Transient Cerebral Ischemia In Mice Research Articles

Long-Term Alterations in Motor Skills, Neurogenesis and Astrocyte Numbers following Transient Cerebral Ischemia in Mice.

Background and Objectives. Neurogenesis is an integral process in post-stroke recovery, involving the recruitment of proliferating neuroblasts from neurogenic niches of the mammal brain. However, the role of neurogenesis in the long-term restoration following ischemic stroke is fragmented. Post-stroke motor dysfunction includes challenges in the proper, coordinated use of hands and is present in roughly two-thirds of human patients. In this study, we investigated chronic behavioral and biochemical alterations after transient cerebral ischemia in adult male mice. Materials and Methods: Twelve-week-old C57BL/6N male mice were used, and fMCAo lasting 60 min was induced. At multiple timepoints after fMCAo induction, a single pellet reaching task was performed. Six months after the procedure, we immunohistochemically determined the number of proliferating neuroblasts (BrdU and DCX-positive) and the number of differentiated astrocytes (GFAP-positive) in both brain hemispheres. Results: The reaching ability of fMCAo mice was impaired from one month to six months after the induction of ischemia. Neuroblast proliferation was increased in the ipsilateral SVZ, whereas GFAP+ cell count was elevated in the hippocampal DG of both hemispheres of the fMCAo group mice. Conclusions: Our current report demonstrates the long-term effects of transient cerebral ischemia on mice functional parameters and neurogenesis progression. Our data demonstrate that transient cerebral ischemia promotes a long-lasting regenerative response in the ipsilateral brain hemisphere, specifically in the neurogenic SVZ and DG regions.

Poststroke Lung Infection by Opportunistic Commensal Bacteria Is Not Mediated by Their Expansion in the Gut Microbiota.

Respiratory and urinary tract infections are frequent complications in patients with severe stroke. Stroke-associated infection is mainly due to opportunistic commensal bacteria of the microbiota that may translocate from the gut. We investigated the mechanisms underlying gut dysbiosis and poststroke infection. Using a model of transient cerebral ischemia in mice, we explored the relationship between immunometabolic dysregulation, gut barrier dysfunction, gut microbial alterations, and bacterial colonization of organs, and we explored the effect of several drug treatments. Stroke-induced lymphocytopenia and widespread colonization of lung and other organs by opportunistic commensal bacteria. This effect correlated with reduced gut epithelial barrier resistance, and a proinflammatory sway in the gut illustrated by complement and nuclear factor-κB activation, reduced number of gut regulatory T cells, and a shift of gut lymphocytes to γδT cells and T helper 1/T helper 17 phenotypes. Stroke increased conjugated bile acids in the liver but decreased bile acids and short-chain fatty acids in the gut. Gut fermenting anaerobic bacteria decreased while opportunistic facultative anaerobes, notably Enterobacteriaceae, suffered an expansion. Anti-inflammatory treatment with a nuclear factor-κB inhibitor fully abrogated the Enterobacteriaceae overgrowth in the gut microbiota induced by stroke, whereas inhibitors of the neural or humoral arms of the stress response were ineffective at the doses used in this study. Conversely, the anti-inflammatory treatment did not prevent poststroke lung colonization by Enterobacteriaceae. Stroke perturbs homeostatic neuro-immuno-metabolic networks facilitating a bloom of opportunistic commensals in the gut microbiota. However, this bacterial expansion in the gut does not mediate poststroke infection.

Possible Role of Nitric Oxide in Anxiety Following Transient Cerebral Ischemia in Mice

The possible role of nitric oxide (NO) in anxiety following transient cerebral ischemia by a 10-min bilateral carotid occlusion was examined in mice. Two days after the ischemia, mice showed a significant decrease in time spent on the open arms in the elevated plus-maze test; and likewise, they showed shortened social interaction time in the social interaction test, suggesting the induction of anxiety. Such anxiety behavior, however, was diminished 7 days after the treatment in both tests. A nonselective nitric oxide synthase (NOS) inhibitor, Nw-nitro-L-arginine methyl ester (L-NAME), and a selective inducible NOS (iNOS) inhibitor, S-ethylisothiourea (EIT), given twice after reperfusion, produced an anxiolytic effect in the elevated plus-maze test 2 days after the ischemia, while only the former produced antianxiety in the social interaction test. A relatively selective neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI), failed to decrease the level of anxiety in both tests. These results suggest that the production of NO participates in the anxiogenic behavior by the ischemia. Furthermore, NO generated by endothelial NOS (eNOS) or eNOS with iNOS, with no involvement of nNOS, plays an important role in the anxiety induced by the ischemia. Thus, we conclude that 10-min bilateral carotid occlusion provides a useful exploratory animal model for anxiety following transient cerebral ischemia.

Abstract TP92: Microglial Kv1.3 Channels Are Rapidly Downregulated Following Transient Ischemic Stroke in Mice

Background: Microglia and CNS-infiltrating macrophages (CNS MPs) perform opposing deleterious pro-inflammatory and protective functions in ischemic stroke. Kv1.3 channels regulate pro-inflammatory microglial functions and are promising therapeutic targets to curb neuroinflammation. Since Kv1.3 channel expression in CNS MPs following ischemic stroke is unknown, we characterized Kv1.3 expression in CNS MPs at different times following ischemic stroke. Methods: In the 30-min transient middle cerebral artery occlusion (MCAO) mouse model, we performed flow cytometric assays of cell surface Kv1.3 expression (fluorescent labeled Kv1.3 blocking peptide ShK-F6CA) and phycoerythrin-microsphere phagocytosis on acutely isolated CNS MPs from ipsi- and contralateral hemispheres (including sham surgery controls) at 30 min, 24h, 48h, 72h and 7d time points. Among CD11b + CNS MPs, resting microglia (CD45 low Ly6c low ), activated microglia (C45 high Ly6c low ) and infiltrating macrophages (CD45 high Ly6c high ) were monitored. By quantitative PCR, we measured Kv1.3, Kir2.1 potassium channel and COX2 gene expression in CD11b + CNS MPs. Results: After 48h following MCAO, proportions of activated microglia and infiltrating macrophages were increased ipsilaterally. Kv1.3 channels were highly expressed in CD45 high Ly6c low activated microglia at baseline but significantly declined after 48 hours ipsilaterally with same trend contralaterally. Kv1.3 mRNA in CNS MPs also showed an overall decreasing trend ipsilateral to MCAO. No changes in phagocytic properties were observed following MCAO. Conclusions: Despite increased proportions of activated microglia and infiltrating macrophages, a precipitous downregulation of Kv1.3 channel expression occurs in CD45 high activated microglia after 48 hours following transient cerebral ischemia in mice. Early microglial Kv1.3 channel blockade effects on neuroinflammation following ischemic stroke need to be determined.

MicroRNA-424 protects against focal cerebral ischemia and reperfusion injury in mice by suppressing oxidative stress.

We previously showed that the microRNA miR-424 protects against permanent cerebral ischemic injury in mice by suppressing microglia activation. This study investigated the role of miR-424 in transient cerebral ischemia in mice with a focus on oxidative stress-induced neuronal injury. Transient cerebral ischemia was induced in C57/BL6 mice by middle cerebral artery occlusion for 1 hour followed by reperfusion (ischemia/reperfusion). The miR-424 level in the peri-infarct cortex was quantified. Mice were also administered miR-424 angomir by intracerebroventricular injection. Cerebral infarct volume, neuronal apoptosis, and levels of oxidative stress markers and antioxidants were evaluated. In an in vitro experiment, primary cortical neurons were exposed to H2O2 and treated with miR-424 angomir, nuclear factor erythroid 2-related factor 2 siRNA, and superoxide dismutase (SOD) inhibitor; cell activity, lactate dehydrogenase release, malondialdehyde level, and manganese (Mn)SOD activity were then evaluated. MiR-424 levels in the peri-infarct cortex increased at 1 and 4 hours then decreased 24 hours after reperfusion. Treatment with miR-424 decreased infarct volume and inhibited neuronal apoptosis after ischemia/reperfusion, reduced reactive oxygen species and malondialdehyde levels in the cortex, and increased the expression and activation of MnSOD as well as the expression of extracellular SOD and the redox-sensitive transcription factor nuclear factor erythroid 2-related factor. In neuronal cultures, miR-424 treatment abrogated H2O2-induced injury, as evidenced by decreased lactate dehydrogenase leakage and malondialdehyde level and increased cell viability and MnSOD activity; the protective effects of miR-424 against oxidative stress were reversed by nuclear factor erythroid 2-related factor knockdown and SOD inhibitor treatment. MiR-424 protects against transient cerebral ischemia/reperfusion injury by inhibiting oxidative stress.

EAAC1 gene deletion alters zinc homeostasis and enhances cortical neuronal injury after transient cerebral ischemia in mice

The excitatory amino acids glutamate and cysteine are actively transported into neurons from the extracellular space by the high affinity glutamate transporter EAAC1. The astrocyte glutamate transporters, GLT1 and GLAST, are the primary mediators of glutamate clearance. EAAC1 has a limited role in this function. However, uptake of cysteine into neurons via EAAC1 contributes to neuronal antioxidant function by providing cysteine substrate for glutathione synthesis. Mice in which the EAAC1 gene has been deleted were seen to have enhanced susceptibility to neuronal oxidative stress and developed brain atrophy and cognitive function decline with aging. The aim of the current study was to evaluate if EAAC1 confers protection against ischemic events. Young adult CD-1 wild-type or EAAC1−/− mice were subjected to 30min of bilateral common carotid artery occlusion and evaluated for neuronal death and zinc translocation. The intensity of TSQ fluorescence in the cytoplasm of cortical neurons in the EAAC1−/− mice was significantly higher than wild-type mice, indicating that the cortical neurons of EAAC1−/− mice contain higher cytoplasmic concentrations of labile (or free) zinc. Zinc translocation into cortical neurons was also enhanced in EAAC1−/− mice. Three days after ischemia, Fluoro-Jade B staining revealed that EAAC1−/− mice had more than twice as many degenerating neurons as wild-type mice. N-acetylcysteine, a membrane-permeant cysteine pro-drug, normalized basal zinc levels, reduced TSQ (+) neurons and reduced ischemic neuronal death in the EAAC1−/− mice when delivered in a pre-treatment fashion. Taken together, this study implicates EAAC1-dependent cysteine uptake as an endogenous source of enhancing antioxidant function and zinc homeostasis in neurons in the ischemic brain.

MCP-1/CCR-2-double-deficiency severely impairs the migration of hematogenous inflammatory cells following transient cerebral ischemia in mice

Monocyte chemoattractant protein-1 (MCP-1) and its receptor CCR-2 are known to play a major role in inflammatory responses after cerebral ischemia. Mice deficient in either MCP-1 or CCR-2 have been reported to develop smaller infarct sizes and show decreased numbers of infiltrating inflammatory cells. In the present study we used green fluorescent protein (GFP) transgenic mice to investigate the effect of MCP-1/CCR-2-double deficiency on the recruitment of inflammatory cells in a model of both, mild and severe cerebral ischemia. We show that MCP-1/CCR-2-double deficiency virtually entirely abrogates the recruitment of hematogenous macrophages and significantly reduces neutrophil migration to the ischemic brain 4 and 7days following focal cerebral ischemia. This argues for a predominant role of the MCP-1/CCR-2 axis in chemotaxis of monocytes despite a wide redundancy in the chemokine-receptor-system. Chemokine analysis revealed that even candidates known to be involved in monocyte and neutrophil recruitment like MIP-1α, CXCL-1, C5a, G-CSF and GM-CSF showed a reduced and delayed or even a lack of relevant compensatory response in MCP-1−/−/CCR-2−/−-mice. Solely, chemokine receptor 5 (CCR-5) increased early in both, but rose above wildtype levels at day 7 in MCP-1−/−/CCR-2−/−-animals, which might explain the higher number of activated microglial cells compared to control mice. Our study was, however, not powered to investigate infarct volumes. Further studies are needed to clarify whether these mechanisms of inflammatory cell recruitment might be essential for early infarct development and final infarct size and to evaluate potential therapeutic implications.

Proteomic approach with LCMS-IT-TOF identified an increase of Rab33B after transient focal cerebral ischemia in mice

BackgroundSeveral proteins are known to be markedly expressed in the brain during cerebral ischemia; however, the changes in protein profiles within the ischemic brain after an ischemic insult have not been fully elucidated. We studied the changes in the ischemic brain proteome after focal cerebral ischemia, induced by middle cerebral artery occlusion (MCAO) in mice.MethodsLCMS-IT-TOF mass spectrometry was used to detect the changes in ischemic brain protein patterns after MCAO. We evaluated the protein expression detected in the ischemic area, by western blotting and immunohistochemistry.ResultsNine unique proteins were identified from the ischemic area at 10 h after ischemic insult. Among these proteins, we focused on Rab33b, a member of RAS oncogene family and we found that Rab33b was up-regulated in the ischemic striatum and the number of Rab33B-positive cells increased in a time-dependent manner. Rab33B colocalized with Iba-1 positive microglia in the ischemic area.ConclusionThese findings suggest that LCMS-IT-TOF is useful for identifying changes in proteins after cerebral ischemia and that Rab33B is partially related to the pathogenesis of transient cerebral ischemia in mice.

The Reck tumor suppressor protein alleviates tissue damage and promotes functional recovery after transient cerebral ischemia in mice

The extracellular matrix (ECM) is important for both structural integrity and functions of the brain. Matrix metalloproteinases (MMPs) play major roles in ECM-remodeling under both physiological and pathological conditions. Reversion-inducing cysteine-rich protein with Kazal motifs (Reck) is a membrane-anchored MMP-regulator implicated in coordinated regulation of pericellular proteolysis. Although patho-physiological importance of MMPs and another group of MMP-regulators, tissue inhibitor of metalloproteinases, in brain ischemia has been demonstrated, little is known about the role of Reck in this process. In this study, we found that Reck is up-regulated in hippocampus and penumbra of subventricular zone after transient cerebral ischemia in mice. Most of the Reck-positive cells found at day 2 after ischemia are positive for Nestin as well as Ki67 and localized to the CA2 region of the hippocampus. At day 7 after ischemia, the Reck-positive cells increased in number, extended processes, expressed the reactive astrocyte marker GFAP and the neuronal marker NF200, and were widely distributed in the hippocampus. In the mutant mice carrying single functional Reck allele (Reck+/-), tissue damage and cell death after cerebral ischemia were augmented, the recovery of long-term potentiation in the hippocampus was compromised, NR2C subunit of NMDA receptor was up-regulated, gelatinolytic activity of MMPs were up-regulated and laminin-immunoreactivity was reduced. Our data implicate Reck in protection of ECM/tissue integrity and promotion of functional recovery in the brain after transient cerebral ischemia.

Protection of hippocampal neurogenesis by TAT-Bcl-x L after cerebral ischemia in mice

Endogenous neurogenesis persists in the subgranular zone (SGZ) of the adult rodent brain. Cerebral ischemia stimulates endogenous neurogenesis involving proliferation, migration and differentiation of SGZ-derived neural precursor cells (NPC). However, the biological meaning of this phenomenon is limited by poor survival of NPC. In order to study the effects of an acute neuroprotective treatment on hippocampal endogenous neurogenesis after transient cerebral ischemia in mice, we applied a fusion protein consisting of the TAT domain of the HI virus with the anti-apoptotic Bcl-x L. Intravenous injection of TAT-Bcl-x L resulted in reduced hippocampal cell injury for up to 4 weeks after stroke as assessed by TUNEL and NeuN staining. This was in line with a TAT-Bcl-x L-mediated reduced postischemic microglia activation. Analysis of endogenous hippocampal cell proliferation revealed an increased number of BrdU + cells in the TAT-Bcl-x L group 4 weeks after stroke compared to animals treated with saline and TAT-HA (negative control). Cell proliferation in non-ischemic sham operated animals was not affected by TAT-Bcl-x L. Twenty-eight days after stroke co-expression of BrdU + cells with the immature neuronal marker doublecortin was significantly increased in TAT-Bcl-x L animals. Although TAT-Bcl-x L treatment also resulted in an increased number of BrdU + cells expressing the mature neuronal marker NeuN, the total amount of these cells was low. These data show that TAT-Bcl-x L treatment yields both postischemic sustained hippocampal neuroprotection and increased survival of NPC rather than an induction of endogenous neurogenesis itself.

TAT-Bcl-x L improves survival of neuronal precursor cells in the lesioned striatum after focal cerebral ischemia

Cerebral ischemia activates endogenous neurogenesis in the subventricular zone (SVZ) and the dentate gyrus. Consecutively, SVZ-derived neural precursors migrate towards ischemic lesions. However, functional relevance of activated neurogenesis is limited by poor survival of new-born precursors. We therefore employed the HI-virus-derived fusion protein TAT-Bcl-x L to study the effects of acute anti-apoptotic treatment on endogenous neurogenesis and functional outcome after transient cerebral ischemia in mice. TAT-Bcl-x L treatment led to significantly reduced acute ischemic cell death (128 ± 23 vs. 305 ± 65 TUNEL + cells/mm 2 in controls) and infarct volumes resulting in less motor deficits and improved spatial learning. It significantly increased survival of doublecortin (Dcx)-positive neuronal precursors (389 ± 96 vs. 213 ± 97 Dcx + cells in controls) but did not enhance overall post-ischemic cell proliferation or lesion-specific neuronal differentiation 28 days after ischemia. Our data demonstrate that post-stroke TAT-Bcl-x L-treatment results in acute neuroprotection, improved functional outcome, and enhanced survival of lesion-specific neuronal precursor cells after cerebral ischemia in mice.

Neovascularization promoted by mononuclear cell transplantation after transient cerebral ischemia in mice

Objectives: Ischemic stroke can cause major disability and a deteriorated quality of life. The purposes of this study were to examine whether neovascularization might be promoted by the intra-arterial transplantation of mononuclear cells (MNCs) in ischemic murine brain and to determine whether the new endothelial cells are derived from the donor or from the recipient.Methods: Transient middle cerebral artery occlusion was accomplished using the intraluminal filament technique. Immediately after the embolus was withdrawn, MNCs harvested from C57BL/6J mice (wild-MNCs) or transgenic mice ubiquitously expressing green fluorescent protein (EGFP-MNCs) were infused intra-arterially. The brains of the mice were then sectioned and stained with anti-CD31 antibody to detect the endothelial cells.Results: At 30 minutes after operation, transplanted EGFP-MNCs were found in the lung, spleen, and liver but not in the brain. At 6 weeks after operation, CD31-positive cells were evident in the murine brain transplanted with MNCs. Furthermore, the CD31-positive cells were stained with anti-GFP antibody in EGFP mice transplanted with wild-MNCs but not in wild-type mice transplanted with EGFP-MNCs.Discussion: This animal model resembles the typical clinical course of acute cerebral infarction followed by thrombolytic therapies in humans and could be used to examine new therapies after intra-arterial thrombolysis. In cerebral ischemia, just as in limb ischemia, transplanted MNCs did not differentiate into endothelial cells but instead promoted the formation of new vessels.Conclusion: In murine brain reperfused post ischemia, intra-arterial MNC transplantation promotes neovascularization. Furthermore, the new endothelial cells are derived from the recipient cells.

Stroke Outcomes in Mice Lacking the Genes for Neuronal Heme Oxygenase-2 and Nitric Oxide Synthase

Heme oxygenase-2 (HO-2) has been suggested to be a cytoprotective enzyme in a variety of in vivo experimental models. HO-2, the constitutive isozyme, is enriched in neurons and, under normal conditions, accounts for nearly all of brain HO activity. HO-2 deletion (HO-2-/-) leads to increased neurotoxicity in cultured brain cells and increased damage following transient cerebral ischemia in mice. Moreover, pharmacologic inhibition of HO activity significantly augments focal ischemic damage in wildtype (WT) mice, but does not further exacerbate it in HO-2-/- mice. The HO system shares some similarities with nitric oxide synthase (NOS), notably their syntheses of carbon monoxide (CO) and nitric oxide (NO), respectively, which are diffusible gases with numerous biological actions, including neurotransmission and vasodilation. While deletion of HO-2 results in greater stroke damage, the pharmacologic inhibition of neuronal nitric oxide synthase (nNOS), or its gene deletion, confers neuroprotection in animal models of transient cerebral ischemia. To investigate the interactions, the outcome of focal cerebral ischemia-reperfusion in double knockout (HO-2-/- X nNOS-/-) mice lacking both genes was compared to control WT mice. Wildtype and double knockout male mice underwent intraluminal middle cerebral occlusion for 2 hours, followed by reperfusion for 22 hours. Outcomes in neurologic deficits and infarct size were determined. No difference was observed between WT and double knockout mice in the volume of infarction, neurologic signs, decrease in relative cerebral blood flow during ischemia, or core body temperature. The results suggest that the deleterious action of nNOS would counteract the role of HO-2 in neuroprotection.

Differential alterations in the expression and activity of matrix metalloproteinases 2 and 9 after transient cerebral ischemia in mice.

Abnormal expression and activity of matrix metalloproteinases (MMPs) may contribute to the pathophysiology of cerebral disease such as ischemic injury. In this study, we compared the cellular localization, expression, and activity of MMP-2 and -9 in relation to the evolution of neuronal damage 24 and 72 h after transient global ischemia. In response to ischemia, there was a generalized increase in cellular MMP-2 immunoreactivity at 24-h reperfusion (in neurons, glia and vessels) whereas at 72-h reperfusion the increase in MMP-2 was predominantly in glia. These glial alterations contributed to a significant increase in pro MMP-2 levels in ischemic regions (P < 0.01) as measured by zymography. In contrast, MMP-9 was predominantly upregulated in neurons and this was significantly different to shams at 24- and 72-h reperfusion after ischemia (P < 0.05). Notably, a dramatic increase in proteolytic activity in neurons was observed 24 h after ischemia and this response was absent at 72 h post-ischemia. The present data are supportive of a role for MMPs in contributing to neuronal injury after ischemia.

Possible role of nitric oxide in anxiety following transient cerebral ischemia in mice.

The possible role of nitric oxide (NO) in anxiety following transient cerebral ischemia by a 10-min bilateral carotid occlusion was examined in mice. Two days after the ischemia, mice showed a significant decrease in time spent on the open arms in the elevated plus-maze test; and likewise, they showed shortened social interaction time in the social interaction test, suggesting the induction of anxiety. Such anxiety behavior, however, was diminished 7 days after the treatment in both tests. A nonselective nitric oxide synthase (NOS) inhibitor, N( omega)-nitro-L-arginine methyl ester (L-NAME), and a selective inducible NOS (iNOS) inhibitor, S-ethylisothiourea (EIT), given twice after reperfusion, produced an anxiolytic effect in the elevated plus-maze test 2 days after the ischemia, while only the former produced antianxiety in the social interaction test. A relatively selective neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI), failed to decrease the level of anxiety in both tests. These results suggest that the production of NO participates in the anxiogenic behavior by the ischemia. Furthermore, NO generated by endothelial NOS (eNOS) or eNOS with iNOS, with no involvement of nNOS, plays an important role in the anxiety induced by the ischemia. Thus, we conclude that 10-min bilateral carotid occlusion provides a useful exploratory animal model for anxiety following transient cerebral ischemia.

Protective effect of Oren-gedoku-to (Huang-Lian-Jie-Du-Tang) against impairment of learning and memory induced by transient cerebral ischemia in mice

The protective effect of Oren-gedoku-to (OGT; Huang-Lian-Jie-Du-Tang), a traditional Chinese medicine, against impairment of learning and memory induced by transient cerebral ischemia was investigated in mice. The cerebral ischemia caused a reduction of step-down latency and an increase of step-down errors in the passive avoidance task. Pretreatment with oral administration of OGT (2, 4 or 8 g of herbs per kg) once daily for 5 days prolonged the step-down latency significantly and decreased the step-down errors as compared with those of sham-operated controls. In the Morris water maze test, the cerebral ischemia caused an increase in the latency until finding the platform in the training trial and a decrease in the percentage of swimming in the quadrant of the former platform in the probe trial. Oren-gedoku-to (OGT; 2, 4 and 8 g/kg, p.o.) shortened the latency of escaping markedly onto the platform in the training trial and increased the percentage of crossing the former platform quadrant in the probe trial. A reference drug, tacrine (0.5 and 1.0 mg/kg, p.o.), prevented the reduction of step-down latency in the passive avoidance task and shortened the escape latency in the Morris water maze task. Furthermore, OGT significantly protected against cerebral ischemia-induced reduction in the acetylcholine (ACh) content of the cerebral cortex, hippocampus and striatum. These results indicate that the protective effects of OGT against the impairment of learning and memory induced by transient cerebral ischemia may be associated with preventing the decrease in the ACh content of the mouse brain.

Salvianolic Acid B Protects the Memory Functions against Transient Cerebral Ischemia in Mice

The objective of this work was to study the protective effects of salvianolic acid B (Sal B) on the dysfunctions of learning and memory induced by transient cerebral ischemia in mice. The mechanisms of its actions also were researched both in vivo and in vitro. The model of dysfunction of learning and memory induced by transient cerebral ischemia in mice was used. One trail passive avoidance tests were used to evaluate the learning and memory functions and experiments in vitro were employed to observe the antioxidative effects of Sal B. Cerebral transient ischemia would impair the function of memory in mice. In step down test, the error number and latency were 2.63 and 120.5 in control group and were 1.35 and 234.4 respectively in sham operated group (p < 0.05). In Sal B treated groups, the error number was less and latency was longer significantly than those of control group. Meanwhile, 3 and 10 mg kg−1 of Sal B iv. reduced the malondialdehyde contents in cortex, hippocampus and striatum of cerebral transient ischemia rat in vivo. Sal B 10–100 nmol L−1 also inhibited lipid-peroxidation and scavenged free hydroxyl radicals in vitro. As conclusion, Sal B ameliorated learning and memory dysfunctions induced by cerebral transient ischemia. Its actions might be related to its antioxidant activity.

U-50,488H, a κ-opioid receptor agonist, markedly prevents memory dysfunctions induced by transient cerebral ischemia in mice

Transient ischemia produced marked memory dysfunctions in mice on three different tasks, spontaneous alternation, elevated plus-maze and passive avoidance, as tested 1, 1–2, and 2–3 days after ischemic insult, respectively. U-50,488H, a κ-opioid receptor, agonist, administered 20 min before ischemic insult markedly prevented the impairment of spontaneous alternation, the prolongation of transfer latency in elevated-plus maze and the shortening of step-through latency in passive avoidance induced by transient ischemia. The protective effect of U-50,488H (30 mg/kg) on ischemia-induced memory dysfunctions observed in the three tasks was almost completely reversed by pretreatment with nor-binaltorphimine (4 μg, i.c.v.), a κ-selective opioid antagonist. Although U-50,488H (30 mg/kg) did not affect body temperature in sham mice, it blocked hypothermia induced by ischemic insult. These results suggest that the protective effect of U-50,488H on memory dysfunctions in the ischemic mice is associated with the activation of κ-opioid receptors and is not based upon hypothermia.

Dynorphin A-(1–13) potently prevents memory dysfunctions induced by transient cerebral ischemia in mice

The effect of dynorphin A-(1–13), an endogenous κ-opioid receptor agonist, on memory dysfunctions induced by transient cerebral ischemia in mice was investigated by using three different tasks, namely, spontaneous alteration, elevated plus-maze performance, and passive avoidance behavior. Transient ischemia produced a marked memory dysfunction in mice, as assessed in the three tasks, which were carried out consecutively 1 to 3 days after the ischemic insult. The i.c.v. injection of dynorphin A-(1–13) before the ischemic insult potently prevented the impairment of spontaneous alternations, the prolongation of transfer latency in the elevated plus-maze and the shortening of step-through latency in the passive avoidance task induced by transient ischemia. Dynorphin A-(1–13) (10 μg), however, did not affect the body temperature of the sham-operated or the ischemic mice. The protective effect of dynorphin A-(1–13) (10 μg) on ischemia-induced memory dysfunctions observed in the three tasks was almost completely reversed by pretreatment with nor-binaltorphimine (4 μg, i.c.v.), a κ-selective opioid receptor antagonist. These results suggest that dynorphin A-(1–13) prevents memory dysfunctions in ischemic mice through the activation of κ-opioid receptors.