Focal Cerebral Ischemia In Rats Research Articles

Abstract WP235: Docosanoids Protect Inflammation-induced Brain Damage And Lead To Neurological Recovery In Experimental Stroke

Objective: Cerebral ischemia initiates lipid peroxidation, uncompensated oxidative stress, and neuronal damage. We investigated the neuroprotective bioactivity of docosanoid (DOC) mediators: Neuroprotectin D1 (NPD1), Resolvin D1 (RvD1), and their combination (NPD1+RvD1) against experimental ischemic stroke. These lipid mediators are biosynthesized “on-demand” in response to stroke onset to resolve neuroinflammation and restore homeostasis. This study investigated the dose-response and therapeutic window in a model of focal cerebral ischemia. Methods: Male SD rats received 2h of middle cerebral artery occlusion (MCAo). Neurological score was evaluated on days 1, 2, 3, and 7, followed by an ex vivo MRI on day 7. In dose-response study, rats were treated with NPD1 (111, 222, and 333μg/kg), RvD1 (111, 222, and 333μg/kg), NPD1 + RvD1 or vehicle (n=4-5 per group). All treatments were administered IV at 3h after the onset of MCAo. In the therapeutic window study, vehicle, NPD1 (222 μg/kg), RvD1 (222 μg/kg), NPD1+RvD1 were administered IV at 3, 4, 5, and 6 h after onset of MCAo. Results: No adverse behavioral side effects were observed after drugs administration. NPD1 (111, 222, and 333μg/kg) treatment alone greatly improved neurological scores (by 32-35%) and RvD1 (111, 222, and 333μg/kg) alone (by 31-35%) vs vehicle group on day 7. The neuroprotective effect was enhanced using the NPD1+ RvD1, which improved total neurological scores on days 1-7 by 25-40% vs vehicle. Ischemic core, penumbra, and total lesion volumes (computed from T2WI) were reduced with NPD1+RvD1 by 69, 70, and 67% vs vehicle. Therapeutic window: treatments with NPD1 and RvD1 alone when administered at 3h improved behavior on day 7 by 35%. Combinatory NPD1+RvD1 treatment improved behavior when administered at 3h (by 56%), 4h (by 23%), 5h (by 26%), and 6 h (by 28%), vs vehicle. Conclusion: We show that NPD1, RvD1 alone provides high-grade neuroprotection against inflammation and elicit neurological recovery in the MCAo model. Combination therapy with NPD1 and RvD1 is more effective than the single therapy when administered up to 6h after focal cerebral ischemia in rats. Thus, we hypothesize that these treatments may potentially be used in treating focal ischemic stroke in the clinical setting.

Gastrodin injection alleviates lung injury caused by focal cerebral ischemia in rats through NGF/TrkA pathway-mediated activation of the anti-inflammatory pathway

To investigate the therapeutic mechanism of gastrodin injection for alleviating lung injury caused by focal cerebral ischemia in rats and the role of the NGF-TrkA pathway in mediating this effect. Forty SD rats were equally randomized into normal group, sham-operated group, model group and gastrodin group, and in the latter two groups, rat models of focal cerebral ischemia were established by embolization of the right middle cerebral artery. After successful modeling, the rats were treated with intraperitoneal injection of gastrodin injection at the daily dose of 10 mg/kg for 14 days. After the treatment, the wet/dry weight ratio of the lung tissue was determined, the pathological changes in the lung tissue were observed using HE staining, and the levels of IL-10 and TNF-α in the arterial blood were detected with ELISA. The expressions of NF-κB p65 and TNF-α in the lung tissue were detected with Western blotting, and the expressions of NGF and TrkA were detected using immunohistochemical staining and Western blotting. Compared with the normal control and sham-operated groups, the rats in the model group showed obvious inflammatory lung injury, significantly increased wet/ dry weight ratio of the lungs (P < 0.01), increased TNF-α level in arterial blood (P < 0.01), and significantly up-regulated protein expressions of NF-κB p65 (P < 0.01), TNF-α (P < 0.01), NGF (P < 0.05) and TrkA(P < 0.05) in the lung tissue. Treatment with gastrodin injection obviously alleviated lung inflammation, decreased the wet/dry weight ratio of the lungs (P < 0.05), and significantly lowered TNF-α level (P < 0.01) and increased IL-10 level in the arterial blood in the rat models (P < 0.01); gastrodin injection also significantly decreased the protein expressions of NF-κB p65 and TNF-α (P < 0.05) and up-regulated the expressions of NGF and TrkA in the lung tissue of the rats (P < 0.05). The NGF/TrkA pathway may participate in cerebral ischemia-induced inflammatory lung injury, which can be obviously alleviated by gastrodin through the activation of the anti-inflammatory pathway mediated by the NGF/TrkA pathway.

RETRACTED ARTICLE: Inhibition of Long non-coding RNA zinc finger antisense 1 improves functional recovery and angiogenesis after focal cerebral ischemia via microRNA-144-5p/fibroblast growth factor 7 axis

Statement of Retraction Tong Li, Bai Ling Qing, Yan Deng, Xian Ting Que, Cheng Zhi Wang, Hua Wen Lu, Shao Hua Wang & Zi Jun Wang (2022) Inhibition of Long non-coding RNA zinc finger antisense 1 improves functional recovery and angiogenesis after focal cerebral ischemia via microRNA-144-5p/fibroblast growth factor 7 axis, Bioengineered, 13:1, 1702-1716, DOI: 10.1080/21655979.2021.2018093 Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed. We have been informed in our decision-making by our editorial policies and the COPE guidelines. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

NEP1‑40 promotes myelin regeneration via upregulation of GAP‑43 and MAP‑2 expression after focal cerebral ischemia in rats.

Axon regeneration after lesions to the central nervous system (CNS) is largely limited by the presence of growth inhibitory molecules expressed in myelin. Nogo-A is a principal inhibitor of neurite outgrowth, and blocking the activity of Nogo-A can induce axonal sprouting and functional recovery. However, there are limited data on the expression of Nogo-A after CNS lesions, and the mechanism underlying its influences on myelin growth remains unknown. The aim of the present study was to observe the time course of Nogo-A after cerebral ischemia/reperfusion in rats using immunohistochemistry and western blot techniques, and to test the effect of its inhibitor Nogo extracellular peptide 1–40 (NEP1-40) on neural plasticity proteins, growth-associated binding protein 43 (GAP-43) and microtubule associated protein 2 (MAP-2), as a possible mechanism underlying myelin suppression. A classic model of middle cerebral artery occlusion (MCAO) was established in Sprague-Dawley rats, which were divided into three groups: i) MCAO model group; ii) MCAO + saline group; and iii) MCAO + NEP1-40 group. Rats of each group were divided into five subgroups by time points as follows: days 1, 3, 7, 14 and 28. Animals that only received sham operation were used as controls. The Nogo-A immunoreactivity was located primarily in the cytoplasm of oligodendrocytes. The number of Nogo-A immunoreactive cells significantly increased from day 1 to day 3 after MCAO, nearly returning to the control level at day 7, increased again at day 14 and decreased at day 28. Myelin basic protein (MBP) immunoreactivity in the ipsilateral striatum gradually decreased from day 1 to day 28 after ischemia, indicating myelin loss appeared at early time points and continuously advanced during ischemia. Then, intracerebroventricular infusion of NEP1-40, which is a Nogo-66 receptor antagonist peptide, was administered at days 1, 3 and 14 after MCAO. It was observed that GAP-43 considerably increased from day 1 to day 7 and then decreased to a baseline level at day 28 compared with the control. MAP-2 expression across days 1–28 significantly decreased after MCAO. Administration of NEP1-40 attenuated the reduction of MBP, and upregulated GAP-43 and MAP-2 expression at the corresponding time points after MCAO compared with the MCAO + saline group. The present results indicated that NEP1-40 ameliorated myelin damage and promoted regeneration by upregulating the expression of GAP-43 and MAP-2 related to neuronal and axonal plasticity, which may aid with the identification of a novel molecular mechanism of restriction in CNS regeneration mediated by Nogo-A after ischemia in rats.

Effects of electroacupuncture on myelin-related protein in corpus callosum of rats with focal cerebral ischemia

To observe the effect of electroacupuncture(EA)on the expression of myelin basic protein (MBP), axon growth inhibitor Nogo-A and Nogo receptor (NgR) in corpus callosum of rats with focal cerebral ischemia, so as to explore the mechanism of EA underlying improving ischemic white matter injury. Fourty male SD rats were randomly divided into normal, sham operation, model and EA groups, with 10 rats in each group. The focal cerebral ischemia rat model was established by occlusion of the middle cerebral artery (MCAO). EA was applied to "Baihui"(GV20) and "Zusanli"(ST36) on the left side for 30 min, once daily for 14 days. Neurological function score and the adhensive removal test were used to evaluate neurological deficit severity; Hematoxylin-esion staining was used to observe the pathological changes in myelin of corpus callosum and luxol fast blue（LFB） staining was used to observe the myelin of corpus callosum. Immunohistochemical staining and Western blot were used to detect the expressions of MBP、Nogo-A and NgR in the ischemic corpus callosum. After MCAO, the neurological function score was significantly increased (P<0.05), the time required for contact with tape and tape removal was longer (P<0.001), the intensity of LFB staining and the expression of MBP decreased, while the veside area and the expression of Nogo-A and its receptor NgR increased (P<0.01, P<0.05) in the model group relevant to the normal and sham operation groups. The fiber arrangement of the corpus callosum on the ischemic side was disordered and a large amount of myelin sheath was lost in the model group. Following the treatment, the neurological deficit score of EA group was gradually decreased and significantly decreased on the 3rd, 7th and 14th day (P<0.05), and the time to remove the adhesive tape was shortened at the 7th and 14th day (P<0.001). The shape of the corpus callosum in the EA group was close to normal, and the myelin structure was relatively complete. The intensity of LFB staining and the expression of MBP was increased (P<0.05, P<0.01) while the expression of Nogo-A and its receptor NgR were decreased in the EA group relevant to the model group (P<0.01). EA can play a protective role in myelin of the corpus callosum after cerebral ischemia, which may be related to down-regulating the expressions of Nogo-A and NgR.

Alpinia oxyphylla Miq extract reduces cerebral infarction by downregulating JNK-mediated TLR4/T3JAM- and ASK1-related inflammatory signaling in the acute phase of transient focal cerebral ischemia in rats

BackgroundPost-ischemic inflammation is a crucial component in stroke pathology in the early phase of cerebral ischemia–reperfusion (I/R) injury. Inflammation caused by microglia, astrocytes, and necrotic cells, produces pro-inflammatory mediators and exacerbates cerebral I/R injury. This study evaluated the effects of the Alpinia oxyphylla Miq [Yi Zhi Ren (YZR)] extract on cerebral infarction at 1 day after 90 min of transient middle cerebral artery occlusion (MCAo) and investigated the molecular mechanisms underlying the regulation of c-Jun N-terminal kinase (JNK)-mediated inflammatory cascades in the penumbral cortex. Rats were intraperitoneally injected with the YZR extract at the doses of 0.2 g/kg (YZR-0.2 g), 0.4 g/kg (YZR-0.4 g), or 0.8 g/kg (YZR-0.8 g) at MCAo onset.ResultsYZR-0.4 g and YZR-0.8 g treatments markedly reduced cerebral infarction, attenuated neurological deficits, and significantly downregulated the expression of phospho-apoptosis signal-regulating kinase 1 (p-ASK1)/ASK1, tumor necrosis factor receptor-associated factor 3 (TRAF3), TRAF3-interacting JNK-activating modulator (T3JAM), ionized calcium-binding adapter molecule 1 (Iba1), p-JNK/JNK, inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α, toll-like receptor 4 (TLR4), glial fibrillary acidic protein (GFAP), nuclear factor-kappa B (NF-κB), and interleukin-6 in the penumbral cortex at 1 day after reperfusion. SP600125 (SP), a selective JNK inhibitor, had the same effects. Furthermore, Iba1- and GFAP-positive cells were colocalized with TLR4, and colocalization of GFAP-positive cells was found with NF-κB in the nuclei.ConclusionYZR-0.4 g and YZR-0.8 g treatments exerted beneficial effects on cerebral ischemic injury by downregulating JNK-mediated signaling in the peri-infarct cortex. Moreover, the anti-infarction effects of YZR extract treatments were partially attributed to the downregulation of JNK-mediated TLR4/T3JAM- and ASK1-related inflammatory signaling pathways in the penumbral cortex at 1 day after reperfusion.

Electroacupuncture Pretreatment Prevents Cognitive Impairment Induced by Cerebral Ischemia-Reperfusion via Adenosine A1 Receptors in Rats.

A previous study has demonstrated that pretreatment with electroacupuncture (EA) induces rapid tolerance to focal cerebral ischemia. In the present study, we investigated whether adenosine receptor 1 (A1 R) is involved in EA pretreatment-induced cognitive impairment after focal cerebral ischemia in rats. Two hours after EA pretreatment, focal cerebral ischemia was induced by middle cerebral artery occlusion for 120 min in male Sprague-Dawley rats. The neurobehavioral score, cognitive function [as determined by the Morris water maze (MWM) test], neuronal number, and the Bax/Bcl-2 ratio was evaluated at 24 h after reperfusion in the presence or absence of CCPA (a selective A1 receptor agonist), DPCPX (a selective A1 receptor antagonist) into left lateral ventricle, or A1 short interfering RNA into the hippocampus area. The expression of the A1 receptor in the hippocampus was also investigated. The result showed that EA pretreatment upregulated the neuronal expression of the A1 receptor in the rat hippocampus at 90 min. And EA pretreatment reversed cognitive impairment, improved neurological outcome, and inhibited apoptosis at 24 h after reperfusion. Pretreatment with CCPA could imitate the beneficial effects of EA pretreatment. But the EA pretreatment effects were abolished by DPCPX. Furthermore, A1 receptor protein was reduced by A1 short interfering RNA which attenuated EA pretreatment-induced cognitive impairment.

Eprosartan: A closer insight into its neuroprotective activity in rats with focal cerebral ischemia-reperfusion injury.

Eprosartan (EPRO), an angiotensin receptor type-1 (AT-1) blocker, exhibited neuroprotective activities in ischemic stroke resulting from focal cerebral ischemia in rats. The current study aimed to clarify the neuroprotective role of EPRO in middle carotid artery occlusion (MCAO)-induced ischemic stroke in rats. Fifty-six male Wistar rats were divided into four groups (n = 14 per group): sham-operated group, sham receiving EPRO (60 mg/kg/day, po) group, ischemia-reperfusion (IR) group, and IR receiving EPRO (60 mg/kg/day, po) group. MCAO led to a remarkable impairment in motor function together with stimulation of inflammatory and apoptotic pathways in the hippocampus of rats. After MCAO, the AT1 receptor in the brain was stimulated, resulting in activation of Janus kinase 2/signal transducers and activators of transcription 3 signaling generating more neuroinflammatory milieu and destructive actions on the hippocampus. Augmentation of caspase-3 level by MCAO enhanced neuronal apoptosis synchronized with neurodegenerative effects of oxidative stress biomarkers. Pretreatment with EPRO opposed motor impairment and decreased oxidative and apoptotic mediators in the hippocampus of rats. The anti-inflammatory activity of EPRO was revealed by downregulation of nuclear factor-kappa B and tumor necrosis factor-β levels and (C-X-C motif) ligand 1 messenger RNA (mRNA) expression. Moreover, the study confirmed the role of EPRO against a unique pathway of hypoxia-inducible factor-1α and its subsequent inflammatory mediators. Furthermore, upregulation of caveolin-1 mRNA level was also observed along with decreased oxidative stress marker levels and brain edema. Therefore, EPRO showed neuroprotective effects in MCAO-induced cerebral ischemia in rats via attenuation of oxidative, apoptotic, and inflammatory pathways.

Contribution of Human Trophoblast Progenitor Cells to Neurogenesis in Rat Focal Cerebral Ischemia Model

ABSTRACT Objective : A decrease in the blood flow below a current level in the brain results in ischemia. Studies demonstrated that human trophoblast progenitor cells (hTPCs) contribute to the treatment of many diseases. Therefore, hTPCs might be a promising source to repair ischemia in cerebral ischemia models. For this purpose, we evaluated the expression of many neurogenesis markers by performing hTPC transplantation after focal cerebral ischemia in rats. Methods : hTPCs, isolated from the term placentae, were characterized by immunofluorescent staining and differentiated into neuron-like cells. Differentiation was confirmed with immunostaining of GFAP and NeuN proteins. Cerebral ischemia models were generated in rats via middle cerebral artery occlusion and, after 24 hours, hTPCs were injected via the tail vein. Animals were sacrificed on day 3 or day 11. Immunohistochemical analysis was performed with proteins associated with neurogenesis and neuronal development, such as DLX2, DLX5, LHX6, NGN1, and NGN2, Olig1, Olig2, and PDGFRα. Results : According to our results, hTPCs may alleviate ischemic damage in the brain and contribute to the neurogenesis after ischemia. Conclusions : Based on our findings, this topic should be further investigated as the hTPC-based therapies may be a reliable source that can be used in the treatment of stroke and ischemia.

Kellerin from Ferula sinkiangensis exerts neuroprotective effects after focal cerebral ischemia in rats by inhibiting microglia-mediated inflammatory responses

Ethnopharmacological relevanceFerula sinkiangensis K. M. Shen is a traditional Chinese medicine that has a variety of pharmacological properties relevant to neurological disorders and inflammations. Kellerin, a novel compound extracted from Ferula sinkiangensis, exerts a strong anti-neuroinflammatory effect by inhibiting microglial activation. Microglial activation plays a vital role in ischemia-induced brain injury. However, the potential therapeutic effect of kellerin on focal cerebral ischemia is still unknown. Aim of the studyTo explore the effect of kellerin on cerebral ischemia and clarify its possible mechanisms, we applied the middle cerebral artery occlusion (MCAO) model and the LPS-activated microglia model in our study. Materials and methodsNeurological outcome was examined according to a 4-tiered grading system. Brain infarct size was measured using TTC staining. Brain edema was calculated using the wet weight minus dry weight method. Neuron damage and microglial activation were observed by immunofluorescence in MCAO model in rats. In in vitro studies, microglial activation was examined by flow cytometry and the viability of neuronal cells cultured in microglia-conditioned medium was measured using MTT assay. The levels of pro-inflammatory cytokines were measured by qRT-PCR and ELISA. The proteins involved in NF-κB signaling pathway were determined by western blot. Intracellular ROS was examined using DCFH-DA method and NADPH oxidase activity was measured using the NBT assay. ResultsWe found that kellerin improved neurological outcome, reduced brain infarct size and decreased brain edema in MCAO model in rats. Under the pathologic conditions of focal cerebral ischemia, kellerin alleviated neuron damage and inhibited microglial activation. Moreover, in in vitro studies of LPS-stimulated BV2 cells kellerin protected neuronal cells from being damaged by inhibiting microglial activation. Kellerin also reduced the levels of pro-inflammatory cytokines, suppressed the NF-κB signaling pathway, and decreased ROS generation and NADPH oxidase activity. ConclusionsOur discoveries reveal that the neuroprotective effects of kellerin may largely depend on its inhibitory effect on microglial activation. This suggests that kellerin could serve as a novel anti-inflammatory agent which may have therapeutic effects in ischemic stroke.

GJ-4 ameliorates memory impairment in focal cerebral ischemia/reperfusion of rats via inhibiting JAK2/STAT1-mediated neuroinflammation

Ethnopharmacological relevanceGardenia jasminoides J. Ellis (Fructus Gardenia) is a traditional Chinese medicine with diverse pharmacological functions, such as anti-inflammation, anti-depression, as well as improvement of cognition and ischemia brain injury. GJ-4 is a natural extract from Gardenia jasminoides J. Ellis (Fructus Gardenia) and has been proved to improve memory impairment in Alzheimer's disease (AD) mouse model in our previous studies. Aim of the studyThis study aimed to evaluate the therapeutic effects of GJ-4 on vascular dementia (VD) and explore the potential mechanisms. Material and methodsIn our experiment, a focal cerebral ischemia and reperfusion rat model was successfully developed by the middle cerebral artery occlusion and reperfusion (MCAO/R). GJ-4 (10 mg/kg, 25 mg/kg, 50 mg/kg) and nimodipine (10 mg/kg) were orally administered to rats once a day for consecutive 12 days. Learning and memory behavioral performance was assayed by step-down test and Morris water maze test. The neurological scoring test was performed to evaluate the neurological function of rats. 2,3,5-Triphenyltetrazolium chloride (TTC) staining and Nissl staining were respectively employed to determine the infarct condition and neuronal injury of the brain. Iba1 immunohistochemistry was used to show the activation of microglia. Moreover, the synaptic damage and inflammatory level were detected by Western blot. ResultsGJ-4 could significantly improve memory impairment, cerebral infraction, as well as neurological deficits of VD rats induced by MCAO/R. Further research indicated VD-induced neuronal injury was alleviated by GJ-4. In addition, GJ-4 could protect synapse of VD rats by upregulating synaptophysin (SYP) expression, post synaptic density 95 protein (PSD95) expression, and downregulating N-Methyl-D-Aspartate receptor 1 (NMDAR1) expression. Subsequent investigation of the underlying mechanisms identified that GJ-4 could suppress neuroinflammatory responses, supported by inhibited activation of microglia and reduced expression of inflammatory proteins, which ultimately exerted neuroprotective effects on VD. Further mechanistic study indicated that janus kinase 2 (JAK2)/signal transducer and activator of transcription 1 (STAT1) pathway was inhibited by GJ-4 treatment. ConclusionThese results suggested that GJ-4 might serve as a potential drug to improve VD. In addition, our study indicated that inhibition of neuroinflammation might be a promising target to treat VD.

Catalpol protects vascular structure and promotes angiogenesis in cerebral ischemic rats by targeting HIF-1α/VEGF

BackgroundThe initial factor in the occurrence, development, and prognosis of cerebral ischemia is vascular dysfunction in the brain, and vascular remodeling of the brain is the key therapeutic target and strategy for ischemic tissue repair. Catalpol is the main active component of the radix of Rehmannia glutinosa Libosch, and it exhibits potential pleiotropic protective effects in many brain-related diseases, including stroke. PurposeThe present study was designed to investigate whether catalpol protects vascular structure and promotes angiogenesis in cerebral ischemic rats and to identify its possible mechanisms in vivo and in vitro. Study designCerebral ischemic rats and oxygen-glucose deprivation-exposed brain microvascular endothelial cells were used to study the therapeutic potential of catalpol in vivo and in vitro. MethodsFirst, neurological deficits, histopathological morphology, infarct volume, vascular morphology, vessel density, and angiogenesis in focal cerebral ischemic rats were observed to test the potential treatment effects of catalpol. Then, oxygen-glucose deprivation-exposed brain microvascular endothelial cells were used to mimic the pathological changes in vessels during ischemia to study the effects and possible mechanisms of catalpol in protecting vascular structure and promoting angiogenesis. ResultsThe in vivo results showed that catalpol reduced neurological deficit scores and infarct volume, protected vascular structure, and promoted angiogenesis in cerebral ischemic rats. The in vitro results showed that catalpol improved oxygen-glucose deprivation-induced damage and promoted proliferation, migration, and in vitro tube formation of brain microvascular endothelial cells. The HIF-1α (hypoxia-inducible factor 1α)/VEGF (vascular endothelial growth factor) pathway was activated by catalpol both in the brains of cerebral ischemic rats and in primary brain microvascular endothelial cells, and the activating effects of catalpol were inhibited by SU1498. ConclusionThe results of both the in vivo and in vitro studies proved that catalpol protects vascular structure and promotes angiogenesis in focal cerebral ischemic rats and that the mechanism is dependent on HIF-1α/VEGF.

Tanshinone IIA Promotes Axonal Regeneration in Rats with Focal Cerebral Ischemia Through the Inhibition of Nogo-A/NgR1/RhoA/ROCKII/MLC Signaling.

PurposeThe aim of this study was to evaluate the neuroprotective effect of tanshinone IIA (TSA) on focal cerebral ischemia in rats and to investigate whether it was associated with Nogo-A/NgR1/RhoA/Rho-associated protein kinase 2 (ROCKII)/myosin light chain (MLC) signaling.MethodsIn this study, focal cerebral ischemia animal model was used. Neurological deficit scores and infarction volume were investigated to evaluate the neuroprotection of TSA. Hematoxylin-eosin staining, Nissl staining, and immunofluorescence staining were conducted to detect ischemic changes in brain tissue and changes in neurofilament protein 200 (NF200) and growth-associated protein-43 (GAP-43) expression, respectively. Western blotting and qRT-PCR analyses were used to detect the expression levels of NF200, GAP-43 and Nogo-A/NgR1/RhoA/ROCKII/MLC pathway-related signaling molecules.ResultsTSA treatment can improve the survival rate of rats, reduce the neurological score and infarct volume, and reduce neuron damage. In addition, TSA also increased axon length and enhanced expression of NF200 and GAP-43. Importantly, TSA significantly attenuated the expression of Nogo-A, NgR1, RhoA, ROCKII, and p-MLC, and thus inhibiting the activation of this signaling pathway.ConclusionTSA promoted axonal regeneration by inhibiting the Nogo-A/NgR1/RhoA/ROCKII/MLC signaling pathway, thereby exerting neuroprotective effects in cerebral ischemia rats, which provided support for the clinical application of TSA in stroke treatment.

Morphometric Analysis and Pattern of Protein and Gene Expression of Apoptosis in Focal Cerebral Ischemia in Rats and the Neuroprotetive Action of Hypothermia and Ketoprofen

Morphometric Analysis and Pattern of Protein and Gene Expression of Apoptosis in Focal Cerebral Ischemia in Rats and the Neuroprotetive Action of Hypothermia and Ketoprofen

CD151 Alleviates Early Blood-Brain Barrier Dysfunction After Experimental Focal Brain Ischemia in Rats.

Preservation of the blood-brain barrier (BBB) function is a potential protective strategy against cerebral ischemic injuries. CD151 has a beneficial effect in maintaining vascular stability and plays a role in pro-angiogenesis. Both vascular stability and angiogenesis can affect BBB function. Therefore, we aimed to examine the action of CD151 in regulating BBB permeability after cerebral ischemic injury in the present study. Using a transient focal cerebral ischemia (tFCI) rat model, we established that CD151 overexpression in the brain mitigated the leakage of endogenous IgG at 6-24h after tFCI in vivo. Moreover, we found that CD151 can decrease the diffusion of macromolecules through monolayer brain microvessel endothelial cells (BMVECs) after glucose and oxygen deprivation (OGD)-reoxygenation in vitro. Furthermore, overexpression of CD151 in BMVECs suppressed OGD-reoxygenation-induced F-actin formation and RhoA activity. However, while preserving BBB integrity after tFCI, CD151 overexpression did not affect the post-stroke outcomes. Taken together, the present study demonstrated that CD151 overexpression in the brain protects BBB permeability at early phase after tFCI. CD151 may be a potential target for early BBB protection in ischemic stroke.

Optogenetic translocation of protons out of penumbral neurons is protective in a rodent model of focal cerebral ischemia

BackgroundIntracellular acidosis in the ischemic penumbra can contribute to further cell death, effectively enlarging the infarct core. Restoring the acid-base balance may enhance tissue survivability after cerebral ischemia. ObjectiveThis study investigated whether translocating protons out of penumbral neurons could mitigate tissue acidification and induce neuroprotection in a rodent model of acute cerebral ischemia. MethodsWe modulated the penumbral neurons via a light-driven pump to translocate protons out (i.e., archaerhodopsin/ArchT group) or into (i.e., channelrhodopsin-2/ChR2 group) neurons after focal cerebral ischemia in rats. Intracellular pH values were imaged via neutral red (NR) fluorescence and cerebral blood flow (CBF) was monitored through laser speckle contrast imaging (LSCI). Global CBF responses to electrical stimulation of the hindlimbs were obtained 24 h and 48 h after ischemia to assess neurological function. Behavioral and histological outcomes were evaluated 48 h after ischemia. A control group without gene modification was included. ResultsThe reduction of relative pH (RpH), the amplitude of negative peak of hypoemic response (RNP) and the hemispheric lateralization index (LI) in ArchT group were significantly less than those of the ChR2 or control group. Moreover, RpH was strongly correlated with RNP (r = 0.60) and LI (r24h = 0.80, r48h = 0.59). In addition, behavioral and histological results supported a neuroprotective effect of countering neuronal acidosis in penumbra through optogenetic stimulation. Conclusion(s)These results indicate that countering intracellular acidosis by optogenetically translocating protons out of penumbral neurons during the acute ischemic stage could induce protection after ischemic brain injury.

Brozopine Inhibits 15-LOX-2 Metabolism Pathway After Transient Focal Cerebral Ischemia in Rats and OGD/R-Induced Hypoxia Injury in PC12 Cells.

The goal of this study was to elucidate the mechanisms of protection of Sodium (±)-5-bromo-2-(α-hydroxypentyl) benzoate (trade name: Brozopine, BZP) against cerebral ischemia in vivo and in vitro. To explore the protective effect of BZP on focal cerebral ischemia-reperfusion injury, we evaluated the effects of various doses of BZP on neurobehavioral score, cerebral infarction volume, cerebral swelling in MCAO rats (ischemia for 2 h, reperfusion for 24 h). In addition, the effects of various doses of BZP on OGD/R-induced-PC12 cells injury (hypoglycemic medium containing 30 mmol Na2S2O4 for 2 h, reoxygenation for 24 h) were evaluated. Four in vivo and in vitro groups were evaluated to characterize targets of BZP: Control group, Model group, BZP group (10 mg/kg)/BZP group (30 μmol/L), C8E4 group (10 mg/kg)/C8E4 group (30 μmol/L). An ELISA kit was used to determine the levels of 15-HETE (a 15-LOX-2 metabolite) in vivo and in vitro. Rat nuclear factor κB subunit p65 (NF-κB p65), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and intercellular adhesion molecule-1 (ICAM-1) were also quantified in vivo and in vitro. The results showed that BZP improved focal cerebral ischemia-reperfusion injury in rats and PC12 cells treated with Na2S2O4 in dose/concentration-dependent manners through inhibition of production of 15-HETE and expression of NF-κB, IL-6, TNF-α, and ICAM-1. In conclusion, BZP exerted protective effects against cerebral ischemia via inhibition of 15-LOX-2 activity.

Abstract WP159: FPS-ZM1 Alleviates Neuroinflammation in Focal Cerebral Ischemia Rats via Blocking the RAGE/DIAPH1/NF-kappa B Pathway

Introduction: The receptor for advanced glycation end-products (RAGE), belonging to the cell surface immunoglobulin superfamily, is a multi-ligands receptor. It is reported that RAGE plays a crucial role in neuroinflammation and neurodegeneration diseases. Objective: In this study, we investigate the pathophysiological effect of RAGE and the therapeutically effect of its specific antagonist FPS-ZM1 in focal cerebral ischemia rats to provide an experimental evidence for the future clinical translational research. Methods: Sprague-Dawley male rats were anesthetized to receive the permanent middle cerebral artery occlusion (MCAO) or sham operation. After the operation, four groups of rats, the MCAO+vehicle, MCAO+FPS-ZM1, sham+vehicle and sham+FPS-ZM1 groups, were treated with FPS-ZM1 or vehicle as indicated above. One week later, neurological function was evaluated and then brain tissues were collected, along with 2,3,5-triphenyltetrazolium chloride staining, Nissl staining, TUNEL staining, Western blot and immunohistochemistry. Results: Treatment with FPS-ZM1 significantly attenuated the neurological deficit and reduced the infarct areas and necrotic neurons number. More interestingly, FPS-ZM1 decreased the ischemia-induced elevated levels of pro-inflammatory cytokines, inhibited the cell apoptosis and prevented the deposition of amyloid-beta and advanced glycation end products. FPS-ZM1 blocked the increase in RAGE levels and specifically, in levels of DIAPH1, the key cytoplasmic hub for RAGE ligand-mediated activation of cellular signaling. Downstream of RAGE/ DIAPH1, the increase in the protein level of phosphorylated NF-kappa B was also reversed by FPS-ZM1 in focal cerebral ischemia rats. Conclusion: These results illuminate that FPS-ZM1 treatment reduce neuroinflammation in focal cerebral ischemia rats and suggest that the RAGE/DIAPH1/NF-kappa B signaling pathway may contribute to alleviation of neuroinflammation by FPS-ZM1 in focal cerebral ischemia rats.

Epigallocatechin gallate alleviates neuronal cell damage against focal cerebral ischemia in rats.

Cerebral ischemia is a neurological disorder that causes permanent disability and is sometimes fatal. Epigallocatechin gallate (EGCG) is a natural polyphenol that exerts beneficial antioxidant and anti-inflammatory effects. The aim of this study was to investigate the neuroprotective effects of EGCG against cerebral ischemia. Middle cerebral artery occlusion was surgically initiated to induce focal cerebral ischemia in adult male rats. EGCG (50 mg/kg) or vehicle was intraperitoneally injected just prior to middle cerebral artery occlusion (MCAO) induction. Neuronal behavior tests were performed 24 hr after MCAO. Brain tissues were isolated to evaluate infarct volume, histological changes, apoptotic cell death, and caspase-3 and poly ADP-ribose polymerase (PARP) levels. MCAO injury led to serious functional neurological deficits and increased infarct volume. Moreover, it induced histopathological lesions and increased the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the cerebral cortex. However, EGCG improved MCAO-induced neurological deficits and reduced infarct volume, alleviated histopathological changes, and decreased TUNEL-positive cells in the cerebral cortex of MCAO rats. Western blot analysis showed increases of caspase-3 and PARP expression levels in MCAO rats with vehicle, whereas EGCG administration alleviated these increases after MCAO injury. These results demonstrate that EGCG exerts a neuroprotective effect by regulating caspase-3 and PARP proteins during cerebral ischemia. In conclusion, we suggest that EGCG acts as a potent neuroprotective agent by modulating the apoptotic signaling pathway.

Effect of 3-Aminobenzamide on the Ultrastructure of Astrocytes and Microvessels After Focal Cerebral Ischemia in Rats.

The disruption of blood–brain barrier (BBB) is a critical event in the formation of brain edema during early phases of ischemic brain injury. Poly(ADP-ribose) polymerase (PARP) activation, which contributes to BBB damage, has been reported in ischemia–reperfusion and traumatic brain injury. Here, we investigated the effect of 3-aminobenzamide (3-AB), a PARP-1 inhibitor, on the ultrastructure of BBB. Male Sprague Dawley rats were suffered from 90 minutes of middle cerebral artery occlusion, followed by 4.5 hours or 22.5 hours of reperfusion (R). The vehicle or 3-AB (10 mg/kg) was administered intraperitoneally (ip) 60 minutes after lacking of blood. Tissue Evans Blue (EB) levels, ultrastructures of astrocytes and microvessels, and areas of perivascular edema were examined in penumbra and core, at I 1.5 hours /R 4.5 hours and I 1.5 hours /R 22.5 hours, respectively. The severity of ultrastructural changes was graded with a scoring system in each group. We showed that 3-AB treatment significantly decreased tissue EB levels and ultrastructural scores, attenuated damages in astrocytes and microvessels, and reduced areas of perivascular edema. In conclusion, PARP inhibition may provide a novel therapeutic approach to ischemic brain injury.