurological deficits were determined during 35 days after ischemia. Brain atrophy, cavitas volume, neuron loss, and densities of microglia and astrocytes were determined at the end of the experiment, and their changes were compared between AQP4-/- and AQP4+/+ mice.3. Then we clarified the properties of brain cryoinjury at acute and later phases. At 1, 7 and 14 days after cryoinjury, lesion volume, neuron loss, and densities of microglia and astrocytes were determined. The changes in AQP4-/- and AQP4+/+ mice were compared.4. In AQP4-/- mice, we also determined the excitatory toxicity. The brain injury was induced by microinjection of NMD A into the cortex. The injured area was determined by toluidine blue staining, degenerated neurons were detected by Fluoro-Jade B staining, and increased blood-brain barrier (BBB) permeability was evaluated by IgG immunostaining.MethodsFocal cerebral ischemia was induced by middle cerebral arterial occlusion (MCAO). Brain cold injury (cryoinjury) was induced according to the reported method with some modifications. We also established brain excitotoxic injury model by NMDA micro injection (150 nmol in 0.5μl) into the cerebral cortex.Brain injury area was determined by frozen sections stained with toluidine blue. Blood brain barrier permeability was determined by IgG immunohistochemistry. The survival neurons, microglia, neutrophils and astrocyte were determined by immunohistochemistry; Necrotic neuron was determined by Fluoro-Jade B staining. The CysLT1 and CysLT2 receptor protein expression were determined by Western blot analysis, and the cellular distribution of the receptor was assessed by double immunofluorescence.Results1. AQP4 deficiency aggravates acute injury and inflammation in the brain after focal cerebral ischemiaCompared to AQP4+/+ mice, AQP4-/- mice showed more neuron loss, more severe microglial activation and neutrophil infiltration, but less astrocyte proliferation in the brain after MCAO. In addition, the protein expression of both CysLT1 and CysLT2 receptors was up-regulated in the ischemic brain, and the up-regulation was more pronounced in AQP4-/- mice. The increased CysLT1 and CysLT2 receptors were primarily localized in neurons and neutrophils in the ischemic core, in microglia in the boundary zone, but not in astrocytes 24 and 72 h after MCAO. Those localized in microglia and neutrophils were enhanced in AQP4-/- mice. The results suggest that AQP4 may play an inhibitory role in post-ischemic inflammation.2. AQP4 deficiency aggravates chronic brain injury after focal cerebral ischemiaCompared to AQP4+/+ mice, the survival rate and body weight gain were reduced over 35 days after MCAO in AQP4V-/- mice. The brain atrophy and cavity volume in AQP4-/- mice were aggravated at 35 days following MCAO, and neuronal loss worsened, compared to those in AQP4+/+ mice. Besides, microglial cells density increased more, but astrocyte proliferation was attenuated in AQP4-/- mice. Therefore, AQP4 deficiency worsens delayed lesions after MCAO, perhaps due to the more microgliosis and less astrocyte proliferation in the late phase.3. AQP4 deficiency attenuates acute lesions but aggravates delayedlesions after cryoinjury to mouse brainLesion volume and neuron loss were milder in AQP4-/- mice than in AQP4+/+ mice 24 h after cryoinjury. In contrast, lesion volume and neuron loss were worsened, microglial density increased more, and astrocyte proliferation and glial scar formation were attenuated 7 and 14 days after cryoinjury in AQP4-/- mice. Thus, AQP4 deficiency ameliorates acute lesions, while it worsens delayed lesion perhaps due to microgliosis in the late phase.4. AQP4 deficiency aggravates NMDA-induced brain injuryCompared with AQP4+/+ mice, AQP4-/- mice exhibited increased cortical lesion area, aggravated neuron degeneration, and increased BBB disruption after NMDA microinjection. Therefore, AQP4 may play a protective role in NMDA-induced brain injury in mice.Conclusions:1. After focal cerebral ischemia, AQP4 deficiency aggravates brain ischemic injury and inflammation in both acute and chronic phases. AQP4-/- mice exhibited more severe cellular inflammation and enhanced expression of the inflammation-related CysLT1 and CysLT2 receptors after MCAO. AQP4 deficiency also aggravates chronic brain injury, reducing survival rate and weight gain as well as increasing brain atrophy and cavity formation, but glial scar formation was attenuated after focal cerebral ischemia. This suggests that AQP4 may be important in post-ischemic injury/inflammation and recovery process in both acute and chronic phases. Astrocyte dysfunction duo to AQP4 deficiency may contribute to these responses. 2. AQP4 deficiency has a dual role in brain cryoinjury. AQP4-/- mice exhibited milder acute lesions and more severe delayed lesions and associated microglial inflammation after cryoinjury. This may result from astrocyte dysfunction due to AQP4 deficiency.3. AQP4 deficiency also aggravates excitatory toxicity. AQP4-/- mice exhibited increased cortical lesion area, aggravated neuron degeneration, and increased BBB disruption after NMDA microinjection. AQP4 may play a protective role in NMDA-induced brain injury in mice.4. Taking together, AQP4 deficiency induces astrocyte dysfunction and reduces the ability to limit inflammation or other lesions; this may be one of causes for aggravated brain injury in AQP4-deficent mice. In other words, AQP4 is an important factor for maintaining astrocyte function limiting injuries.