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.

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.

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.

IRE1α inhibition attenuates neuronal pyroptosis via miR-125/NLRP1 pathway in a neonatal hypoxic-ischemic encephalopathy rat model.

BACKGROUND: Inhibition of inositol-requiring enzyme-1 alpha (IRE1α), one of the sensor signaling proteins associated with endoplasmic reticulum (ER) stress, has been shown to alleviate brain injury and improve neurological behavior in a neonatal hypoxic-ischemic encephalopathy (HIE) rat model. However, there is no information about the role of IRE1α inhibitor as well as its molecular mechanisms in preventing neuronal pyroptosis induced by NLRP1 (NOD-, LRR- and pyrin domain-containing 1) inflammasome. In the present study, we hypothesized that IRE1α can degrade microRNA-125-b-2-3p (miR-125-b-2-3p) and activate NLRP1/caspased-1 pathway, and subsequently promote neuronal pyroptosis in HIE rat model. METHODS: Ten-day old unsexed rat pups were subjected to hypoxia-ischemia (HI) injury, and the inhibitor of IRE1α, STF083010, was administered intranasally at 1 h after HI induction. AntimiR-125 or NLRP1 activation CRISPR was administered by intracerebroventricular (i.c.v) injection at 24 h before HI induction. Immunofluorescence staining, western blot analysis, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), brain infarct volume measurement, neurological function tests, and Fluoro-Jade C staining were performed. RESULTS: Endogenous phosphorylated IRE1α (p-IRE1α), NLRP1, cleaved caspase-1, interleukin-1ß (IL-1ß), and interleukin-18 (IL-18) were increased and miR-125-b-2-3p was decreased in HIE rat model. STF083010 administration significantly upregulated the expression of miR-125-b-2-3p, reduced the infarct volume, improved neurobehavioral outcomes and downregulated the protein expression of NLRP1, cleaved caspase-1, IL-1ß and IL-18. The protective effects of STF083010 were reversed by antimiR-125 or NLRP1 activation CRISPR. CONCLUSIONS: IRE1α inhibitor, STF083010, reduced neuronal pyroptosis at least in part via miR-125/NLRP1/caspase-1 signaling pathway after HI.

Rh-relaxin-2 attenuates degranulation of mast cells by inhibiting NF-κB through PI3K-AKT/TNFAIP3 pathway in an experimental germinal matrix hemorrhage rat model.

BACKGROUND: Mast cells play an important role in early immune reactions in the brain by degranulation and the consequent inflammatory response. Our aim of the study is to investigate the effects of rh-relaxin-2 on mast cells and the underlying mechanisms in a germinal matrix hemorrhage (GMH) rat model. METHODS: One hundred seventy-three P7 rat pups were subjected to GMH by an intraparenchymal injection of bacterial collagenase. Clodronate liposome was administered through intracerebroventricular (i.c.v.) injections 24 h prior to GMH to inhibit microglia. Rh-relaxin-2 was administered intraperitoneally at 1 h and 13 h after GMH. Small interfering RNA of RXFP1 and PI3K inhibitor LY294002 were given by i.c.v. injection. Post-GMH evaluation included neurobehavioral function, Western blot analysis, immunofluorescence, Nissl staining, and toluidine blue staining. RESULTS: Our results demonstrated that endogenous relaxin-2 was downregulated and that RXFP1 level peaked on the first day after GMH. Administration of rh-relaxin-2 improved neurological functions, attenuated degranulation of mast cells and neuroinflammation, and ameliorated post-hemorrhagic hydrocephalus (PHH) after GMH. These effects were associated with RXFP1 activation, increased expression of PI3K, phosphorylated AKT and TNFAIP3, and decreased levels of phosphorylated NF-κB, tryptase, chymase, IL-6, and TNF-α. However, knockdown of RXFP1 and PI3K inhibition abolished the protective effects of rh-relaxin-2. CONCLUSIONS: Our findings showed that rh-relaxin-2 attenuated degranulation of mast cells and neuroinflammation, improved neurological outcomes, and ameliorated hydrocephalus after GMH through RXFP1/PI3K-AKT/TNFAIP3/NF-κB signaling pathway.

Both endoplasmic reticulum and mitochondrial pathways are involved in oligodendrocyte apoptosis induced by capsular hemorrhage.

OBJECTIVE: The white matter injury caused by intracerebral hemorrhage (ICH) includes demyelination and axonal injury. Oligodendrocyte apoptosis is reported to be involved in triggering demyelination. Experimental observations indicate that both endoplasmic reticulum and mitochondrial pathways could mediate cell apoptosis. The purpose of this study was to investigate the demyelination and the possible mechanisms in an autologous blood-injected rat model of internal capsule hemorrhage. METHODS: Transmission electron microscope was applied to examine the pathological changes of myelinated nerve fibers in internal capsule. Western blotting was used to detect the myelin basic protein (MBP) which was an important component of myelin sheath. Double immunofluorescence and Western blotting were used to determine the apoptosis and apoptotic pathways. The levels of caspase-12 (a representative protein of endoplasmic reticulum stress) and cytochrome c (an apoptosis factor released from mitochondria) were assessed in this study. RESULTS: Demyelination occurred on day 1, 3, and 7 after ICH onset. Myelin sheaths of internal capsule nerve fibers were swollen and broken down in ICH groups. MBP expression showed a downregulation after ICH with its minimum value occurred on day 7 post-ICH. Besides, neuron and oligodendrocyte apoptosis were observed at different time intervals post-ICH accompanied with an upregulated caspase-12 expression and enhanced cytochrome c release. CONCLUSIONS: These results suggested that oligodendrocyte and neuron apoptosis may contribute to the demyelination induced by internal capsule hemorrhage and oligodendrocyte apoptosis is positively mediated through both endoplasmic reticulum and mitochondrial pathways.

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.

GW0742 reduces mast cells degranulation and attenuates neurological impairments via PPARß/δ/CD300a/SHP1 pathway after GMH in neonatal rats.

BACKGROUND: Activation of mast cells plays an important role in brain inflammation. CD300a, an inhibitory receptor located on mast cell surfaces, has been reported to reduce the production of pro-inflammatory cytokines and exert protective effects in inflammation-related diseases. Peroxisome proliferator-activated receptor ß/δ (PPARß/δ), a ligand-activated nuclear receptor, activation upregulates the transcription of CD300a. In this study, we aim to investigate the role of PPARß/δ in the attenuation of germinal matrix hemorrhage (GMH)-induced mast cell activation via CD300a/SHP1 pathway. METHODS: GMH model was induced by intraparenchymal injection of bacterial collagenase into the right hemispheric ganglionic eminence in P7 Sprague Dawley rats. GW0742, a PPARß/δ agonist, was administered intranasally at 1 h post-ictus. CD300a small interfering RNA (siRNA) and PPARß/δ siRNA were injected intracerebroventricularly 5 days and 2 days before GMH induction. Behavioral tests, Western blot, immunofluorescence, Toluidine Blue staining, and Nissl staining were applied to assess post-GMH evaluation. RESULTS: Results demonstrated that endogenous protein levels of PPARß/δ and CD300a were decreased, whereas chymase, tryptase, IL-17A and transforming growth factor ß1 (TGF-ß1) were elevated after GMH. GMH induced significant short- and long-term neurobehavioral deficits in rat pups. GW0742 decreased mast cell degranulation, improved neurological outcomes, and attenuated ventriculomegaly after GMH. Additionally, GW0742 increased expression of PPARß/δ, CD300a and phosphorylation of SHP1, decreased phosphorylation of Syk, chymase, tryptase, IL-17A and TGF-ß1 levels. PPARß/δ siRNA and CD300a siRNA abolished the beneficial effects of GW0742. CONCLUSIONS: GW0742 inhibited mast cell-induced inflammation and improved neurobehavior after GMH, which is mediated by PPARß/δ/CD300a/SHP1 pathway. GW0742 may serve as a potential treatment to reduce brain injury for GMH patients.

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.

Edaravone Maintains AQP4 Polarity Via OS/MMP9/ß-DG Pathway in an Experimental Intracerebral Hemorrhage Mouse Model.

Oxidative stress (OS) is the main cause of secondary damage following intracerebral hemorrhage (ICH). The polarity expression of aquaporin-4 (AQP4) has been shown to be important in maintaining the homeostasis of water transport and preventing post-injury brain edema in various neurological disorders. This study primarily aimed to investigate the effect of the oxygen free radical scavenger, edaravone, on AQP4 polarity expression in an ICH mouse model and determine whether it involves in AQP4 polarity expression via the OS/MMP9/ß-dystroglycan (ß-DG) pathway. The ICH mouse model was established by autologous blood injection into the basal nucleus. Edaravone or the specific inhibitor of matrix metalloproteinase 9 (MMP9), MMP9-IN-1, called MMP9-inh was administered 10 min after ICH via intraperitoneal injection. ELISA detection, neurobehavioral tests, dihydroethidium staining (DHE staining), intracisternal tracer infusion, hematoxylin and eosin (HE) staining, immunofluorescence staining, western blotting, Evans blue (EB) permeability assay, and brain water content test were performed. The results showed that OS was exacerbated, AQP4 polarity was lost, drainage function of brain fluids was damaged, brain injury was aggravated, expression of AQP4, MMP9, and GFAP increased, while the expression of ß-DG decreased after ICH. Edaravone reduced OS, restored brain drainage function, reduced brain injury, and downregulated the expression of AQP4, MMP9. Both edaravone and MMP9-inh alleviated brain edema, maintained blood-brain barrier (BBB) integrity, mitigated the loss of AQP4 polarity, downregulated GFAP expression, and upregulated ß-DG expression. The current study suggests that edaravone can maintain AQP4 polarity expression by inhibiting the OS /MMP9/ß-DG pathway after ICH.

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.

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.

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.

Ghrelin attenuates oxidative stress and neuronal apoptosis via GHSR-1α/AMPK/Sirt1/PGC-1α/UCP2 pathway in a rat model of neonatal HIE.

Neuronal apoptosis induced by oxidative stress is one of the major pathological processes involved in neurological impairment after hypoxic-ischemic encephalopathy (HIE). Ghrelin, the unique endogenous ligand for the growth hormone secretagogue receptor-1α (GHSR-1α), could take an anti-apoptotic role in the brain. However, whether ghrelin can attenuate neuronal apoptosis by attenuating oxidative stress after hypoxia-ischemia (HI) insult remains unknown. To investigate the beneficial effects of ghrelin on oxidative stress injury and neuronal apoptosis induced by HI, ten-day old unsexed rat pups were subjected to HI injury and exogenous recombinant human ghrelin(rh-Ghrelin) was administered intranasally at 1 h and 24 h after HI induction. [D-Lys3]-GHRP-6, a selective inhibitor of GHSR-1α and Ex527, a selective inhibitor of GHSR-1α were administered intranasally at 1 h before HI induction respectively. Small interfering ribonucleic acid (siRNA) for GHSR-1α were administered by intracerebroventricular (i.c.v) injection at 24 h before HI induction. Neurological tests, immunofluorescence, MitoSox staining, Fluoro-Jade C staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and western blot experiments were performed. Our results indicated that ghrelin significantly improved neurobehavioral outcomes and reduced oxidative stress and neuronal apoptosis. Moreover, ghrelin treatment significantly promoted phosphorylation of AMPK, upregulated the expression of Sirt1, PGC-1α, UCP2 and the ratio of Bcl2/Bax, while it downregulated cleaved caspase-3 levels. The protective effects of ghrelin were reversed by [D-Lys3]-GHRP-6, GHSR-1α siRNA or Ex527. In conclusion, our data demonstrated that ghrelin reduced oxidative stress injury and neuronal apoptosis which was in part via the GHSR-1α/AMPK/Sirt1/PGC-1α/UCP2 signalling pathway after HI. Ghrelin may be a novel therapeutic target for treatment after neonatasl HI injury.

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.

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.

Protective Effect of Electroacupuncture on Neural Myelin Sheaths is Mediated via Promotion of Oligodendrocyte Proliferation and Inhibition of Oligodendrocyte Death After Compressed Spinal Cord Injury.

Electroacupuncture (EA) has been used worldwide to treat demyelinating diseases, but its therapeutic mechanism is poorly understood. In this study, a custom-designed model of compressed spinal cord injury (CSCI) was used to induce demyelination. Zusanli (ST36) and Taixi (KI3) acupoints of adult rats were stimulated by EA to demonstrate its protective effect. At 14 days after EA, both locomotor skills and ultrastructural features of myelin sheath were significantly improved. Phenotypes of proliferating cells were identified by double immunolabeling of 5-ethynyl-2'-deoxyuridine with antibodies to cell markers: NG2 [oligodendrocyte precursor cell (OPC) marker], 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) (oligodendrocyte marker), and glial fibrillary acidic protein (GFAP) (astrocyte marker). EA enhanced the proliferation of OPCs and CNPase, as well as the differentiation of OPCs by promoting Olig2 (the basic helix-loop-helix protein) and attenuating Id2 (the inhibitor of DNA binding 2). EA could also improve myelin basic protein (MBP) and protect existing oligodendrocytes from apoptosis by inhibiting caspase-12 (a representative of endoplasmic reticulum stress) and cytochrome c (an apoptotic factor and hallmark of mitochondria). Therefore, our results indicate that the protective effect of EA on neural myelin sheaths is mediated via promotion of oligodendrocyte proliferation and inhibition of oligodendrocyte death after CSCI.

Loss of AQP4 polarized localization with loss of ß-dystroglycan immunoreactivity may induce brain edema following intracerebral hemorrhage.

The aquaporin-4 (AQP4) water channel contributes to brain water homeostasis in perivascular and subpial membrane domains of astrocytes where it is concentrated. These membranes form the interface between the neuropil and the extracellular liquid spaces. The brain-selective deletion of the dystroglycan (DG) gene causes a disorganization of AQP4 on the astroglial endfeet. First, we analyzed the expression of AQP4, ß-DG in the brain following intracerebral hemorrhage (ICH) and correlated AQP4 expression with the expression pattern of the ß-DG, which is a component of dystrophin-dystroglycan complex (DDC). Besides, the vessels ultrastructure and brain water content were investigated at different time points post-ICH (day 1, day 3, day 7). We found that AQP4 polarity was disturbed in parallel with the loss of ß-DG in the perihematomal area post-ICH. At day 1 post-ICH, brain edema was obvious and the damage of vascular ultrastructure was the most severe. These results suggest a role for ß-DG in targeting and stabilizing AQP4 channel in astrocytic cells, which may be critical for water homeostasis in brain.