Transient Focal Ischemia In Mice Research Articles

Expression of miR-200c corresponds with increased reactive oxygen species and hypoxia markers after transient focal ischemia in mice

Embolic stroke results in a necrotic core of cells destined to die, but also a peri-ischemic, watershed penumbral region of potentially salvageable brain tissue. Approaches to effectively differentiate between the ischemic and peri-ischemic zones is critical for novel therapeutic discovery to improve outcomes in survivors of stroke. MicroRNAs are a class of small non-coding RNAs regulating gene translation that have region- and cell-specific expression and responses to ischemia. We have previously reported that global inhibition of cerebral microRNA-200c after experimental stroke in mice is protective, however delineating the post-stroke sub-regional and cell-type specific patterns of post-stroke miR-200c expression are necessary to minimize off-target effects and advance translational application. Here, we detail a novel protocol to visualize regional miR-200c expression after experimental stroke, complexed with visualization of regional ischemia and markers of oxidative stress in an experimental stroke model in mice. In the present study we demonstrate that the fluorescent hypoxia indicator pimonidazole hydrochloride, the reactive-oxygen-species marker 8-hydroxy-deoxyguanosine, neuronal marker MAP2 and NeuN, and the reactive astrocyte marker GFAP can be effectively complexed to determine regional differences in ischemic injury as early as 30 min post-reperfusion after experimental stroke, and can be effectively used to distinguish ischemic core from surrounding penumbral and unaffected regions for targeted therapy. This multi-dimensional post-stroke immunofluorescent imaging protocol enables a greater degree of sub-regional mechanistic investigation, with the ultimate goal of developing more effective post-stroke pharmaceutical therapy.

Imaging of reactive oxygen species in focal ischemic mouse brain using a radical trapping tracer [3H]hydromethidine

BackgroundReactive oxygen species (ROS) have been implicated in the pathophysiology of the brain after ischemic stroke. In this study, we investigate the generation of brain ROS after transient focal ischemia in mice using a radical trapping radiotracer, [3H]-labeled N-methyl-2,3-diamino-6-phenyl-dihydrophenanthridine ([3H]hydromethidine), which we recently reported as a ROS imaging probe. We also examined the effect of dimethylthiourea (DMTU), a hydroxyl radical scavenger, on brain ROS generation and infarct volume after transient focal ischemia in mice.Methods[3H]Hydromethidine was intravenously injected into mice at 1, 2, 5, and 7 h after transient middle cerebral artery occlusion (tMCAO), and then, the brain autoradiogram was acquired at 60 min after tracer injection. Brain infarct volumes at 24 h after tMCAO were assessed by 2,3,5-triphenyltetrazolium chloride staining.ResultsAccumulation of radioactivity was observed in the ipsilateral striatum and cortex at 1 h after tMCAO. The increase of radioactivity was attenuated at 2 h after tMCAO and then became maximized at 5 h. The high accumulation of radioactivity remained until 7 h after tMCAO. DMTU treatment significantly attenuated the accumulation of radioactivity in the ipsilateral hemisphere at 1, 5, and 7 h after tMCAO. Brain infarct volumes were also significantly reduced in DMTU-treated mice at 24 h after tMCAO.ConclusionsThese results indicated that [3H]hydromethidine is a useful radiotracer for detecting in vivo brain ROS generation such as hydroxyl radical after ischemic injury.

Abstract 3494: LOXblock-1 Provides Efficient, Specific, And Long-lasting Neuroprotection Against Transient Focal Ischemia In Mice By Inhibiting 12/15-Lipoxygenase

Background and Purpose - Acute phase stroke treatment options are currently very limited. We have previously demonstrated 12/15-lipoxygenase (12/15-LOX) to initiate an apoptotic cell death cascade, contributing to neuronal cell death, but also vascular leakage and edema formation in the ischemic brain. The current study was designed to test if a novel inhibitor LOXBlock-1, can specifically reduce 12/15-LOX activity in vivo, and provide efficient and long-lasting protection against stroke-induced injury. Methods - In vitro protection by LOXBlock-1 was tested in HT22 cells. A cell-based reporter assay determined activation of HIF1a and NRF2. The lipoxygenase metabolite 12-HETE was measured using a commercial EIA kit, and confirmed by HPLC/mass spectrometry. We injected LOXBlock-1 intraperitoneally in mice subjected to middle cerebral artery occlusion (MCAO). Immunohistochemistry was used to detect MDA-modified lysine in the ischemic brain, TTC and H&E stains were used to determine infarct size. Neurological severity score and corner test were used to evaluate behavioral outcome. Intracerebral hemorrhage was induced by collagenase injection. Results - Nanomolar LOXBlock-1 protected HT22 cells against oxidative glutamate toxicity. LOXBlock-1 reduced 12-HETE levels, without activating HIF1a- and NRF2-dependent pathways. One day after MCAO, MDA-modified lysine co-localized with 12/15-LOX in the peri-infarct cortex, and LOXBlock-1 reduced hemispheric swelling, levels of 12-HETE, and infarct size. After 14 days, infarct size remained reduced, and behavioral outcome was improved. LOXBlock-1 did not increase collagenase-induced hemorrhage. Conclusion - 12/15-LOX inhibition by LOXBlock-1 provides effective and long-lasting protection against transient focal ischemia in mice, without worsening hemorrhage. LOXBlock-1 may be a suitable acute phase stroke treatment.

Gabapentin reduces infarct volume but does not suppress peri-infarct depolarizations

Spreading depression (SD) is an intense depolarization wave implicated in brain injury. In focal ischemia, recurrent peri-infarct depolarization (PID) waves akin to SD worsen the ischemic injury by exacerbating the blood flow-metabolism mismatch. We recently showed that gabapentin suppresses SD. We, therefore, tested gabapentin on PIDs and stroke outcome. Gabapentin pretreatment (200 mg/kg, intravenously) reduced the infarct volume by 23% after transient focal ischemia in mice. However, the frequency and duration of PIDs were not suppressed when recorded for 2 hours during ischemia, suggesting that gabapentin reduces infarct volume independent of PID suppression.

P90RSK activation contributes to cerebral ischemic damage via phosphorylation of Na+/H+ exchanger isoform 1

Excessive activation of Na+/H+ exchanger isoform 1 (NHE-1) plays a role in cerebral ischemic injury. The current study investigated whether NHE-1 protein in ischemic brains is regulated by extracellular signal-regulated kinase (ERK)/90-kDa ribosomal S6 kinase (p90(RSK)) signaling pathways. A transient focal ischemia in mice was induced by a 60-min-occlusion of the middle cerebral artery followed by reperfusion for 3, 10, or 60 min (Rp). Expression of phosphorylated ERK 1/2 was significantly elevated in the ipsilateral hemispheres at 3-10 min Rp and reduced by 60 min Rp. An increase in phosphorylation of p90(RSK), a known NHE-1 kinase, was also detected at 3-10 min Rp, which was accompanied with a transient elevation of NHE-1 phosphorylation (p-NHE-1). Stimulation of p90(RSK) in ischemic neurons was downstream of ERK activation because inhibition of MEK1 (MAP kinase/ERK kinase) with its inhibitor U0126 blocked phosphorylation of p90(RSK). Moreover, direct inhibition of p90(RSK) by its selective inhibitor fluoromethyl ketone not only reduced p-NHE-1 expression but also ischemic infarct volume. Taken together, our study revealed that reperfusion triggers a transient stimulation of the ERK/p90(RSK) pathway. p90(RSK) activation contributes to cerebral ischemic damage in part via activation of NHE-1 protein.

The usefulness of operant conditioning procedures to assess long-lasting deficits following transient focal ischemia in mice

In this study, we examined a number of short and long-term sensorimotor, behavioural and cognitive consequences of an experimental ischemia induced by a 60-min right middle cerebral artery occlusion (MCAO) in 129S2 mice. During 14 days after surgery, a classical sensorimotor assessment was conducted using hanging wire test, negative geotaxis test, grip strength test, accelerated rotarod test and locomotor activity-meter. In order to provide a technique for the assessment of more resistant consequences of ischemia on fine psychomotor control, the peak procedure (a modified version of the operant fixed-interval schedule of reinforcement) was used. This procedure also helped to objectify temporal perception in mice five weeks following surgery. On several sensorimotor tests, ischemic mice showed some degree of impairment which rapidly tended to improve after stroke, a profile of results substantially consistent with previous studies. Five weeks post-surgery, ischemic mice tested with the peak procedure exhibited a moderate but yet significant temporal regulation impairment along with a reduced response rate compared to control mice. The present results suggest that the peak procedure and other derived operant schedules of reinforcement may provide useful and sensitive tools for the long-term assessment of both behavioural and cognitive aspects of the consequences of an experimental ischemia.

Protein Kinase A-Dependent Suppression of Reactive Oxygen Species in Transient Focal Ischemia in Adrenomedullin-Deficient Mice

This study was designed to examine the effect of adrenomedullin deficiency on cerebral infarction and the relationship between adrenomedullin and cyclic AMP-protein kinase A pathway in regulating reactive oxygen species (ROS). Adrenomedullin heterozygous and wild-type mice were subjected to 60-mins focal ischemia. We used adrenomedullin heterozygous mice because adrenomedullin homozygotes die in utero. Infarct volume, neurologic deficit scores, and immunohistochemical analyses were evaluated at several time points after ischemia. The infarct volume and neurologic deficit scores were significantly worse in adrenomedullin heterozygous mice. Significant accumulation of inducible nitric oxide, oxidative DNA damage, and lipid peroxidation was noted after reperfusion in adrenomedullin heterozygous mice. Treatment of wild-type mice with H89, a protein kinase A inhibitor, resulted in increased infarct size, and worsening of neurologic deficit score and other parameters to levels comparable to those of adrenomedullin heterozygous mice. In contrast, cilostazol, which increases cyclic AMP, rescued neurologic deficit and ROS accumulation in adrenomedullin heterozygous mice. This study showed that adrenomedullin downregulation results in increase in ROS after transient focal ischemia in mice. The results also indicated that adrenomedullin has an important function against ischemic injury through the cyclic AMP-protein kinase A pathway.

Region-specific, longitudinal assessment of functional recovery of reperfused brain following transient focal ischemia in mice using bicuculline-induced brain activation

Region-specific, longitudinal assessment of functional recovery of reperfused brain following transient focal ischemia in mice using bicuculline-induced brain activation

Expression of tumor necrosis factor α in nerve fibers and oligodendrocytes after transient focal ischemia in mice

The expression of tumor necrosis factor α (TNFα) increases and participates in several central nervous system (CNS) disorders. However, its expression after transient middle cerebral artery occlusion (tMCAO) in mice is not fully discussed yet. Therefore, we examined gene expression and protein localization of TNFα in brain using real-time polymerase chain reaction (PCR) and immunostaining after 1 h tMCAO in mice. After 1 h of ischemic conditions, we observed an increase in the expression of TNFα mRNA from basal level. While the expression decreased immediately to control level after reperfusion, it increased again significantly at 24 and 48 h after tMCAO. TNFα-like immunoreactivity (TNFα-LI) was slightly detected in fibrous structures of the neurons before ischemia. After ischemia, TNFα-LI spread widely to the soma of neurons and became more abundant in the nerve fibers, including axonal and dendritic processes. Moreover, TNFα-LI was also expressed in the oligodendrocytes and, occasionally, in microglia/macrophages, but not in astrocytes 24 h after tMCAO. These results suggest that TNFα shows biphasic expression that corresponds with ischemia and reperfusion, and might play a role in various cells to regulate CNS disorders such as neuronal and oligodendritic cell death after transient ischemia.

Adenovirus-Mediated Bcl-X L Expression Using a Neuron-Specific Synapsin-1 Promoter Protects against Disseminated Neuronal Injury and Brain Infarction Following Focal Cerebral Ischemia in Mice

The effects of an adenovirus-mediated Bcl-X L expression, driven by a neuron-specific human synapsin-1 promoter, on the degree of injury, were examined after transient focal ischemia in mice. Therefore, injections of vehicle, of an adenoviral E1-deleted control vector (Ad-dE1), or a Bcl-X L vector (Ad-Syn-Bcl-X L) were stereotactically made in the striatum. Seven days later, focal ischemia was induced either by 30 min or 2 h of intraluminal thread occlusion. In line with previous data, 30 min of middle cerebral artery (MCA) occlusion reproducibly resulted in disseminated neuronal injury of the striatum, as revealed by cresyl violet and TUNEL 3 days after ischemia. The degree of cell injury was significantly reduced in Ad-Syn-Bcl-X L treated as compared with Ad-dE1 and vehicle-treated animals. On the other hand, 2 h of MCA occlusion produced reproducible infarcts both in vehicle and Ad-dE1 treated animals 24 h after ischemia. The infarct area at the level of the striatum was significantly decreased by Ad-Syn-Bcl-X L treatment. The present data demonstrate that an adenoviral Bcl-X L expression with a neuron-specific synapsin-1 promoter provides a powerful tool, which not only diminishes disseminated neuronal injury, but also protects against tissue infarction.

Induction of peripherin expression in subsets of brain neurons after lesion injury or cerebral ischemia

Peripherin is a type III intermediate filament predominantly expressed in neurons having direct axonal projections toward peripheral structures. Here, we report that brain injuries can trigger expression of peripherin and the formation of peripherin accumulations in neurons that are normally silent for this gene. Stab lesions made with nitrocellulose implants induced within 4 days the formation of peripherin accumulations, devoid of neurofilament proteins, in thalamic neurites at the site of the lesion. The local administration of interleukin-6 or leukemia inhibitory factor at the site of the stab lesion extended the expression pattern of peripherin to other neuronal subsets in areas of the cortex and/or of the hippocampus adjacent to injury. We also show that transient focal ischemia in mice, a model of stroke, can trigger within 72 h the formation of neuronal peripherin accumulations in neurons of the cortex, thalamus and hippocampus. This new type of potentially noxious intermediate filament protein accumulations, composed of peripherin, may be of relevance to many brain degenerative disorders with occurrence of proinflammatory cytokines.

Inhibition of glial glutamate transporter GLT-1 augments brain edema after transient focal cerebral ischemia in mice

Excessively released glutamate is neurotoxic. Glutamate transporters maintain the extracellular level of glutamate by uptake into glia or neurons. We examined the role of GLT-1, a glial glutamate transporter, in brain damage resulting from transient focal ischemia in mice. Heterozygous gene deletion of GLT-1 significantly augmented brain swelling resulting from 1 h of middle cerebral artery occlusion and 24 h reperfusion. In addition, this gene deletion significantly increased brain water contents in ischemic hemisphere at 6 h after reperfusion. Moreover, intraperitoneal injection of dihydrokainate (10 mg/kg), a specific inhibitor of GLT-1, augmented brain swelling. These data suggest that GLT-1 limits brain edema resulting from ischemia.

GTPase RhoB: An Early Predictor of Neuronal Death after Transient Focal Ischemia in Mice

Applying the recently developed DNA array technique to a murine stroke model, we found that the gene coding for RhoB, a member of the family of GTPases that regulate a variety of signal transduction pathways, is upregulated in ischemia-damaged neurons. RhoB immunoreactivity precedes DNA single-strand breaks and heralds the evolving infarct, making it an early predictor of neuronal death. Expression of RhoB colocalized with drastic rearrangement of the actin cytoarchitecture indicates a role for Rho in postischemic morphological changes. Apoptosis in a murine hippocampal cell line was also associated with an early increase in RhoB protein. Activation of caspase-3, a crucial step in apoptosis, could be inhibited by cytochalasin D, a substance that counteracts the actin-modulating activity of Rho GTPases, indicating that Rho proteins may have impact on injury-initiated neuronal signal transduction. Our findings make Rho GTPases potential targets for the development of drugs aimed at limiting neuronal death following brain damage.

Evidence for the involvement of Par-4 in ischemic neuron cell death.

After a stroke many neurons in the ischemic brain tissue die by a process called apoptosis, a form of cell death that may be preventable. The specific molecular cascades that mediate ischemic neuronal death are not well understood. The authors recently identified prostate apoptosis response-4 (Par-4) as a protein that participates in the death of cultured hippocampal neurons induced by trophic factor withdrawal and exposure to glutamate. Here, the authors show that Par-4 levels increase in vulnerable populations of hippocampal and striatal neurons in rats after transient forebrain ischemia; Par-4 levels increased within 6 hours of reperfusion and remained elevated in neurons undergoing apoptosis 3 days later. After transient focal ischemia in mice, Par-4 levels were increased 6 to 12 hours after reperfusion in the infarcted cortex and the striatum, and activation of caspase-8 occurred with a similar time course. Par-4 immunoreactivity was localized predominantly in cortical neurons at the border of the infarct area. A Par-4 antisense oligonucleotide protected cultured hippocampal neurons against apoptosis induced by chemical hypoxia and significantly reduced focal ischemic damage in mice. The current data suggest that early up-regulation of Par-4 plays a pivotal role in ischemic neuronal death in animal models of stroke and cardiac arrest.

A2AAdenosine Receptor Deficiency Attenuates Brain Injury Induced by Transient Focal Ischemia in Mice

Extracellular adenosine critically modulates ischemic brain injury, at least in part through activation of the A(1) adenosine receptor. However, the role played by the A(2A) receptor has been obscured by intrinsic limitations of A(2A) adenosinergic agents. To overcome these pharmacological limitations, we explored the consequences of deleting the A(2A) adenosine receptor on brain damage after transient focal ischemia. Cerebral morphology, as well as vascular and physiological measures (before, during, and after ischemia) did not differ between A(2A) receptor knock-out and wild-type littermates. The volume of cerebral infarction, as well as the associated neurological deficit induced by transient filament occlusion of the middle cerebral artery, were significantly attenuated in A(2A) receptor knock-out mice. This neuroprotective phenotype of A(2A) receptor-deficient mice was observed in different genetic backgrounds, confirming A(2A) receptor disruption as its cause. Together with complimentary pharmacological studies, these data suggest that A(2A) receptors play a prominent role in the development of ischemic injury within brain and demonstrate the potential for anatomical and functional neuroprotection against stroke by A(2A) receptor antagonists.

Early decrease of apurinic/apyrimidinic endonuclease expression after transient focal cerebral ischemia in mice.

The authors examined the protein expression of apurinic/apyrimidinic endonuclease (APE/Ref-1), a multifunctional protein in the DNA base excision repair pathway, before and after transient focal ischemia in mice. Immunohistochemistry showed the nuclear expression of APE/Ref-1 in the entire region of the control brains. Nuclear immunoreactivity was decreased as early as 5 minutes after 60 minutes of ischemia in the ischemic core, which was followed by a significant reduction of APE/Ref-1-positive cells in the entire middle cerebral artery territory. Western blot analysis of the sample from the nonischemic brain showed a characteristic 37-kDa band, which was reduced after ischemia. A significant amount of DNA fragmentation was observed at 24 hours, but not at 4 hours, after ischemia. The authors' data provide the first evidence that APE/Ref-1 rapidly decreases after transient focal ischemia, and that this reduction precedes the peak of DNA fragmentation in the brain regions that are destined to show necrosis and apoptosis. Although further examination is necessary to elucidate the direct relationship between the APE/Ref-1 decrease and ischemic necrosis and apoptosis, our results suggest the possibility that rapid decrease of APE/Ref-1 and the failure of the DNA repair mechanism may contribute to necrosis or apoptosis after transient focal ischemia.

Reduced brain edema and infarction volume in mice lacking the neuronal isoform of nitric oxide synthase after transient MCA occlusion.

Infarct volume and edema were assessed after transient focal ischemia in mice lacking neuronal nitric oxide synthase (NOS) gene expression. With use of an 8-0 coated monofilament, the middle cerebral artery (MCA) of mutant (n = 32) and wild-type mice [SV-129 (n = 31), C57Black/6 (n = 18)] were occluded for 3 h and reperfused for up to 24 h. Regional CBF (rCBF), neurological deficits, water content, and infarct volume were examined in all three strains. rCBF, blood pressure, and heart rate did not differ between groups when measured for 1 h after reperfusion. Neurological deficits were less severe in mutant mice after MCA occlusion. Brain water content at 3 h after reperfusion and infarct volume at 24 h after reperfusion were greater in wild-type mice. These data indicate that genetic deletion of neuronal NOS confers resistance to focal ischemic injury in a reperfusion model. The findings agree with previous studies showing that tissue injury is less extensive after both permanent MCA occlusion and global ischemia in mice lacking neuronal NOS gene expression. Hence, NO may play a pivotal role in the pathogenesis of ischemic brain damage.