Effects of different dietary protein levels on intestinal aquaporins in weaned piglets.

This study was conducted to investigate the relationship between changes in intestinal aquaporins (AQPs) in piglets fed diets with different protein levels and nutritional diarrhoea in piglets. Briefly, 96 weaned piglets were randomly divided into four groups fed diets with crude protein (CP) levels of 18%, 20%, 22% and 24%. The small intestines and colons of the weaned piglets were collected, and several experiments were conducted. In the small intestine, AQP4 protein expression was higher in weaned piglets fed the higher-CP diets (22% and 24% CP) than in those fed the 20% CP diet except at 72 h (p < 0.01). At 72 h, the AQP4 protein expression in the small intestine was lower in the 18% group than in the other three groups (p < 0.01). Under 20% CP feeding, AQP2, AQP4 and AQP9 protein expression in the colons of piglets peaked at certain time points. The AQP2 and AQP4 mRNA levels in the colon and the AQP4 and AQP4 mRNA levels in the distal colon were approximately consistent with the protein expression levels. However, the AQP9 mRNA content in the colon was highest in the 18% group, and the AQP2 mRNA content in the distal colon was significantly higher in the 24% group than in the 20% group. AQP2 and AQP4 were expressed mainly around columnar cells in the upper part of the smooth colonic intestinal villi, and AQP9 was expressed mainly on columnar cells and goblet cells in the colonic mucosa. In conclusion, 20% CP is beneficial to the normal expression of AQP4 in the small intestine, AQP2, AQP4 and AQP9 in the colon of weaned piglets, which in turn maintains the balance of intestinal water absorption and secretion in piglets.

Acutely increased aquaporin-4 exhibits more potent protective effects in the cortex against single and repeated isoflurane-induced neurotoxicity in the developing rat brain.

Damage to hippocampus, cerebellum, and cortex associated with cognitive functions due to anesthetic-induced toxicity early in life may cause cognitive decline later. Aquaporin 4 (AQP4), a key protein in waste clearance pathway of brain, is involved in synaptic plasticity and neurocognition. We investigated the effects of single and repeated isoflurane (Iso) anesthesia on AQP4 levels and brain damage. Postnatal-day (P)7 Wistar albino rats were randomly assigned to Iso or Control (C) groups. For single-exposure, pups were exposed to 1.5% Iso in 30% oxygenated-air for 3-h at P7 (Iso1). For repeated-exposure, pups were exposed to Iso for 3 days, 3-h each day, at 1-day intervals (P7 + 9 + 11) starting at P7 (Iso3). C1 and C3 groups received only 30% oxygenated-air. Based on HE-staining and immunoblotting (Bax/Bcl-2, cleaved-caspase3 and PARP1) analyses, Iso exposures caused a higher degree of apoptosis in hippocampus. Anesthesia increased 4-hydroxynonenal (4HNE), oxidative stress marker; the highest ROS accumulation was determined in cerebellum. Increased inflammation (TNF-α, NF-κB) was detected. Multiple Iso-exposures caused more significant damage than single exposure. Moreover, 4HNE and TNF-α contributed synergistically to Iso-induced neurotoxicity. After anesthesia, higher expression of AQP4 was detected in cortex than hippocampus and cerebellum. There was an inverse correlation between increased AQP4 levels and apoptosis/ROS/inflammation. Correlation analysis indicated that AQP4 had a more substantial protective profile against oxidative stress than apoptosis. Remarkably, acutely increased AQP4 against Iso exhibited a more potent neuroprotective effect in cortex, especially frontal cortex. These findings promote further research to understand better the mechanisms underlying anesthesia-induced toxicity in the developing brain.

(-)-Epicatechin gallate prevents inflammatory response in hypoxia-activated microglia and cerebral edema by inhibiting NF-κB signaling.

High-altitude cerebral edema (HACE), a potentially lethal disease, is associated with a time-dependent exposure to altitude-related hypobaric hypoxia (HH) and has reportedly been associated with microglia hyperactivation. Catechins are substances with good antioxidant properties, among which (-)-epigallocatechin gallate (EGCG) may play a neuroprotective role through the inhibition of microglia overactivation; however, the function of its analog- (-)-epicatechin gallate (ECG)-requires further elucidation. The aim of the present study was to investigate whether ECG prevented HACE by inhibiting HH-activated microglia. Primary microglia exposed to lipopolysaccharide (LPS)/ATP were co-treated with EGCG, ECG, and (-)-epigallocatechin, and ECG and EGCG exerted significant anti-inflammatory and neuroprotective effects. ECG inhibited the NF-κB pathway to prevent the activation of microglia induced by 1% O2. In addition, ECG ameliorated the increase in brain water content and aquaporin 4 expression induced by HH in mice. ECG also reduced the number of Iba1+ microglia in the brain, the release of proinflammatory factors, and the recruitment of microglia to blood vessels in HH-exposed mice. The outcomes of the present study revealed that ECG alleviated hypoxic hyperactivated microglia, reduced the neuroinflammation and blood-brain barrier permeability, and prevented HACE by inhibiting NF-κB signaling.

Sulforaphane Upregulates Cultured Mouse Astrocytic Aquaporin-4 Expression through p38 MAPK Pathway.

Protein misfolding and/or aggregation are common pathological features associated with a number of neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson disease (PD). Abnormal protein aggregation may be caused by misfolding of the protein and/or dysfunction of the protein clearance system. Recent studies have demonstrated that the specific water channel protein, aquaporin-4 (AQP4), plays a role in the pathogenesis of neurodegenerative diseases involving protein clearance system. In this study, we aimed to investigate the role of sulforaphane (SFN) in the upregulation of AQP4 expression, along with its underlying mechanism using cultured mouse astrocytes as a model system. At low concentrations, SFN was found to increase cell proliferation and result in the activation of astrocytes. However, high SFN concentrations were found to suppress cell proliferation of astrocytes. In addition, our study found that a 1 µM concentration of SFN resulted in the upregulation of AQP4 expression and p38 MAPK phosphorylation in cultured mouse astrocytes. Moreover, we demonstrated that the upregulation of AQP4 expression was significantly attenuated when cells were pretreated with SB203580, a p38 MAPK inhibitor. In conclusion, our findings from this study revealed that SFN exerts hormesis effect on cultured mouse astrocytes and can upregulate astrocytic AQP4 expression by targeting the p38 MAPK pathway.

Aquaporin-4 Dysregulation in a Controlled Cortical Impact Injury Model of Posttraumatic Epilepsy.

Posttraumatic epilepsy (PTE) is a long-term negative consequence of traumatic brain injury (TBI) in which recurrent spontaneous seizures occur after the initial head injury. PTE develops over an undefined period during which circuitry reorganization in the brain causes permanent hyperexcitability. The pathophysiology by which trauma leads to spontaneous seizures is unknown and clinically relevant models of PTE are key to understanding the molecular and cellular mechanisms underlying the development of PTE. In the present study, we used the controlled-cortical impact (CCI) injury model of TBI to induce PTE in mice and to characterize changes in aquaporin-4 (AQP4) expression. A moderate-severe TBI was induced in the right frontal cortex and video-electroencephalographic (vEEG) recordings were performed in the ipsilateral hippocampus to monitor for spontaneous seizures at 14, 30, 60, and 90 days post injury (dpi). The percentage of mice that developed PTE were 13%, 20%, 27%, and 14% at 14, 30, 60, and 90 dpi, respectively. We found a significant increase in AQP4 in the ipsilateral frontal cortex and hippocampus of mice that developed PTE compared to those that did not develop PTE. Interestingly, AQP4 was found to be mislocalized away from the perivascular endfeet and towards the neuropil in mice that developed PTE. Here, we report for the first time, AQP4 dysregulation in a model of PTE which may carry significant implications for epileptogenesis after TBI.

[The role of aquaporin 4 antibody in the injury of retinal microstructure in neuromyelitis optica spectrum disorders].

Objective: To evaluate the injury of retinal microstructure using optical coherence tomography (OCT) and investigate the role of aquaporin 4 antibody (AQP4 Ab) in this injury process. Methods: Forty patients with neuromyelitis optica spectrum disorders (NMOSD) were retrospectively studied, each of whom reported at least one episode of optic neuritis (ON), namely 59 ON eyes involved in all. All patients were divided into two subgroups based on AQP4 Ab tests including 25 patients (37 ON eyes) with AQP4 positive (Ab(+)/NMOSD group) and 15 patients (22 ON eyes) negative (Ab(-)/NMOSD group). In addition, 10 healthy controls (20 eyes) matched for age and sex (HC group) were analyzed. Spectral domain optical coherence tomography (SD-OCT) was used to quantify peripapillary retinal nerve fiber layer (RNFL). Nonparametric test was used to compare differences between groups. Results: Age distribution and gender ratio were comparable in three groups (P>0.05). Visual acuity in ON eyes of Ab(+)/NMOSD group was worse than that of Ab(-)/NMOSD group (P=0.02). There were no significant differences between Ab(+)/NMOSD and Ab(-)/NMOSD in aspects of disease duration (2.6 vs. 1.9 year), ON episodes (2 vs. 1), longitudinal extensive transverse myelitis (LETM) ratio (48.0% vs. 66.7%), NMOSD specific intracranial lesions ratio (32.0% vs. 53.3%), positive autoimmune antibody ratio (52.0% vs. 20.0%) (P= 0.13, 0.08, 0.25, 0.18, 0.06, respectively). The thickness of temporal, superior, nasal, inferior and average RNFL in ON eyes of both Ab(+)/NMOSD and Ab(-)/NMOSD group were thinner than those in eyes of HC group (all P<0.05). The thickness of superior and inferior RNFL in ON eyes of Ab(+)/NMOSD were 61.0 µm and 62.0 µm, which was thinner than those of Ab(-)/NMOSD 94.5 µm and 97.0 µm (P=0.03 and 0.01, respectively). Conclusions: RNFL reflects the injury of retinal microstructure in NMOSD patients. AQP4 Ab seropositivity is correlated to the severity of RNFL damage, implying the potential role of AQP4 Ab in this pathological process.

Improved long-term outcome after transient cerebral ischemia in aquaporin-4 knockout mice.

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.

Injection of neural progenitor cells attenuates decrease in level of connexin 43 in brain capillaries after cerebral ischemia.

Although functional disruption of the cerebrovasculature, which is called the "neurovascular unit (NVU)", may lead to amplification of ischemia-induced injury, changes in the gap junctional proteins within the NVU and their pathophysiological roles after brain injury remain controversial. We previously demonstrated that the intravenous injection of neural progenitor cells (NPCs) have therapeutic potential for improving the spatial learning dysfunction and depression-like behaviors observed after cerebral ischemia. In this study, we investigated whether severe cerebral ischemia would alter the expression of gap junctional proteins in isolated brain capillaries and examined the effect of intravenous injection of NPCs on the levels of these proteins. Cerebral ischemia induced a sustained decrease in the level of the gap junctional protein connexin 43 (Cx43) in the isolated brain capillaries, whereas the level of aquaporin 4 (AQP-4) was transiently increased. The injection of NPCs increased the level of Cx43 compared that of vehicle in the microsphere embolism (ME) rats, suggesting this decrease to be a possible mechanism for disruption of the astrocyte-endothelial cell interface within the NVU without causing any changes in the level of AQP-4 and N-cadherin. We also demonstrated that some of the intravenously injected NPCs migrated into the blood vessels in the peri-infarct area. These results suggest that the intravenous injection of the NPCs would remodel the NVU after severe cerebral ischemia, which remodeling might be associated with functional improvement following the NPC injection.