Number Of Reactive Astrocytes Research Articles

Nimodipine inhibits spreading depolarization, ischemic injury, and neuroinflammation in mouse live brain slice preparations

Nimodipine is used to prevent delayed ischemic deficit in patients with aneurysmal subarachnoid hemorrhage (aSAH). Spreading depolarization (SD) is recognized as a factor in the pathomechanism of aSAH and other acute brain injuries. Although nimodipine is primarily known as a cerebral vasodilator, it may have a more complex mechanism of action due to the expression of its target, the L-type voltage-gated calcium channels (LVGCCs) in various cells in neural tissue. This study was designed to investigate the direct effect of nimodipine on SD, ischemic tissue injury, and neuroinflammation. SD in control or nimodipine-treated live mouse brain slices was induced under physiological conditions using electrical stimulation, or by subjecting the slices to hypo-osmotic stress or mild oxygen-glucose deprivation (mOGD). SD was recorded applying local field potential recording or intrinsic optical signal imaging. Histological analysis was used to estimate tissue injury, the number of reactive astrocytes, and the degree of microglia activation. Nimodipine did not prevent SD occurrence in mOGD, but it did reduce the rate of SD propagation and the cortical area affected by SD. In contrast, nimodipine blocked SD occurrence in hypo-osmotic stress, but had no effect on SD propagation. Furthermore, nimodipine prevented ischemic injury associated with SD in mOGD. Nimodipine also exhibited anti-inflammatory effects in mOGD by reducing reactive astrogliosis and microglial activation. The results demonstrate that nimodipine directly inhibits SD, independent of nimodipine's vascular effects. Therefore, the use of nimodipine may be extended to treat acute brain injuries where SD plays a central role in injury progression.

Co-transplantation of novel Nano-SDF scaffold with human neural stem cells attenuates inflammatory responses and apoptosis in traumatic brain injury

Traumatic brain injury (TBI) causes long-term disability and mortality worldwide. The prime pathological players in TBI are neuroinflammation and apoptosis. These pathological changes lead to a limited capacity of regeneration after TBI. To alleviate inflammatory responses and apoptosis triggered by TBI, developing bioactive scaffolds conjoined with stem cells is a decisive approach in neural tissue engineering. The aim of this study was to fabricate a novel nano-scaffold made of RADA-16 with a bioactive motif of stromal cell-derived factor-1 α (SDF-1α) and evaluate its effects with stem cell transplantation on inflammatory pathways, reactive gliosis, and apoptosis after TBI. Co-transplantation of Nano-SDF and human neural stem cells (hNSCs) derived from fetus brain in adult rats subjected to TBI led to the improvement of motor activitycompared with the control group. The treated animals with hNSCs+Nano-SDF had a significantly lower expression of toll-like receptor 4 and nuclear factor-kappa B at the injury site than the control animals. A significant reduction in the number of reactive astrocytes was also observed in rats that received hNSCs+Nano-SDF compared with the vehicle and Nano-SDF groups. Furthermore, the TUNEL assay indicated a significant reduction in TUNEL positive cells in the hNSCs+Nano-SDF group compared with the TBI, vehicle, and Nano-SDF groups. These data demonstrated co-transplantation of hNSCs with Nano-SDF can reduce inflammatory responses and cell death after TBI via creating a more supportive microenvironment. Further research is required to establish the therapeutic efficacy of Nano-SDF with stem cells for TBI.

Dihydromyricetin alleviates cerebral ischemia-reperfusion injury by attenuating apoptosis and astrogliosis in peri-infarct cortex

ABSTRACT Objectives In ischemic stroke, reperfusion after thrombolysis is associated with secondary brain damage. Dihydromyricetin (DHM), a flavonoid, has shown neuroprotective effects through anti-oxidant, anti-inflammatory and anti-apoptotic properties. This study investigates the potential of DHM, given postreperfusion in middle cerebral artery occlusion (MCAo) model of stroke in rats. Methods MCAo surgery was performed in male Wistar rats. Reperfusion was performed after 90 min of ischemia. DHM (50 and 100 mg/kg) was administered 10–15 min and 2 h postreperfusion followed by daily dosing for 2 more days. Neurobehavioral parameters and infarct size (TTC staining) were assessed after 72 h. The effective dose (100 mg/kg) was then used to study reduction in infarct size (measured by MRI) and effect on apoptosis (evaluated by protein expression of Bax, Bcl-2 and cleaved caspase-3 and TUNEL assay) in peri-infarct cortex. Furthermore, effects of DHM on neuronal damage and activation of astrocytes were studied by immunofluorescence. Results Poststroke DHM (100 mg/kg) administered for 3 days showed significant improvements in motor-coordination and infarct damage (TTC staining and MRI). MCAo-induced altered apoptotic proteins were normalized to a significant extent in peri-infarct cortex with DHM treatment. Data from TUNEL assay were complementary to the effects on apoptotic proteins. Additionally, DHM caused a significant reduction in the number of reactive astrocytes when compared with the MCAo group. Discussion This study demonstrated the efficacy of subacute DHM treatment in ischemia/reperfusion injury by modulating apoptosis and astrogliosis in the peri-infarct cortex. This suggests the potential of DHM in attenuating disease progression.

Inflammatory Response Leads to Neuronal Death in Human Post-Mortem Cerebral Cortex in Patients with COVID-19.

The recent coronavirus disease of 2019 (COVID-19) pandemic has adversely affected people worldwide. A growing body of literature suggests the neurological complications and manifestations in response to COVID-19 infection. Herein, we explored the inflammatory and immune responses in the post-mortem cerebral cortex of patients with severe COVID-19. The participants comprised three patients diagnosed with severe COVID-19 from March 26, 2020, to April 17, 2020, and three control patients. Our findings demonstrated a surge in the number of reactive astrocytes and activated microglia, as well as low levels of glutathione along with the upregulation of inflammation- and immune-related genes IL1B, IL6, IFITM, MX1, and OAS2 in the COVID-19 group. Overall, the data imply that oxidative stress may invoke a glial-mediated neuroinflammation, which ultimately leads to neuronal cell death in the cerebral cortex of COVID-19 patients.

Chemokines and chemokinesâs receptors in APPPS1 mice

The amyloid precursor protein plus presenilin-1 (APP/PS1) mice are a frequently-used model for Alzheimer’s disease studies (AD). Using behavioral studies, quantitative RT-PCR and Western-blot techniques, significant findings were determined by the expression of proteins involved in inflammation comparing APP/PS1 and Wild type mice. Increased GFAP expression could be associated with the elevation in number of reactive astrocytes. IL-3 is involved in inflammation and ABDF1 intervenes normally in the transport across cell membranes and both were found up-regulated in APP/PS1 mice compared to Wild type mice. We noted for the first time, a CCR8 increase expression with diminution of its CCL1 chemokine, both normally involved in protection from bacterial infection and demyelination. Furthermore, CCR5 expression was decreased and both CCL3 and CCL4 chemokines were highly expressed indicating a possible gliosis and probably an increase in chemotaxis from lymphocytes and T cell generation. Inflammation in AD will be the next step in the therapeutic approach.

Transplantation of R-GSIK scaffold with mesenchymal stem cells improves neuroinflammation in a traumatic brain injury model

Neural tissue engineering has been introduced as a novel therapeutic strategy for traumatic brain injury (TBI). Transplantation of mesenchymal stem cells (MSCs) has been demonstrated to improve functional outcome of brain injury, and RADA4GGSIKVAV (R-GSIK), a self-assembling nano-peptide scaffold, has been suggested to promote the behavior of stem cells. This study was designed to determine the ability of the R-GSIK scaffold in supporting the effects of MSCs on motor function activity and inflammatory responses in an experimental TBI model. A significant recovery of motor function was observed in rats that received MSCs+R-GSIK compared with the control groups. Further analysis showed a reduction in the number of reactive astrocytes and microglial cells in the MSCs and MSCs+R-GSIK groups compared with the control groups. Furthermore, western blot analysis indicated a significant reduction in pro-inflammatory cytokines, such as TLR4, TNF, and IL6, in the MSCs and MSCs+R-GSIK groups compared with the TBI, vehicle, and R-GSIK groups. Overall, this study strengthens the idea that the co-transplantation of MSCs with R-GSIK can increase functional outcomes by preparing a beneficial environment. This improvement may be explained by the immunomodulatory effects of MSCs and the self-assembling nano-scaffold peptide.

Astrocyte ablation induced by La-aminoadipate (L-AAA) potentiates remyelination in a cuprizone demyelinating mouse model.

Chronic demyelination in the central nervous system (CNS) is accompanied by an increase in the number of reactive astrocytes and astrogliosis. There are controversial issues regarding astrocytes and their roles in demyelinating diseases in particular for multiple sclerosis (MS). We aimed to evaluate possible roles for pharmacologic astrocyte ablation strategy using La-aminoadipate (L-AAA) on remyelination in a cuprizone model of demyelination. Male C57BL/6 mice were fed with 0.2% cuprizone for 12weeks followed by 2-week administration of L-AAA through a cannula inserted 1mm above the corpus callosum. Rotarod test showed a significant decrease in the range of motor coordination deficits after ablation of astrocytes in mice receiving cuprizone. Results of Luxol fast blue (LFB) and transmission electron microscopy (TEM) for evaluation of myelin content within the corpus callosum revealed a noticeable rise in the percentage of myelinated areas and in the number of myelinated fibers after L-AAA administration in the animals. Astrocyte ablation reduced protein expressions for GFAP (an astrocyte marker) and Iba-1 (a microglial marker), but increased expression of Olig2 (an oligodendrocyte marker) assessed by immunofluorescence. Finally, expression of genes related to recruitment of microglia (astrocyte chemokines CXCL10 and CXCL12) and suppression of oligodendrocyte progenitor cell (OPC) differentiation (astrocyte peptides ET-1 and EDNRB) showed a considerable decrease after administration of L-AAA (for all p < 0.05). These results are indicative of improved remyelination after ablation of astrocytes possibly through hampering microgliosis and astrogliosis and a further rise in the number of matured Olig2+ cells.

Changes in Chemokines and Chemokine Receptors Expression in a Mouse Model of Alzheimer's Disease.

The amyloid precursor protein plus presenilin-1 (APP/PS1) mice are a frequently-used model for Alzheimer's disease studies (AD). However, the data relevant to which proteins are involved in inflammatory mechanism are not sufficiently well-studied using the AD mouse model. Using behavioral studies, quantitative RT-PCR and Western-blot techniques, significant findings were determined by the expression of proteins involved in inflammation comparing APP/PS1 and Wild type mice. Increased GFAP expression could be associated with the elevation in number of reactive astrocytes. IL-3 is involved in inflammation and ABDF1 intervenes normally in the transport across cell membranes and both were found up-regulated in APP/PS1 mice compared to Wild type mice. Furthermore, CCR5 expression was decreased and both CCL3 and CCL4 chemokines were highly expressed indicating a possible gliosis and probably an increase in chemotaxis from lymphocytes and T cell generation. We also noted for the first time, a CCR8 increase expression with diminution of its CCL1 chemokine, both normally involved in protection from bacterial infection and demyelination. Control of inflammatory proteins will be the next step in understanding the progression of AD and also in determining the mechanisms that can develop in this disease.

Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis

BackgroundNiemann-Pick disease Type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. The pathological mechanisms, underlying NPC1 are not yet completely understood. Especially the contribution of glial cells and gliosis to the progression of NPC1, are controversially discussed. As an analysis of affected cells is unfeasible in NPC1-patients, we recently developed an in vitro model system, based on cells derived from NPC1-patient specific iPSCs. Here, we asked if this model system recapitulates gliosis, observed in non-human model systems and NPC1 patient post mortem biopsies. We determined the amount of reactive astrocytes and the regulation of the intermediate filaments GFAP and vimentin, all indicating gliosis. Furthermore, we were interested in the assembly and phosphorylation of these intermediate filaments and finally the impact of the activation of protein kinase C (PKC), which is described to ameliorate the pathogenic phenotype of NPC1-deficient fibroblasts, including hypo-phosphorylation of vimentin and cholesterol accumulation.MethodsWe analysed glial cells derived from NPC1 patient specific induced pluripotent stem cells, carrying different NPC1 mutations. The amount of reactive astrocytes was determined by means of immuncytochemical stainings and FACS-analysis. Semi-quantitative western blot was used to determine the amount of phosphorylated GFAP and vimentin. Cholesterol accumulation was analysed by Filipin staining and quantified by Amplex Red Assay. U18666A was used to induce NPC1 phenotype in unaffected cells of the control cell line. Phorbol 12-myristate 13-acetate (PMA) was used to activate PKC.ResultsImmunocytochemical detection of GFAP, vimentin and Ki67 revealed that NPC1 mutant glial cells undergo gliosis. We found hypo-phosphorylation of the intermediate filaments GFAP and vimentin and alterations in the assembly of these intermediate filaments in NPC1 mutant cells. The application of U18666A induced not only NPC1 phenotypical accumulation of cholesterol, but characteristics of gliosis in glial cells derived from unaffected control cells. The application of phorbol 12-myristate 13-acetate, an activator of protein kinase C resulted in a significantly reduced number of reactive astrocytes and further characteristics of gliosis in NPC1-deficient cells. Furthermore, it triggered a restoration of cholesterol amounts to level of control cells.ConclusionOur data demonstrate that glial cells derived from NPC1-patient specific iPSCs undergo gliosis. The application of U18666A induced comparable characteristics in un-affected control cells, suggesting that gliosis is triggered by hampered function of NPC1 protein. The activation of protein kinase C induced an amelioration of gliosis, as well as a reduction of cholesterol amount. These results provide further support for the line of evidence that gliosis might not be only a secondary reaction to the loss of neurons, but might be a direct consequence of a reduced PKC activity due to the phenotypical cholesterol accumulation observed in NPC1. In addition, our data support the involvement of PKCs in NPC1 disease pathogenesis and suggest that PKCs may be targeted in future efforts to develop therapeutics for NPC1 disease.

Immediate and delayed hyperbaric oxygen therapy as a neuroprotective treatment for traumatic brain injury in mice.

Traumatic brain injury is the most common cause of death or chronic disability among people under-35-years-old. There is no effective pharmacological treatment currently existing for TBI. Hyperbaric oxygen therapy (HBOT) is defined as the inhalation of pure oxygen in a hyperbaric chamber that is pressurized higher than 1atm. HBOT offers physiological and mechanical effects by inducing a state of increased pressure and hyperoxia. HBOT has been proposed as an effective treatment for moderate traumatic brain injury (mTBI), yet the exact therapeutic window and mechanism that underlies this effect is not completely understood. HBOT was administrated for 4 consecutive days, post a mouse closed head weight drop moderate TBI (mTBI) in 2 different time lines: immediate treatment - initiated 3h post-injury and delayed treatment - initiated 7days post-injury. Behavioral cognitive tests and biochemical changes were assessed. The results were similar for both the immediate and the delayed treatments. mTBI mice exhibited impairment in learning abilities, whereas mTBI mice treated with HBO displayed significant improvement compared with the mTBI group, performing similar to the sham groups. mTBI mice had a decline in myelin basic protein, an increase in neuronal loss (NeuN staining), and an increase in the number of reactive astrocytes (GFAP). The HBO treated mice in both groups did not exhibit these changes and remained similar to the sham group. The delayed HBOT has a potential to serve as a neuroprotective treatment for mTBI with a long therapeutic window. Further research is needed for fully understanding the cellular changes.

Effects of fentanyl on pain and motor behaviors following a collagenase-induced intracerebral hemorrhage in rats.

PurposeIntracerebral hemorrhage (IH) and cephalalgia are common consequences of traumatic brain injury. One of the primary obstacles for patient recovery is the paucity of treatments to support an appropriate analgesic protocol. The present study aimed to assess pain and motor behaviors following different doses of fentanyl on a rat model of IH.MethodsTwenty-one male Sprague Dawley rats underwent a stereotaxic surgery to produce a collagenase-induced IH in the right caudoputamen nucleus. The control group (n=6) received saline subcutaneously (SC), and experimental groups received either 5 (n=6), 10 (n=6), or 20 (n=3) µg/kg of fentanyl SC, 2 hours following surgery and on 2 subsequent days. Only 3 animals received 20 µg/kg because this dose caused catalepsy for 15–20 minutes following the injection. The rat grimace scale, a neurological examination, balance beam test, and rotarod test were performed for 5 consecutive days postoperatively to evaluate pain and motor performance. At the end of the experimentation, the brains were evaluated to determine hematoma volume, and the number of reactive astrocytes and necrotic neurons.ResultsWhen compared to controls, the grimace scale showed that 5 µg/kg fentanyl significantly alleviated pain on day 2 only (P<0.01) and that 10 µg/kg alleviated pain on days 1 (P<0.01), 2 (P<0.001), and 3 (P<0.01). For the rotarod test, only the 10 µg/kg group showed significant decreases in performance on days 5 (P<0.05) and 6 (P<0.02). The neurological examination was not significantly different between the groups, but only the hopping test showed poor recuperation for the 5 and 10 µg/kg fentanyl group when compared to saline (P<0.01). No differences were found between the groups for the balance beam test, the histopathological results.ConclusionFentanyl, at a dose of 10 µg/kg SC, provides substantial analgesia following a collagenase-induced IH in rats; however, it can alter motor performance following analgesic treatments.

Cuprizone Model as a Tool for Preclinical Studies of the Efficacy of Multiple Sclerosis Diagnosis and Therapy.

To study demyelination and remyelination processes and their response to different drugs, a protocol for modeling multiple sclerosis using the copper chelator cuprizone was developed. Magnetic resonance imaging confirmed the presence of demyelination lesions on week 4 of 0.6% cuprizone-containing diet. Immunohistochemical staining with polyclonal antibodies to glial fibrillary acidic protein (pAb GFAP) confirmed the increase in the number of reactive astrocytes on week 4 of diet and during remyelination (week 2 after diet). Analysis of neurophysiological functions in mice with cuprizone-induced demyelination revealed motor and behavioral deficits. This model can be used as a tool for preclinical studies of the efficiency of multiple sclerosis diagnostic and therapy.

A Notch Closer to Understanding Multiple Sclerosis

Inhibiting endothelin signaling decreases Notch activation and enables oligodendrocyte maturation in MS-like lesions.

Thymoquinone Attenuates Astrogliosis, Neurodegeneration, Mossy Fiber Sprouting, and Oxidative Stress in a Model of Temporal Lobe Epilepsy

Temporal lobe epilepsy (TLE) is a rather common and difficult-to-treat variant of epilepsy. Nearly one third of people with epilepsy do not respond effectively to currently available anticonvulsants. In this study, we evaluated the protective effect of thymoquinone (TQ), the main constituent of black seed with antioxidant and anti-inflammatory effects, in the intrahippocampal kainate model of TLE in rat. Following kainate injection, seizure activity was observed that was significantly diminished by TQ pretreatment at a dose of 10 mg/kg, p.o. Intrahippocampal kainate also increased malondialdehyde (MDA), nitrite, and nitrate levels and decreased activity of superoxide dismutase and TQ only significantly attenuated MDA. In addition, intrahippocampal kainate caused a significant reduction of neurons in CA1, CA3 and the hilar regions, and TQ significantly attenuated these changes. Timm histochemistry showed a marked mossy fiber sprouting (MFS) in the dentate gyrus of kainate-lesioned rats, and TQ significantly lowered MFS intensity. Meanwhile, a number of reactive astrocytes (astrogliosis) increased significantly in the kainate group, and TQ pretreatment significantly decreased it. These data suggest that TQ pretreatment could attenuate seizure activity and lipid peroxidation, lower hippocampal neuronal loss and MFS, and mitigate astrogliosis in kainate model of TLE.

Increased Ictal Discharge Frequency and Neocortex Gliosis in Lateral Temporal Lobe Epilepsy

Medial temporal lobe epilepsy (TLE) with hippocampal sclerosis has both a scalp EEG initial ictal discharge frequency, which is faster, and also an intracranial EEG onset site that is more restricted to the hippocampus, than lateral TLE without hippocampal sclerosis. This study was performed to determine if lateral TLE patients have either intracranial EEG neocortical initial ictal frequencies or measures of lateral neocortex (LNC) histopathology that differ from patients whose seizures start in medial or multiple temporal lobe areas. Thirty-six TLE patients undergoing ictal depth and subdural strip electrode recordings were studied to determine the initial ictal discharge site (epileptogenic zone) within the temporal lobe and neocortical ictal frequency. In 25 patients, the number of reactive astrocytes in the neocortex and other measures of pathologic assessment of LNC were assessed. The initial neocortical ictal frequency was significantly faster when the initial ictal discharge was in the LNC ± medial paleocortex than either when it was in the hippocampus ± medial paleocortex or when it occurred simultaneously over the entire temporal lobe. Intracortical and Chaslin gliosis were both significantly greater when the initial ictal discharge was limited to the LNC than when it was in the hippocampus and/or medial paleocortex. Temporal lobe seizures originating in neocortex had a faster initial neocortical ictal frequency than seizures arising either medially in the hippocampus or widely over the whole temporal lobe. Epileptogenic zones limited to temporal neocortex were associated with greater intraneocortical and Chaslin gliosis compared with zones confined to medial structures.

Sequel of spontaneous seizures after kainic acid-induced status epilepticus and associated neuropathological changes in the subiculum and entorhinal cortex

Injection of the seaweed toxin kainic acid (KA) in rats induces a severe status epilepticus initiating complex neuropathological changes in limbic brain areas and subsequently spontaneous recurrent seizures. Although neuropathological changes have been intensively investigated in the hippocampus proper and the dentate gyrus in various seizure models, much less is known about changes in parahippocampal areas. We now established telemetric EEG recordings combined with continuous video monitoring to characterize the development of spontaneous seizures after KA-induced status epilepticus, and investigated associated neurodegenerative changes, astrocyte and microglia proliferation in the subiculum and other parahippocampal brain areas. The onset of spontaneous seizures was heterogeneous, with an average latency of 15 ± 1.4 days (range 3–36 days) to the initial status epilepticus. The frequency of late spontaneous seizures was higher in rats in which the initial status epilepticus was recurrent after its interruption with diazepam compared to rats in which this treatment was more efficient. Seizure-induced neuropathological changes were assessed in the subiculum by losses in NeuN-positive neurons and by Fluoro-Jade C staining of degenerating neurons. Neuronal loss was already prominent 24 h after KA injection and only modestly progressed at the later intervals. It was most severe in the proximal subiculum and in layer III of the medial entorhinal cortex and distinct Fluoro-Jade C labeling was observed there in 75% of rats even after 3 months. Glutamatergic neurons, labeled by in situ hybridization for the vesicular glutamate transporter 1 followed a similar pattern of cell losses, except for the medial entorhinal cortex and the proximal subiculum that appeared more vulnerable. Glutamate decarboxylase65 (GAD65) mRNA expressing neurons were generally less vulnerable than glutamate neurons. Reactive astrocytes and microglia were present after 24 h, however, became prominent only after 8 days and remained high after 30 days. In the proximal subiculum, parasubiculum and entorhinal cortex the number of microglia cells was highest after 30 days. Although numbers of reactive astrocytes and microglia were reduced again after 3 months, they were still present in most rats. The time course of astrocyte and microglia proliferation parallels that of epileptogenesis.

Is endothelial dysfunction of cerebral small vessel responsible for white matter lesions after chronic cerebral hypoperfusion in rats?

Cerebral white matter (WM) lesions contribute to cognitive impairment and motor dysfunction in the elderly. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are two important adhesion molecules that are upregulated during endothelial activation. Data from recent studies have suggested that ICAM-1 levels are related to progression of white matter hyperintensities (WMH) on MRI. In the present study, we hypothesized that ICAM-1 and VCAM-1 are involved in the endothelial dysfunction and the subsequent WM lesions after chronic cerebral hypoperfusion. Rats underwent bilateral common carotid artery ligation. They were divided into the lipoic acid group and the saline (vehicle) group. RT-PCR and double immunofluorescence for ICAM-1, VCAM-1, endothelial cells (staining positive for von Willebrand factor, vWF), reactive astrocytes (GFAP staining) and activated microglia/macrophages/(CD11b/c staining) were analyzed at baseline and at 1, 3, 7, 14 and 28 days after hypoperfusion. The severity of the WM lesions in the corpus callosum, internal capsule, and external capsule of both hemispheres was graded by luxol fast blue staining. RT-PCR and double immunofluorescence analysis of white matter from rats that had received lipoic acid (100 mg/kg/day) for 28 days exhibited markedly reduced expression of ICAM-1 and VCAM-1 over endothelial cells compared with that of rats receiving saline. In the rats treated with lipoic acid, the WM lesions after chronic cerebral hypoperfusion were significantly less severe, and the number of reactive astrocytes and activated microglia/macrophages (CD11b/c staining) were also significantly lower as compared with the saline-treated rats. These findings indicate that endothelial dysfunction plays a critical role in overexpression of ICAM-1 and VCAM-1, glial cell activation and WM lesions after chronic cerebral hypoperfusion and suggest the potential value of lipoic acid as a therapeutic tool in cerebrovascular WM lesions. Our results also provide support for endothelial activation being involved in early pathogenesis of WM lesions and suggest that therapies that stabilize the endothelium may have a role in preventing WM lesions progression.

Efficacy of Rho-kinase Inhibition in Promoting Cell Survival and Reducing Reactive Gliosis in the Rodent Retina

To analyze the outcomes of Rho-kinase (ROCK) inhibition on retinal cell survival and glial reactivity under adverse conditions. Organotypic cultures of mouse retinas were incubated with the specific ROCK-inhibitor H-1152P for 24 to 48 hours under serum deprivation. Cell damage was determined by ethidium homodimer-1 uptake and caspase-3 cleavage. Immunohistochemistry and Western blot were performed to detect reactive gliosis and to confirm the specificity of H-1152P. The cytokine profile of the culture medium was analyzed using a membrane-based array. H-1152P was administered intravitreally into rats before optic nerve crush (ONC) and the extent of apoptosis and reactive gliosis was determined after 7 days. Cell damage in cultured retinas was significantly reduced in response to 1 microM H-1152P, particularly in the ganglion cell layer. This was associated with a decrease in the levels of glial fibrillary acidic protein (GFAP) isoforms and the number of reactive astrocytes, Müller cells, and microglia. The release of proinflammatory cytokines including TNF-alpha, interferon-gamma, and IL-6 was also reduced, which likely contributed to the significantly lower toxicity of the conditioned media collected from retinas incubated with H-1152P. H-1152P (1 microM) suppressed the ROCK-dependent phosphorylation of adducin without a considerable interference with the protein kinase A/C-mediated phosphorylation events, indicating the specificity of the inhibitor for ROCK. H-1152P also resulted in a significant decrease in the extent of apoptosis and reactive gliosis after ONC. These results demonstrate the neuroprotective effect of H-1152P-mediated ROCK-inhibition on retinal cells under stress, which may rely partly on the attenuation of glial cell reactivity.

Mapping of Rat Hippocampal Neurons With NeuN After Ischemia/Reperfusion and Ginkgo Biloba Extract (EGb 761) Pretreatment

1. The neuroprotective effect of Ginkgo biloba extract (EGb 761) against transient forebrain ischemia following 7 days of reperfusion was studied in male Wistar rats after four-vessel occlusion for 20 min. 2. NeuN, a neuronal specific nuclear protein was used for immunohistochemical detection of surviving pyramidal neurons in the hippocampus, as well as counterstaining with hematoxylin in the same sections for detection of neurons that underwent delayed neuronal death and for glial nuclei staining. GFAP immunohistochemistry was used for detection of astrocytes in the studied area of CA1 region. 3. In the group of rats pretreated 7 days with Ginkgo biloba extract (EGb 761), following 20 min of ischemia and 7 days of reperfusion without EGb 761, increased number of NeuN immunoreactive cells were counted in the most vulnerable CA1 pyramidal layer of hippocampus. On the other hand, the group of rats with 7 days of EGb 761 pretreatment following 20 min of ischemia and 7 days of reperfusion with EGb 761 showed decreased number of surviving NeuN immunoreactive CA1 pyramidal cells in comparison with the first above-mentioned experimental group. 4. Increased number of reactive astrocytes immunolabeled for GFAP (Glial fibrilary acidic protein) was observed in both experimental groups in the stratum oriens and stratum lacunosum and moleculare. 5. Twenty minutes of ischemia is lethal for most population of CA1 pyramidal cell layer. Our results showed that prophylactic oral administration of Ginkgo biloba extract (EGb 761) in the dose 40 mg/kg/day during the 7 days protects the most vulnerable CA1 pyramidal cells against 20 min of ischemia.

Sigma Receptor Activation Reduces Infarct Size at 24 Hours After Permanent Middle Cerebral Artery Occlusion in Rats

The only available treatment for embolic stroke is recombinant tissue plasminogen activator, which must be administered within three hours of stroke onset. We examined the effects of 1,3-di-o-tolyguanidine (DTG), a high affinity sigma receptor agonist, as a potential treatment for decreasing infarct area at delayed time points. Rats were subjected to permanent embolic middle cerebral artery occlusion (MCAO) and allowed to recover before receiving subcutaneous injections of 15 mg/kg of DTG at 24, 48, and 72 hours. At 96 hours the rats were euthanized, and brains harvested and sectioned. Infarct areas were quantified at the level of the cortical/striatal and cortical/hippocampal regions in control (MCAO-only) and DTG treated animals using a marker for neurodegeneration, Fluoro-Jade. DTG treatment significantly reduced infarct area in both cortical/striatal and cortical/hippocampal regions by >80%, relative to control rats. These findings were confirmed by immunohistochemical experiments using the neuronal marker, mouse anti-neuronal nuclei monoclonal antibody (NeuN), which showed that application of DTG significantly increased the number of viable neurons in these regions. Furthermore, DTG blocked the inflammatory response evoked by MCAO, as indicated by decreases in the number of reactive astrocytes and activated microglia/macrophages detected by immunostaining for glial fibrillary acidic protein (GFAP) and binding of isolectin IB4, respectively. Thus, our results demonstrate that the sigma receptor-selective agonist, DTG, can enhance neuronal survival when administered 24 hr after an ischemic stroke. In addition, the efficacy of sigma receptors for stroke treatment at delayed time points is likely the result of combined neuroprotective and anti-inflammatory properties of these receptors.