AQP8 Modulates Mitochondrial H2O2 Transport to Influence Glioma Proliferation.

BACKGROUND: Aquaporin-8 (AQP8) is involved in impacting glioma proliferation and can effect tumour growth by regulating Intracellular reactive oxygen species (ROS) signalling levels. In addition to transporting H2O2, AQP8 has been shown to affect ROS signaling, but evidence is lacking in gliomas. In this study, we aimed to investigate how AQP8 affects ROS signaling in gliomas. MATERIALS AND METHODS: We constructed A172 and U251 cell lines with AQP8 knockdown and AQP8 rescue by CRISPR/Cas9 technology and overexpression of lentiviral vectors. We used CCK-8 and flow cytometry to test cell proliferation and cycle, immunofluorescence and Mito-Tracker CMXRos to observe the distribution of AQP8 expression in glioma cells, Amplex and DHE to study mitochondria release of H2O2, mitochondrial membrane potential (MMP) and NAD+/NADH ratio to assess mitochondrial function and protein blotting to detect p53 and p21 expression. RESULT: We found that AQP8 co-localised with mitochondria and that knockdown of AQP8 inhibited the release of H2O2 from mitochondria and led to increased levels of ROS in mitochondria, thereby impairing mitochondrial function. We also discovered that AQP8 knockdown resulted in suppression of cell proliferation and was blocked at the G0/G1 phase with increased expression of mitochondrial ROS signalling-related p53/p21. CONCLUSIONS: This finding provides further evidence for mechanistic studies of AQP8 as a prospective target for the treatment of gliomas.

The phosphokinase activity of IRE1ɑ prevents the oxidative stress injury through miR-25/Nox4 pathway after ICH.

BACKGROUND: Endoplasmic reticulum (ER) stress and oxidative stress are the major pathologies encountered after intracerebral hemorrhage (ICH). Inositol-requiring enzyme-1 alpha (IRE1α) is the most evolutionarily conserved ER stress sensor, which plays a role in monitoring and responding to the accumulation of unfolded/misfolded proteins in the ER lumen. Recent studies have shown that ER stress is profoundly related to oxidative stress in physiological or pathological conditions. The purpose of this study was to investigate the role of IRE1α in oxidative stress and the potential mechanism. METHODS: A mouse model of ICH was established by autologous blood injection. The IRE1α phosphokinase inhibitor KIRA6 was administrated intranasally at 1 h after ICH, antagomiR-25 and agomiR-25 were injected intraventricularly at 24 h before ICH. Western blot analysis, RT-qPCR, immunofluorescence staining, hematoma volume, neurobehavioral tests, dihydroethidium (DHE) staining, H2O2 content, brain water content, body weight, Hematoxylin and Eosin (HE) staining, Nissl staining, Morris Water Maze (MWM) and Elevated Plus Maze (EPM) were performed. RESULTS: Endogenous phosphorylated IRE1α (p-IRE1α), miR-25-3p, and Nox4 were increased in the ICH model. Administration of KIRA6 downregulated miR-25-3p expression, upregulated Nox4 expression, promoted the level of oxidative stress, increased hematoma volume, exacerbated brain edema and neurological deficits, reduced body weight, aggravated spatial learning and memory deficits, and increased anxiety levels. Then antagomiR-25 further upregulated the expression of Nox4, promoted the level of oxidative stress, increased hematoma volume, exacerbated brain edema and neurological deficits, whereas agomiR-25 reversed the effects promoted by KIRA6. CONCLUSION: The IRE1α phosphokinase activity is involved in the oxidative stress response through miR-25/Nox4 pathway in the mouse ICH brain.

Geranylgeranylacetone-induced heat shock protein70 expression reduces retinal ischemia-reperfusion injury through PI3K/AKT/mTOR signaling.

Retinal ischemia-reperfusion (I/R) injury is a common pathophysiological stress state connected to various diseases, including acute glaucoma, retinal vascular obstruction, and diabetic retinopathy. Recent studies have suggested that geranylgeranylacetone (GGA) could increase heat shock protein70 (HSP70) level and reduce retinal ganglion cells (RGCs) apoptosis in a rat retinal I/R model. However, the underlying mechanism remains unclear. Moreover, the injury caused by retinal I/R includes not only apoptosis but also autophagy and gliosis, and the effects of GGA on autophagy and gliosis have not been reported. Our study established a retinal I/R model by anterior chamber perfusion pressuring to 110 mmHg for 60 min, followed by 4 h of reperfusion. The levels of HSP70, apoptosis-related proteins, GFAP, LC3-II, and PI3K/AKT/mTOR signaling proteins were determined by western blotting and qPCR after treatment with GGA, HSP70 inhibitor quercetin (Q), PI3K inhibitor LY294002, and mTOR inhibitor rapamycin. Apoptosis was evaluated by TUNEL staining, meanwhile, HSP70 and LC3 were detected by immunofluorescence. Our results demonstrated that GGA-induced HSP70 expression significantly reduced gliosis, autophagosome accumulation, and apoptosis in retinal I/R injury, indicating that GGA exerted protective effects on retinal I/R injury. Moreover, the protective effects of GGA mechanistically relied on the activation of PI3K/AKT/mTOR signaling. In conclusion, GGA-induced HSP70 overexpression has protective effects on retinal I/R injury by activating PI3K/AKT/mTOR signaling.

Bumetanide Rescues Aquaporin-4 Depolarization via Suppressing ß-Dystroglycan Cleavage and Provides Neuroprotection in Rat Retinal Ischemia-Reperfusion Injury.

Aquaporin-4 (AQP4) regulates retinal water homeostasis and participates in retinal oedema pathophysiology. ß-dystroglycan (ß-DG) is responsible for AQP4 polarization and can be cleaved by matrix metalloproteinase-9 (MMP9). Retinal oedema induced by ischemia-reperfusion (I/R) injury is an early complication. Bumetanide (BU) has potential efficacy against cytotoxic oedema. Our study investigated the effects of ß-DG cleavage on AQP4 and the roles of BU in a rat retinal I/R injury model. The model was induced by applying 110 mm Hg intraocular pressure to the anterior eye chamber. BU and U0126 (a selective ERK inhibitor) were intraperitoneally administered 15 and 30 min, respectively, before I/R induction. Rhodamine isothiocyanate extravasation detection, quantitative real-time PCR, transmission electron microscopy, hematoxylin-eosin staining, immunofluorescence staining, western blotting, and TUNEL staining were performed. AQP4 lost its polarization in the retinal perivascular domain as a result of ß-DG cleavage. BU rescued AQP4 depolarization, suppressed AQP4 protein expression, attenuated retinal cytotoxic oedema, and downregulated ß-DG and AQP4 mRNA expression. BU suppressed glial responses and mitochondria-mediated apoptotic protein expression, including that of Caspase-3 and Cyto C, raised the Bcl-2/Bax ratio, and lowered the number of apoptotic cells in the retina. Both BU and U0126 downregulated p-ERK and MMP9 expression. Thus, BU treatment suppressed ß-DG cleavage, recovered AQP4 polarization partially via inhibiting ERK/MMP9 signaling pathway, and possess potential neuroprotective efficacy in the rat retinal ischemia-reperfusion injury model.

ER Stress is Involved in Mast Cells Degranulation via IRE1α/miR-125/Lyn Pathway in an Experimental Intracerebral Hemorrhage Mouse Model.

The degranulation of mast cells accounts for the development of neuroinflammation following intracerebral hemorrhage (ICH). Inhibition of IRE1α, a sensor signaling protein related to endoplasmic reticulum stress, has been shown to exert anti-inflammatory effects in several neurological diseases. The objective of this study was to investigate the effects of IRE1α inhibition on mast cells degranulation in an ICH mouse model and to explore the contribution of miR-125/Lyn pathway in IRE1α-mediated mast cells degranulation. Male mice were subjected to ICH by intraparenchymal injection of autologous blood. STF083010, an inhibitor of IRE1α, was administered intranasally at 1 h after ICH induction. AntimiR-125 was delivered by intracerebroventricular (i.c.v.) injection prior to ICH induction to elucidate the possible mechanisms. Western blot analysis, immunofluorescence staining, neurological test, hematoma volume, brain water content, toluidine blue staining and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) were performed. Endogenous phosphorylated IRE1α (p-IRE1α), tryptase, interleukin-17A (IL-17A), tumor necrosis factor α (TNF-α) and tryptase mRNA were increased in time dependent manner while miR-125b-2-3p was decreased after ICH. Inhibition of IRE1α, with STF083010, remarkably reduced brain water content, improved neurological function, decreased hematoma volume, upregulated the expression of miR-125b-2-3p, decreased the number of mast cells, and downregulated the protein expression of Lyn kinase, XBP1s (spliced X-box binding protein-1), tryptase, IL-17A and TNF-α. The downregulation of Lyn kinase, tryptase, IL-17A, TNF-α, and decreased mast cells number were reversed by antimiR-125. The present findings demonstrate that IRE1α inhibition attenuates mast cells degranulation and neuroinflammation, at least partially, through IRE1α/miR-125/Lyn signaling pathway after ICH.

Neuroglobin expression and function in the temporal cortex of bilirubin encephalopathy rats.

Bilirubin encephalopathy (BE) is a neurological syndrome in newborns, mainly caused by neuronal injury due to excessive oxidative stress produced by unconjugated bilirubin (UCB). Neuroglobin (NGB) can protect the brain by removing oxidative stress species, but its expression and significance in BE are not clear. To address this question, the neonatal BE model was established by injecting UCB into the cerebellomedullary cistern of 7-day-old SD rats. Rats were divided into a sham and BE 6 hr group, BE 12 hr group, BE 24 hr group, and BE 7 d group according to UCB action times. Hematoxylin/eosin and Nissl staining, and electron microscopy were employed to observe the pathological and ultrastructural changes of nerve cells in each group. Immunofluorescence staining was used to detect NGB expression sites and cell types. Western blotting and quantitative PCR served to detect NGB expression and test the mitochondrial apoptosis signal pathway. The results confirm that UCB can lead to pathological damage and ultrastructural changes in rats' temporal cortex, increasing the expression of apoptosis-related proteins Bax, Bcl-2, Cyt c, Caspase-3, and neuronal NGB. UCB promotes NGB expression with an increase in action time and reach a peak at 12 hr. In summary, brain damage induced by UCB will cause an increase in NGB expression, the increasing NGB can inhibit neuron apoptosis in early BE phases. Therefore, promoting the expression of endogenous NGB, to act as a neuroprotective agent may be a potential treatment strategy for BE.

Honokiol exerts protective effects on neural myelin sheaths after compressed spinal cord injury by inhibiting oligodendrocyte apoptosis through regulation of ER-mitochondrial interactions.

OBJECTIVE: To investigate the effect of honokiol on demyelination after compressed spinal cord injury (CSCI) and it's possible mechanism. DESIGN: Animal experiment study. SETTING: Institute of Neuroscience of Chongqing Medical University. INTERVENTIONS: Total of 69 Sprague-Dawley (SD) rats were randomly divided into 3 groups: sham group (n=15), honokiol group (n=27) and vehicle group (n=27). After established CSCI model by a custom-made compressor successfully, the rats of sham group were subjected to the limited laminectomy without compression; the rats of honokiol group were subjected to CSCI surgery and intraperitoneal injection of 20 mg/kg honokiol; the rats of vehicle group were subjected to CSCI surgery and intraperitoneal injection of an equivalent volume of saline.Outcome measures: The locomotor function of each group was assessed using the Basso, Beattie and Bresnahan (BBB) rating scale. The pathological changes of myelinated nerve fibers of spinal cord in 3 groups were detected by osmic acid staining and transmission electron microcopy (TME). Immunofluorescence and Western blot were used to research the experessions of active caspase-3, caspase-12, cytochrome C and myelin basic protein (MBP) respectively. RESULTS: In the vehicle group, the rats became paralyzed and spastic after injury, and the myelin sheath became swollen and broken down along with decreased number of myelinated nerve fibers. Western blot analysis manifested that active caspase-3, caspase-12 and cytochrome C began to increase 1 d after injury while the expression of MBP decreased gradually. After intervened with honokiol for 6 days, compared with the vehicle group, the locomotor function and the pathomorphological changes of myelin sheath of the CSCD rats were improved with obviously decreased expression of active caspase-3, caspase-12 and cytochrome C. CONCLUSIONS: Honokiol may improve locomotor function and protect neural myelin sheat from demyelination via prevention oligodendrocytes (OLs) apoptosis through mediate endoplasmic reticulum (ER)-mitochondria pathway after CSCI.

Small Interfering RNA Targeting DMP1 Protects Mice Against Blood-Brain Barrier Disruption and Brain Injury After Intracerebral Hemorrhage.

Dentin matrix protein 1 (DMP1) is an extracellular matrix phosphoprotein that is known to facilitate mineralization of collagen in bone and promote osteoblast/odontoblast differentiation. Blood-brain barrier (BBB) disruption is the major pathogenesis in secondary brain injury after intracerebral hemorrhage (ICH). This study aimed to investigate the expression pattern of DMP1 in the mouse brain and explore the role of DMP1 in BBB disruption and brain injury in a mouse model of ICH. Mice were subjected to autologous blood injection-induced ICH. Immunofluorescence staining, western blot analysis, neurobehavioral tests, brain water content measurements, Evans blue permeability assay, and transmission electron microscopy were performed. Small interfering RNA targeting DMP1 (DMP1 siRNA) was administered at 72 h prior to ICH. Results showed that DMP1 is expressed extensively in the mouse brain, and is upregulated in the ICH model. Administration of DMP1 siRNA effectively ameliorated BBB disruption, attenuated brain edema, and improved neurological function after ICH. Moreover, the expression of zonula occludens-1 (ZO-1) and occludin were upregulated, and matrix metalloproteinase-9 (MMP-9) was downregulated in the ICH model. DMP1 siRNA administration reversed the expression of ZO-1, occludin, and MMP-9. These results demonstrated that DMP1 upregulation plays an essential role in inducing BBB disruption and brain injury after ICH. The inhibition of DMP1 could be a potential therapeutic strategy for ICH treatment.

Changes in the prefrontal cortex after the hippocampus was injected with Aß25-35 via the P35/P25-CDK5-Tau hyperphosphorylation signaling pathway.

Alzheimer's disease (AD) is one of the common neurodegenerative illnesses in aging populations around the world. Recently, psychiatric symptoms are becoming increasingly important in recognizing the manifestations of AD in addition to cognitive impairment. Some studies suggest that the prefrontal cortex (PFC) is closely related to apathy/depression, and a network may exist between the CA1 of hippocampus and PFC. However, whether the injection of Aß2535 into hippocampi may result in PFC abnormalities in AD model rats is unclear. In this study, it was investigated the changes in the PFCs after the hippocampal injection via the P35/P25 - Cyclin-dependent kinase5 (CDK5) - Tau hyperphosphorylation signaling pathway. Our results demonstrated that rats injected with Aß25-35 showed decreased learning and memory ability, and increased depression-like behaviors compared with uninjected controls and saline-injected shams. P35/P25, CDK5, Tau[pS199], and Tau[pS202] are significantly elevated in the PFCs and hippocampi after Aß25-35 was injected into the hippocampi. Furthermore, P35/P25-CDK5 complexes were detected in vivo by immunofluorescence and co-immunoprecipitation. Therefore, the relative expression of proteins associated with the P35/P25-CDK5 pathway showed the same changes in the hippocampi and PFCs after Aß25-35 injection. These findings demonstrate a potential mechanism for prefrontal-mediated cognitive impairment and the psychiatric symptoms of AD.

Poldip2 mediates blood-brain barrier disruption and cerebral edema by inducing AQP4 polarity loss in mouse bacterial meningitis model.

BACKGROUND: Specific highly polarized aquaporin-4 (AQP4) expression is reported to play a crucial role in blood-brain barrier (BBB) integrity and brain water transport balance. The upregulation of polymerase δ-interacting protein 2 (Poldip2) was involved in aggravating BBB disruption following ischemic stroke. This study aimed to investigate whether Poldip2-mediated BBB disruption and cerebral edema formation in mouse bacterial meningitis (BM) model occur via induction of AQP4 polarity loss. METHODS AND RESULTS: Mouse BM model was induced by injecting mice with group B hemolytic streptococci via posterior cistern. Recombinant human Poldip2 (rh-Poldip2) was administered intranasally at 1 hour after BM induction. Small interfering ribonucleic acid (siRNA) targeting Poldip2 was administered by intracerebroventricular (i.c.v) injection at 48 hours before BM induction. A specific inhibitor of matrix metalloproteinases (MMPs), UK383367, was administered intravenously at 0.5 hour before BM induction. Western blotting, immunofluorescence staining, quantitative real-time PCR, neurobehavioral test, brain water content test, Evans blue (EB) permeability assay, transmission electron microscopy (TEM), and gelatin zymography were carried out. The results showed that Poldip2 was upregulated and AQP4 polarity was lost in mouse BM model. Both Poldip2 siRNA and UK383367 improved neurobehavioral outcomes, alleviated brain edema, preserved the integrity of BBB, and relieved the loss of AQP4 polarity in BM model. Rh-Poldip2 upregulated the expression of MMPs and glial fibrillary acidic protein (GFAP) and downregulated the expression of ß-dystroglycan (ß-DG), zonula occludens-1 (ZO-1), occludin, and claudin-5; whereas Poldip2 siRNA downregulated the expression of MMPs and GFAP, and upregulated ß-DG, ZO-1, occludin, and claudin-5. Similarly, UK383367 downregulated the expression of GFAP and upregulated the expression of ß-DG, ZO-1, occludin, and claudin-5. CONCLUSION: Poldip2 inhibition alleviated brain edema and preserved the integrity of BBB partially by relieving the loss of AQP4 polarity via MMPs/ß-DG pathway.

Role of rno-miR-124-3p in regulating MCT1 expression in rat brain after permanent focal cerebral ischemia.

This study aimed to assess the role of microRNAs (miRNAs) in regulating monocarboxylate transporter-1 (MCT1) expression in rat brain after permanent focal cerebral ischemia to identify a new target for early treatment of cerebral ischemia. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO) in rats. Morphology and protein expression levels of MCT1 were assessed by immunofluorescence and Western blotting. Using bioinformatics and double luciferase reporter assays, rno-miR-124-3p was selected as a direct target for rat MCT1. Expression of rno-miR-124-3p after pMCAO was detected. Then, rats were treated with rno-miR-124-3p agomir via lateral ventricle injection, and after 6 h or 24 h ischemia, rno-miR-124-3p expression and gene and protein expression of MCT-1 were detected by qRT-PCR and Western blotting. Brain infarction was identified by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Results showed that pMCAO induced brain infarction and increased the expression of MCT1. The levels of rno-miR-124-3p after pMCAO were in contrast to those of MCT1 protein in ischemic region, while declined after 3, 6 and 12 h of pMCAO in ischemic penumbra. After administration of rno-miR-124-3p agomir, MCT1 mRNA and protein levels were increased after 6 h of pMCAO, while decreased after 24 h of pMCAO. Meanwhile, rno-miR-124-3p levels increased after both times. TTC staining showed treatment with rno-miR-124-3p agomir reduced brain infarction. The role of rno-miR-124-3p in regulating MCT1 was as a positive regulator after 6 h of pMCAO, while a negative regulator after 24 h of pMCAO, however, both activities had protective effects against cerebral ischemia.

Curcumin Ameliorates Memory Deficits by Enhancing Lactate Content and MCT2 Expression in APP/PS1 Transgenic Mouse Model of Alzheimer's Disease.

Curcumin is a natural product with several anti-Alzheimer's disease (AD) neuroprotective properties. This study aimed to investigate the effects of curcumin on memory deficits, lactate content, and monocarboxylate transporter 2 (MCT2) in APP/PS1 mouse model of AD. APP/PS1 transgenic mice and wild-type (WT) C57BL/6J mice were used in the present study. Spatial learning and memory of the mice was detected using Morris water-maze test. Cerebral cortex and hippocampus lactate contents were detected using lactate assay. MCT2 expression in the cerebral cortex and hippocampus was examined by immunohistochemistry and Western blotting. Results showed that spatial learning and memory deficits were improved in curcumin-treated APP/PS1 mouse group compared with those in APP/PS1 mice group. Brain lactate content and MCT2 protein level were increased in curcumin-treated APP/PS1 mice than in APP/PS1 mice. In summary, our findings indicate that curcumin could ameliorate memory impairments in APP/PS1 mouse model of AD. This phenomenon may be at least partially due to its improving effect on the lactate content and MCT2 protein expression in the brain. Anat Rec, 302:332-338, 2019. © 2018 Wiley Periodicals, Inc.

p53-Mediated oligodendrocyte apoptosis initiates demyelination after compressed spinal cord injury by enhancing ER-mitochondria interaction and E2F1 expression.

Oligodendrocyte apoptosis mediated demyelination is a pathological change characteristic of compressed spinal cord injury (CSCI). However, the mechanism of demyelination due to oligodendrocyte apoptosis is not known. In this study, after successfully establishing a rat CSCI model using a custom-made compressor, we investigated the pathological changes, MBP expression, as well as apoptosis-related protein (p53, active caspase-3) expression to determine whether or not apoptosis and demyelination occurred after injury. To understand the possible mechanism of oligodendrocyte apoptosis, caspase-12 and cytochrome C were analyzed to explore the relationship between oligodendrocyte apoptosis and endoplasmic reticulum(ER)-mitochondria interaction. The transcription factor, E2F1, was also detected by immunofluorescence and Western blot assays. The results showed that CSCI increased the expression levels of p53, E2F1 and active caspase-3 followed by the swelling and breakdown of myelin sheaths. The number of myelinated nerve fibers also decreased with down-regulated expression of MBP. Expression levels of caspase-12 and cytochrome C were enhanced along with a reduction in the number of oligodendrocytes. After treatment of CSCI in rats with Pifithrin-µ(PFT-µ), a specific inhibitor of p53, pathomorphological changes of myelin sheath improved significantly. Expression levels of E2F1, active caspase-3, caspase-12 and cytochrome C were down-regulated, consistent with reduced the number of apoptotic oligodendrocytes. These results demonstrated that over-expression of p53 could mediate oligodendrocyte apoptosis thus resulting in demyelination in two ways; by enhancing ER-mitochondria interaction and by triggering the E2F1 mediated apoptosis pathway.

LYTAK1, a TAK1 inhibitor, suppresses proliferation and epithelial­mesenchymal transition in retinal pigment epithelium cells.

The proliferation of retinal pigment epithelium (RPE) cells following epithelial­mesenchymal transition (EMT) is critical in proliferative vitreoretinopathy (PVR), which results in retinal detachment and the loss of vision. The current study was conducted to examine the importance of transforming growth factor ß­1 (TGF­ß1)­activated kinase 1 (TAK1) inhibitor (LYTAK1) in regulating EMT and the proliferation of RPE cells. RPE cells were pre-treated with increasing concentrations of LYTAK1 prior to treatment with TGF­ß1 for 24 h. The effect of LYTAK1 on RPE cell proliferation was examined using a Cell Counting kit­8 assay. The expression levels of TAK1, smooth muscle actin, fibronectin, p-Smad2, p-Smad3, nuclear factor (NF)-κB p65 and IκB kinase α were detected by western blotting. LYTAK1 suppressed the proliferation and migration of RPE cells. Additionally, LYTAK1 significantly prevented TGF­ß1­induced EMT by decreasing the levels of fibronectin and α­smooth muscle actin. It was demonstrated that the effects of LYTAK1 were via the Smad signaling pathway. The present study also determined, that the underlying mechanism of the effects of LYTAK1 on EMT in RPE cells involves downregulation of the NF­κB signaling pathway. In conclusion, TAK1 transcription factor was shown to be important in TGF­ß1­induced EMT in human RPE cells. Thus, the results of this study aid in elucidating the pathogenesis of human PVR. In addition, this study suggests that specific inhibition by LYTAK1 may provide a novel approach for the treatment and prevention of PVR.

Changes in lactate content and monocarboxylate transporter 2 expression in Aß25₋35-treated rat model of Alzheimer's disease.

Decreased brain energy metabolism is correlated with cognitive impairment in Alzheimer's disease (AD). Accumulating evidence indicates that lactate and monocarboxylate transporters (MCTs) participate in brain energy metabolism. To date, changes in lactate level and expression of MCTs in AD remain unclear. This study was conducted to detect the changes in lactate content and expression of MCT2 in Aß25-35-treated rat model of AD. Sprague-Dawley rats were randomly divided into control and model groups, which received bilateral intrahippocampal injections of saline and Aß25-35, respectively. Cognitive functions were detected by Morris water-maze test. Lactate content in the cerebral cortex and hippocampus was measured by absorbance assay. The MCT2 level in the brain was examined by immunohistochemistry and Western blot. Morris water-maze test showed that the model group exhibited impaired learning and memory compared with the control group. Lactate content in the cerebral cortex and hippocampus was decreased in the model group compared with that in the control group. Immunohistochemistry and Western blot showed that the expression of MCT2 in the model group significantly decreased compared with that in the control group. Results indicate that decreased lactate content and downregulated MCT2 expression in the cerebral cortex and hippocampus reflected impaired energy metabolism in the brain, which may participate in the pathologic progression of AD.

Internalization of aquaporin-4 after collagenase-induced intracerebral hemorrhage.

Brain edema formation following intracerebral hemorrhage (ICH) appears to be related with aquaporin-4 (AQP4), which is critically involved in brain volume homeostasis and water balance. Despite its importance, the regulation of AQP4 expression involved in transmembrane water movements still remains rudimentary. Many studies suggest that the internalization of several membrane-bound proteins, including AQP4, may occur with or without lysosomal degradation. Previously, we investigated the internalization of AQP4 in retinal ischemic-reperfusion model. Here, we test the hypothesis that AQP4 is internalized post-ICH and then degraded in the lysosome. The results demonstrated that both AQP4 and the mannose-6-phosphate receptor (MPR) co-localized in perihematomal region at 6 hr post-ICH. In addition, AQP4 and lysosomal-associated membrane protein 1 (LAMP1) also co-localized in perihematomal region, with co-expression increasing followed by a gradual decrease at different time windows post-ICH (6, 12, 24, 48, and 72 hr). After ICH, the Evans blue leakage happened very early at 1 hr and the brain swelling occurred at 3 hr. Moreover, we also found the AQP4 mRNA and AQP4 protein were increased post-ICH. These results suggest that AQP4 is internalized and the lysosome is involved in degrading the internalized AQP4 post-ICH. Both the AQP4 internalization and lysosomal degradation may provide biophysical insights regarding the potential of new treatments for brain edema.

An applied anatomical study on the external laryngeal nerve loop and the superior thyroid artery in the neck surgical region.

This study was conducted to investigate the topographic relationship between the external laryngeal nerve (ELN) loop and the superior thyroid artery (STA), in order to provide the anatomical foundations for protecting the ELN during surgery. In the present study, 48 adult human cadavers were dissected and analyzed. For the 21 (21.9%) low-position ELN loops observed, the neurovascular relationship between the STA and the nerve was classified into four types: (1) the artery overlapped the nerve; (2) the artery passed through the ELN loop; (3) the muscular branch of the ELN loop and the laryngeal branch of the STA coursed together; and (4) the branches of the STA and the ELN loop were interlaced. Our study suggested that the patterns of ELN loops are so complicated that they have not been statistically defined in any previous study, which should be kept in mind when attempting to protect the nerve from injury. Also, because of the variable morphology of the ELN loop and its complicated topographic relationship to the STA, the vessels should be individually isolated and then ligated during thyroidectomy. When ligating the laryngeal branch of the STA during larynx surgery, special attention should be paid to avoiding damage to the muscular branch of the ELN/ELN loop.

Expression of aquaporin8 in human astrocytomas: correlation with pathologic grade.

Aquaporin8 (AQP8), a member of the aquaporin (AQP) protein family, is weakly distributed in mammalian brains. Previous studies on AQP8 have focused mainly on the digestive and the reproductive systems. AQP8 has a pivotal role in keeping the fluid and electrolyte balance. In this study, we investigated the expression changes of AQP8 in 75 cases of human brain astrocytic tumors using immunohistochemistry, Western blotting, and reverse transcription polymerase chain reaction. The results demonstrated that AQP8 was mainly distributed in the cytoplasm of astrocytoma cells. The expression levels and immunoreactive score of AQP8 protein and mRNA increased in low-grade astrocytomas, and further increased in high-grade astrocytomas, especially in glioblastoma. Therefore, AQP8 may contribute to the proliferation of astrocytomas, and may be a biomarker and candidate therapy target for patients with astrocytomas.

Lysosomal degradation of retinal glial AQP4 following its internalization induced by acute ocular hypertension.

The membrane-bound water channel aquaporin-4 plays a significant role in the regulation of water movement within the retina. In retinal ischemia-reperfusion injury, changes in the expression and localization of aquaporin-4 have been reported. Previous studies also suggest that the internalization of several membrane-bound proteins, including aquaporin-4, may occur with or without lysosomal degradation. In this study, the internalization of aquaporin-4 was detected in the ischemic rat retina via double immunofluorescence labeling. Specifically, both aquaporin-4 and the mannose-6-phosphate receptor co-localized post-ischemic injury (10, 30 and 60 min). The same results were found during a 12-h reperfusion window (2, 4 and 8 h, respectively) following 60 min of ischemia. Moreover, the co-expression of aquaporin-4 and lysosomal-associated membrane protein-1 was observed at 1-12 h of reperfusion, with co-expression increasing followed by a gradual decrease. These combined findings suggest that AQP4 is internalized in the ischemic-reperfused retina, and the lysosome is involved in degrading the internalized aquaporin-4 during the reperfusion phase. Both the internalization of aquaporin-4 and its lysosomal degradation may serve as valuable therapeutic targets for managing ischemic-reperfused retinal injury.