Inhibition of transient receptor potential vanilloid 4 decreases the expressions of caveolin‐1 and caveolin‐2 after focal cerebral ischemia and reperfusion in rats

This study aimed to investigate the effects of transient receptor potential vanilloid 4 (TRPV4) inhibition on blood–brain barrier (BBB) integrity and the expressions of caveolae structural proteins caveolin‐1 and caveolin‐2 in rats with focal cerebral ischemia and reperfusion. BBB permeability was assessed by Evans blue extravasation. The mRNA and protein expressions of caveolin‐1 and caveolin‐2 were determined by RT‐PCR, Western blot and immunohistochemistry assays. We found that BBB permeability significantly increased and reaches its peak at 72 h of reperfusion in cerebral ischemia‐reperfusion rats and is able to be ameliorated by administration of HC‐067047, an antagonist of TRPV4. Additionally, it shows a significant upregulation of caveolin‐1 and caveolin‐2 expression in cerebral microvessels of ischemic tissue. However, treatment with HC‐067047 was shown to downregulate caveolin‐1 and caveolin‐2 expression during cerebral ischemia‐reperfusion. This study demonstrates that inhibition of TRPV4 ameliorates BBB leakage induced by ischemia‐reperfusion injury through the downregulation of caveolin‐1 and caveolin‐2.     

Inflammatory cytokine tumor necrosis factor α suppresses neuroprotective endogenous erythropoietin from astrocytes mediated by hypoxia‐inducible factor‐2α

Interest in erythropoietin (EPO) as a neuroprotective mediator has grown since it was found that systemically administered EPO is protective in several animal models of disease. However, given that the blood–brain barrier limits EPO entry into the brain, alternative approaches that induce endogenous EPO production in the brain may be more effective clinically and associated with fewer untoward side‐effects. Astrocytes are the main source of EPO in the central nervous system. In the present study we investigated the effect of the inflammatory cytokine tumor necrosis factor α (TNFα) on hypoxia‐induced upregulation of EPO in rat brain. Hypoxia significantly increased EPO mRNA expression in the brain and kidney, and this increase was suppressed by TNFα in vivo. In cultured astrocytes exposed to hypoxic conditions for 6 and 12 h, TNFα suppressed the hypoxia‐induced increase in EPO mRNA expression in a concentration‐dependent manner. TNFα inhibition of hypoxia‐induced EPO expression was mediated primarily by hypoxia‐inducible factor (HIF)‐2α rather than HIF‐1α. The effects of TNFα in reducing hypoxia‐induced upregulation of EPO mRNA expression probably involve destabilization of HIF‐2α, which is regulated by the nuclear factor (NF)‐κB signaling pathway. TNFα treatment attenuated the protective effects of astrocytes on neurons under hypoxic conditions via EPO signaling. The effective blockade of TNFα signaling may contribute to the maintenance of the neuroprotective effects of EPO even under hypoxic conditions with an inflammatory response.     

Induction of interleukin‐1β by activated microglia is a prerequisite for immunologically induced fatigue

We previously reported that an intraperitoneal (i.p.) injection of synthetic double‐stranded RNA, polyriboinosinic:polyribocytidylic acid (poly‐I:C), produced prolonged fatigue in rats, which might serve as a model for chronic fatigue syndrome. The poly‐I:C‐induced fatigue was associated with serotonin transporter (5‐HTT) overexpression in the prefrontal cortex (PFC), a brain region that has been suggested to be critical for fatigue sensation. In the present study, we demonstrated that microglial activation in the PFC was important for poly‐I:C‐induced fatigue in rats, as pretreatment with minocycline, an inhibitor of microglial activation, prevented the decrease in running wheel activity. Poly‐I:C injection increased the microglial interleukin (IL)‐1β expression in the PFC. An intracerebroventricular (i.c.v.) injection of IL‐1β neutralising antibody limited the poly‐I:C‐induced decrease in activity, whereas IL‐1β (i.c.v.) reduced the activity in a dose‐dependent manner. 5‐HTT expression was enhanced by IL‐1β in primary cultured astrocytes but not in microglia. Poly‐I:C injection (i.p.) caused an increase in 5‐HTT expression in astrocytes in the PFC of the rat, which was inhibited by pretreatment with minocycline (i.p.) and rat recombinant IL‐1 receptor antagonist (i.c.v.). Poly‐I:C injection (i.p.) led to a breakdown of the blood–brain barrier and enhanced Toll‐like receptor 3 signaling in the brain. Furthermore, direct application of poly‐I:C enhanced IL‐1β expression in primary microglia. We therefore propose that poly‐I:C‐induced microglial activation, which may be at least partly caused by a direct action of poly‐I:C, enhances IL‐1β expression. Then, IL‐1β induces 5‐HTT expression in astrocytes, resulting in the immunologically induced fatigue.     

α-Tocopherol and ubiquinones in rat brain subjected to decapitation ischemia

Levels of α-tocopherol (α-Toc), reduced ubiquinones (QH₂) and oxidized ubiquinones (Q) were assayed in rat forebrain subjected to decapitation ischemia. Post-decapitation levels of α-Toc decreased by 16% at 3 min and 20% at 15 min. Increases in Q₉H₂ (83%) and in Q₁₀H₂ (107%) were observed immediately following decapitation; thereafter their levels began to decrease and approached to the pre-ischemic values at 15 min. In contrast, Q₉ and Q₁₀ tended to increase continuously during ischemia. The data indicate that complete ischemia results in distinct changes in the recebral content of lipid-soluble antioxidants. The decrease of α-Toc may make the brain prone to peroxidative attack when cerebral tissue is subsequently reoxygenated.     

Permeability of blood–brain barrier in macaque model of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐induced Parkinson disease

Brain bioavailability of drugs developed to address central nervous system diseases is classically documented through cerebrospinal fluid collected in normal animals, i.e., through an approximation as there are fundamental differences between cerebrospinal fluid and tissue contents. The fact that disease might affect brain availability of drugs is almost never considered at this stage although several conditions are associated with blood–brain barrier damage. Building upon our expertise in Parkinson's disease translational research, the present study addressed this gap comparing plasma and cerebrospinal fluid bioavailability of l ?3,4‐dihydroxyphenylalanine, carbamazepine, quinidine, lovastatin, and simvastatin, in healthy and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐treated macaques, the gold standard model of Parkinson's disease. The drugs were selected based upon their differential transport across the blood–brain barrier. Interestingly, brain bioavailability of quinidine was decreased while others were unaffected. Pharmacokinetics and pharmacodynamics experiments of drugs addressing Parkinson's disease might thus be performed in healthy animals unless the drugs are known to interact with the organic cation transporter. Synapse 70:231–239, 2016 . © 2016 Wiley Periodicals, Inc.     

Tumor necrosis factor‐α potentiates long‐term potentiation in the rat dentate gyrus after acute hypoxia

An inadequate supply of oxygen in the brain may lead to an inflammatory response through neuronal and glial cells that can result in neuronal damage. Tumor necrosis factor‐α (TNF‐α) is a proinflammatory cytokine that is released during acute hypoxia and can have neurotoxic or neuroprotective effects in the brain. Both TNF‐α and interleukin‐1β (IL‐1β) have been shown by a number of research groups to alter synaptic scaling and also to inhibit long‐term potentiation (LTP) in the hippocampus when induced by specific high‐frequency stimulation (HFS) protocols. This study examines the effects of TNF‐α on synaptic transmission and plasticity in hippocampal slices after acute hypoxia using two HFS protocols. Field excitatory postsynaptic potentials were elicited in the medial perforant pathway of the dentate gyrus. Exogenous TNF‐α (5 ng/ml) attenuated LTP induced by theta burst stimulation but had no effect on LTP induced by a more prolonged HFS. Pretreatment with lipopolysaccharide (100 ng/ml) or TNF‐α but not IL‐1β (4 ng/ml) prior to a 30‐min hypoxic insult resulted in a significant enhancement of LTP post hypoxia when induced by the HFS. Anti‐TNF, 3,6′‐dithiothalidomide (a TNF‐α synthesis inhibitor), and SB203580 (a p38 MAPK inhibitor) significantly reduced this effect. These results indicate an important modulatory role for elevated TNF‐α levels on LTP in the hippocampus after an acute hypoxic event. © 2015 Wiley Periodicals, Inc.     

Lymphocyte migration through the blood–brain barrier (BBB) in feline immunodeficiency virus infection is significantly influenced by the pre‐existence of virus and tumour necrosis factor (TNF)‐α within the central nervous system (CNS): studies using an in vitro feline BBB model

Aims: In human immunodeficiency virus infection, macrophage‐tropic and lymphotropic viruses exist in the host. Central nervous system (CNS) infection is an early and ongoing event, important to understand when developing strategies to treat infection. Some knowledge exists on macrophage‐tropic virus interactions with the blood–brain barrier (BBB), and the aim of this study was to investigate lymphotropic lentivirus interactions with the BBB. Methods: Interactions of the lymphotropic feline immunodeficiency virus (FIV) with an in vitro model of the feline BBB were evaluated in scenarios to mimic in vivo infections. Results: Cell‐free FIV crossed the BBB in very low quantities, and in the presence of tumour necrosis factor (TNF)‐α, BBB integrity was unaffected. However, cell‐associated FIV readily crossed the BBB, but BBB integrity was not significantly altered. Transmigration of uninfected and infected lymphocytes increased in response to TNF‐α, accompanied by a moderate disruption of barrier integrity and an upregulation of vascular cell adhesion molecule‐1 rather than intercellular adhesion molecule‐1. Significant enhancement of migration and disruption of BBB tight junctions occurred when infected cells and TNF‐α were added to the brain side of the BBB and this enhancement was not mediated through additional TNF‐α production. Conclusions: Small quantities of virus in the brain together with TNF‐α have the potential to stimulate greater cell and viral entry into the CNS and this is likely to involve important factors other than further TNF‐α production. Lymphotropic lentivirus entry to the CNS is governed by many factors similar to macrophage‐tropic strains.     

Hypoxia‐inducible factor 1α may be a marker for vasculitic neuropathy

Neuromuscular biopsy is still an essential method for diagnosing vasculitic neuropathy, although its diagnostic sensitivity is at most 60%. Our objective was to examine the expression of hypoxia‐inducible factor 1α (HIF‐1α) in peripheral nerves and to evaluate its usefulness in diagnosing vasculitic neuropathy, especially for discrimination from other axonal neuropathies. Forty‐one patients with vasculitic neuropathy consisting of 20 definite, 14 probable and seven possible diagnoses, 15 patients with metabolic neuropathy, five with motor neuron disease and six with chronic inflammatory demyelinating polyneuropathy were included. Nerve biopsy specimens were immunohistochemically examined for HIF‐1α and various cell markers. Distinct immunoreactivity (IR) was observed in nuclei of endoneurial cells in 54% (22/41) of vasculitic patients, while specimens from metabolic neuropathies showed less nuclear IR and the difference of mean density of HIF‐1α‐positive nuclei was significant. Two patients with possible vasculitis who showed HIF‐1α‐positive nuclei in endoneurium, were later confirmed to have vasculitis by skin biopsies. Most of the cells expressing HIF were demonstrated to be Schwann cells. There was a trend in the vasculitic patients with early phase nerve damage to display higher endoneurial HIF‐1α‐IR. HIF‐1α may be an immunohistochemical marker for vasculitic neuropathy, especially when the observed section contains no vasculitic lesions.     

Magnetic resonance imaging as a tool to image neuroinflammation in a rat model of Parkinson's disease – phagocyte influx to the brain is promoted by bilberry‐enriched diet

Neuroinflammation is a chronic event in neurodegenerative disorders. In the rat model of Parkinson's disease, including a striatal injection of the neurotoxin 6‐hydroxydopamine (6‐OHDA), antioxidant treatment affects the inflammatory process. Despite a heavy accumulation of microglia early after the injury, dopamine nerve fibre regeneration occurs. It remains unclear why this heavy accumulation of microglia is found early after the lesion in antioxidant‐treated animals, or even more, what is the origin of these microglia. In this study magnetic resonance imaging (MRI) was used to elucidate whether the inflammatory response was generated from the blood or from activated brain microglia. Superparamagnetic iron oxide (SPIO) nanoparticles were injected intravenously prior to a striatal 6‐OHDA injection to tag phagocytes in the blood. Rats were fed either with bilberry‐enriched or control diet. T2*‐weighted MRI scans were performed 1 week after the lesion, and hypointense areas were calculated from T2*‐weighted images, to monitor the presence of SPIO particles. The results revealed that feeding the animals with bilberries significantly promoted accumulation of blood‐derived immune cells. Gadolinium‐enhanced MRI demonstrated no difference in leakage of the blood–brain barrier independent of diets. To conclude, bilberry‐enriched diet promotes an influx of periphery‐derived immune cells to the brain early after injury.     

Transforming growth factor-β1 exhibits delayed gene expression following focal cerebral ischemia

Transforming growth factor-β1 (TGF-β1) is a pleiotropic peptide growth factor. The expression of TGF-β1 mRNA in the focal ischemic cortex of rats was studied by means of Northern hybridization. A moderately low level of constitutively expressed TGF-β1 mRNA was detected following ohm surgery or in the contralateral (nonlschemic) cortex. A significant increase of TGF-β1 mRNA level in the ischemic cortex was observed at 2 days (3.2-fold increase compared to sham-operated animals, p < 0.01, N = 4) following permanent occlusion of the middle cerebral artery (PMCAO). The elevated TGF-μ1 mRNA expression was plateaued for up to 16 days (3–6-fold increase, p < 0.01) following PMCAO- This temporal profile for TGF-β1 mRNA expression in focal stroke was significantly delayed compared to that of TNF-α, IL-1β and IL-6 MRNA expressions as demonstrated previously which peaked at 12 h and decreased to almost basal levels by S days following PMCAO. Interestingly, the TGF-β1 mRNA expression profile was remarkably parallel with that of monocyte/macrophage accumulation in the ischemic cortex, as well as with the increased formation of extracellular matrix in the focal ischemic brain. These data suggest that TGF-β1 may play a role in anti-inflammatory process and in tissue remodeling following ischernic brain injury.     

Protective effects of peroxisome proliferator‐activated receptors γ coactivator‐1α against neuronal cell death in the hippocampal CA1 subfield after transient global ischemia

Peroxisome proliferator‐activated receptors γ coactivator‐1α (PGC‐1α) may regulate the mitochondrial antioxidant defense system under many neuropathological settings. However, the exact role of PGC‐1α in ischemic brain damage is still under debate. Based on an experimental model of transient global ischemia (TGI), this study evaluated the hypothesis that the activation of PGC‐1α signaling pathway protects hippocampal CA1 neurons against delayed neuronal death after TGI. In Sprague‐Dawley rats, significantly increased content of oxidized proteins in the hippocampal CA1 tissue was observed as early as 30 min after TGI, followed by augmentation of PGC‐1α expression at 1 hr. Expression of uncoupling protein 2 (UCP2) and superoxide dismutases 2 (SOD2) in the hippocampal CA1 neurons was upregulated 4–48 hr after TGI. In addition, knock‐down of PGC‐1α expression by pretreatment with a specific antisense oligodeoxynucleotide in the hippocampal CA1 subfield downregulated the expression of UCP2 and SOD2 with resultant exacerbation of oxidative stress and augmentation of delayed neuronal cell death in the hippocampus after TGI. Overall, our results indicate that PGC‐1α is induced by cerebral ischemia leading to upregulation of UCP2 and SOD2, thereby providing a neuroprotective effect against ischemic brain injury in the hippocampus by ameliorating oxidative stress. © 2009 Wiley‐Liss, Inc.     

Asiatic acid,a pentacyclic triterpene from Centella asiatica,is neuroprotective in a mouse model of focal cerebral ischemia

Asiatic acid, a triterpenoid derivative from Centella asiatica, has shown biological effects such as antioxidant, antiinflammatory, and protection against glutamate‐ or β‐amyloid‐induced neurotoxicity. We investigated the neuroprotective effect of asiatic acid in a mouse model of permanent cerebral ischemia. Various doses of asiatic acid (30, 75, or 165 mg/kg) were administered orally at 1 hr pre‐ and 3, 10, and 20 hr postischemia, and infarct volume and behavioral deficits were evaluated at day 1 or 7 postischemia. IgG (blood–brain barrier integrity) and cytochrome c (apoptosis) immunostaining was carried out at 24 hr postischemia. The effect of asiatic acid on stress‐induced cytochrome c release was examined in isolated mitochondrial fractions. Furthermore, its effects on cell viability and mitochondrial membrane potential were studied in HT‐22 cells exposed to oxygen‐glucose deprivation. Asiatic acid significantly reduced the infarct volume by 60% at day 1 and by 26% at day 7 postischemia and improved neurological outcome at 24 hr postischemia. Our studies also showed that the neuroprotective properties of asiatic acid might be mediated in part through decreased blood–brain barrier permeability and reduction in mitochondrial injury. The present study suggests that asiatic acid may be useful in the treatment of cerebral ischemia. © 2009 Wiley‐Liss, Inc.     

Permeability of injured blood brain barrier for exogenous bFGF and protection mechanism of bFGF in rat brain ischemia

The study aims to explore the protection mechanism of exogenous basic fibroblast growth factor (exo‐bFGF) in brain ischemia. The first part of experiment was to determine the optimal time window for the permeation of exo‐bFGF through damaged blood–brain barrier in rats with permanently occluded middle cerebral arteries. ¹²⁵I labeled bFGF was administered to the rats through the caudal vein. The level of γ‐rays of ¹²⁵I‐bFGF in the ischemic brain were found to increase at 2 h and a high level was maintained for 14 days. The morphology of the basement membrane of capillaries was observed using anti‐blood–brain barrier basement membrane glycoprotein immunohistochemistry. The normal continuous linear or ribbon‐like immunostain of the basement membrane became granular at 0.5 h, gradually faint and finally negative. The newly formed capillaries at the edge of the infarct still showed a negative stain after 14 days. The result suggested the optimal time window of exo‐bFGF began 2 h after insult. The second part of experiment was to observe the dynamic expression of early growth response protein (Egr‐1), endogenous basic fibroblast growth factor (endo‐bFGF) and bFGF receptor (bFGFR) using immunohistochemistry after exo‐bFGF is administered to brain. Egr‐1 was more significantly enhanced in the exo‐bFGF‐used group than in the control group. Endo‐bFGF increased gradually, reaching its peak at 7 days in the control group, while in experiment group, the endo‐bFGF expression showed its first peak at 6 h, indicating that exo‐bFGF could induce earlier and stronger expression of endo‐bFGF. The bFGFR‐group presented an early expression, reaching its maximal level at 3 h, and declining at 6 h. There were no difference in expression of bFGFR between the two groups. The infarct areas reduced from 17% to 24% in the different time intervals. The results suggested that in exo‐bFGF enhanced Egr‐1 protein. Egr‐1 in turn might play an important role in up‐regulating the expression of endo‐bFGF which overlapped with the expression of bFGFR to ensure the combination of ligand and receptor to protect against brain ischemia.     

A critical role of TRPM2 channel in Aβ42‐induced microglial activation and generation of tumor necrosis factor‐α

Amyloid β (Aβ)‐induced neuroinflammation plays an important part in Alzheimer's disease (AD). Emerging evidence supports a role for the transient receptor potential melastatin‐related 2 (TRPM2) channel in Aβ‐induced neuroinflammation, but how Aβ induces TRPM2 channel activation and this relates to neuroinflammation remained poorly understood. We investigated the mechanisms by which Aβ₄₂ activates the TRPM2 channel in microglial cells and the relationships to microglial activation and generation of tumor necrosis factor‐α (TNF‐α), a key cytokine implicated in AD. Exposure to 10–300 nM Aβ₄₂ induced concentration‐dependent microglial activation and generation of TNF‐α that were ablated by genetically deleting (TRPM2 knockout ;TRPM2‐KO) or pharmacologically inhibiting the TRPM2 channel, revealing a critical role of this channel in Aβ₄₂‐induced microglial activation and generation of TNF‐α. Mechanistically, Aβ₄₂ activated the TRPM2 channel via stimulating generation of reactive oxygen species (ROS) and activation of poly(ADPR) polymerase‐1 (PARP‐1). Aβ₄₂‐induced generation of ROS and activation of PARP‐1 and TRPM2 channel were suppressed by inhibiting protein kinase C (PKC) and NADPH oxidases (NOX). Aβ₄₂‐induced activation of PARP‐1 and TRPM2 channel was also reduced by inhibiting PYK2 and MEK/ERK. Aβ₄₂‐induced activation of PARP‐1 was attenuated by TRPM2‐KO and moreover, the remaining PARP‐1 activity was eliminated by inhibiting PKC and NOX, but not PYK2 and MEK/ERK. Collectively, our results suggest that PKC/NOX‐mediated generation of ROS and subsequent activation of PARP‐1 play a role in Aβ₄₂‐induced TRPM2 channel activation and TRPM2‐dependent activation of the PYK2/MEK/ERK signalling pathway acts as a positive feedback to further facilitate activation of PARP‐1 and TRPM2 channel. These findings provide novel insights into the mechanisms underlying Aβ‐induced AD‐related neuroinflammation.     

Deleterious effect of β-estradiol in a rat model of transient forebrain ischemia

Estrogen has demonstrated great potential as a therapeutic agent in focal ischemic brain injury, as exogenous β-estradiol has proven beneficial in a variety of focal stroke models. In contrast, the relatively few studies of estrogen’s efficacy in transient forebrain ischemia have produced inconsistent results. The present study was therefore designed to clarify estrogen’s neuroprotective potential in selective hippocampal neuronal injury resulting from four-vessel occlusion in the rat. Female Wistar rats (normal, ovariectomized, or ovariectomized and estradiol-treated) received 5 or 10 min of ischemia. No differences in hippocampal cell loss were found amongst the groups with 10 min of ischemia. Amongst the groups with 5 min of ischemia, the mildest injury was found in the ovariectomized animals, which lost only 32% of their CA1 pyramidal cells. In comparison, mean cell losses were 54% and 49%, respectively, in intact females and in ovariectomized animals with estradiol replacement. Linear regression analysis demonstrated a highly significant relationship between cell loss and plasma estradiol levels. The mechanism by which exogenous and endogenous estrogen exacerbated the injury is unclear, as estrogen has many neuroprotective effects. On the other hand, many other reported effects of estrogen in hippocampal area CA1 might confer increased sensitivity to ischemia, either by modulating the excitatory effects of glutamate or by modifying the inhibitory effects of GABA. Determining how to modulate the various competing effects of estrogen is of both theoretical and practical importance, as it is now clear that one cannot assume that estrogen administration will always improve outcome in cerebral ischemia.     

Increased frequency of the α‐1‐antichymotrypsin T allele in cerebral amyloid angiopathy

Cerebral amyloid angiopathy (CAA) is a process of unknown etiology characterized by amyloid deposition in the wall of small cerebral and meningeal blood vessels. CAA is also a feature of Alzheimer's disease (AD) and of a subgroup of elderly people. α‐1‐Antichymotrypsin (ACT) is a serum glycoprotein frequently associated with vascular and senile plaque amyloid. The ACT gene is known to have a bi‐allele polymorphism that causes a simple amino acid substitution. In an attempt to clarify the possible role of ACT polymorphism in AD and in cases of CAA, the ACT genotype was investigated in AD, CAA, and intellectually intact controls. Representative brain areas (cerebral cortex, hippocampus, putamen, white matter, and gyrus cinguli) from all cases were studied using classical histologic staining techniques (hematoxylin–eosin (HE), Mallory's thrichromic or alkaline congo red stain), and immunohistochemistry for tau and β‐amyloid proteins. There was a significantly increased T allele and TT genotype frequency in the CAA group, but not in the AD group, suggesting a role for the ACT genotype in the development of vascular lesions. The presence of the apolipoprotein E4 allele (ApoE4) did not correlate with the ACT‐A allele, as previously reported, and appeared to be independent of the risk for developing AD.