Mice and Rats Exhibit Striking Inter-species Differences in Gene Response to Acute Stroke.

Neuroprotection in acute stroke has not been successfully translated from animals to humans. Animal research on promising agents continues largely in rats and mice which are commonly available to researchers. However, controversies continue on the most suitable species to model the human situation. Generally, putative agents seem less effective in mice as compared with rats. We hypothesized that this may be due to inter-species differences in stroke response and that this might be manifest at a genetic level. Here we used whole-genome microarrays to examine the differential gene regulation in the ischemic penumbra of mice and rats at 2 and 6 h after permanent middle cerebral artery occlusion (pMCAO; Raw microarray CEL data files are available in the GEO database with an accession number GSE163654). Differentially expressed genes (adj. p ≤ 0.05) were organized by hierarchical clustering, correlation plots, Venn diagrams and pathway analyses in each species and at each time-point. Emphasis was placed on genes already known to be associated with stroke, including validation by RT-PCR. Gene expression patterns in the ischemic penumbra differed strikingly between the species at both 2 h and 6 h. Nearly 90% of significantly regulated genes and most pathways modulated by ischemia differed between mice and rats. These differences were evident globally, among stroke-associated genes, immediate early genes, genes implicated in stress response, inflammation, neuroprotection, ion channels, and signal transduction. The findings of this study may have significant implications for the choice of species for screening putative stroke therapies.

Plasmin-resistant PSD-95 inhibitors resolve effect-modifying drug-drug interactions between alteplase and nerinetide in acute stroke.

Neuroprotection for acute ischemic stroke is achievable with the eicosapeptide nerinetide, an inhibitor of the protein-protein interactions of the synaptic scaffolding protein PSD-95. However, nerinetide is subject to proteolytic cleavage if administered after alteplase, a standard-of-care thrombolytic agent that nullifies nerinetide's beneficial effects. Here, we showed, on the basis of pharmacokinetic data consistent between rats, primates, and humans, that in a rat model of embolic middle cerebral artery occlusion (eMCAO), nerinetide maintained its effectiveness when administered before alteplase. Because of its short plasma half-life, it can be followed by alteplase within minutes without reducing its neuroprotective effectiveness. In addition, the problem of protease sensitivity is solved by substituting cleavage-prone amino acids from their l- to their d-enantiomeric form. Treatment of rats subjected to eMCAO with such an agent, termed d-Tat-l-2B9c, eliminated protease sensitivity and maintained neuroprotective effectiveness. Our data suggest that both the clinical-stage PSD-95 inhibitor nerinetide and protease-resistant agents such as d-Tat-l-2B9c may be practically integrated into existing stroke care workflows and standards of care.

Deep Brain Stimulation Rescues Memory and Synaptic Activity in a Rat Model of Global Ischemia.

Ischemic stroke is responsible for a large number of neurological deficits including memory impairment. Deep brain stimulation (DBS), a well established therapeutic modality for the treatment of movement disorders, has recently shown potential beneficial effects on memory in animals and patients with Alzheimer's disease. Here, we test DBS for its ability to improve memory impairments by stimulating the entorhinal cortex (EC) in a rat model of global ischemia (GI). Two weeks after GI, adult male rats received high-frequency EC DBS for 1 h, and animals were assessed for changes in locomotor activity, learning, and memory 6 weeks later. GI produced spatial memory impairment that was ameliorated by DBS, with no difference between the group that received DBS for GI (GI-DBS ON group) and nonstroke control groups. Although GI led to a dramatic CA1 neuronal loss that could not be rescued with DBS, stimulation attenuated the reduction of CA1 synaptophysin expression after GI. Further, in vitro slice recordings showed a restoration of typical evoked synaptic dendritic fields in GI-DBS ON animals, indicating that the DBS-induced memory rescue is associated with increased synaptophysin expression and enhanced synaptic function. These results suggest that DBS may ameliorate the functional consequences of cerebral ischemia and point to be a potential new therapeutic approach.SIGNIFICANCE STATEMENT Deep brain stimulation (DBS) is remarkably effective in treating Parkinson's disease and is currently under investigation for the treatment of neuropsychiatric disorders including Alzheimer's disease. Until now, DBS has not been examined for its cognitive benefits in the context of hypoxic-ischemic injuries. Here, we investigated the effect of DBS in a rat model of global ischemia (GI) that mimics the neurological consequences occurring after a cardiac arrest. We show that DBS rescues memory deficits induced by GI and produces changes in synaptic activity in the hippocampus. Novel approaches to improve neurological outcomes after stroke are urgently needed; therefore, the present study highlights a possible role for DBS in the treatment of cognitive impairment associated with ischemia.

Efficacy of the PSD95 inhibitor Tat-NR2B9c in mice requires dose translation between species.

Tat-NR2B9c, a clinical-stage stroke neuroprotectant validated in rats and primates, was recently deemed ineffective in mice. To evaluate this discrepancy, we conducted studies in mice subjected to temporary middle cerebral artery occlusion (tMCAO) for either 30 or 60 min according to the established principles for dose-translation between species. Tat-NR2B9c treatment reduced infarct volume by by 24.5% (p = 0.49) and 26.0% (p = 0.03) for 30 and 60 min tMCAO, respectively, at the rat-equivalent dose of 10 nMole/g, but not at the previously reported 3 nMole/g in mice. Dose translation is thus critical when preclinical experiments are conducted in new species.

Modulation of NMDAR subunit expression by TRPM2 channels regulates neuronal vulnerability to ischemic cell death.

Neuronal vulnerability to ischemia is dependent on the balance between prosurvival and prodeath cellular signaling. In the latter, it is increasingly appreciated that toxic Ca(2+) influx can occur not only via postsynaptic glutamate receptors, but also through other cation conductances. One such conductance, the Transient receptor potential melastatin type-2 (TRPM2) channel, is a nonspecific cation channel having homology to TRPM7, a conductance reported to play a key role in anoxic neuronal death. The role of TRPM2 conductances in ischemic Ca(2+) influx has been difficult to study because of the lack of specific modulators. Here we used TRPM2-null mice (TRPM2(-/-)) to study how TRPM2 may modulate neuronal vulnerability to ischemia. TRPM2(-/-) mice subjected to transient middle cerebral artery occlusion exhibited smaller infarcts when compared with wild-type animals, suggesting that the absence of TRPM2 is neuroprotective. Surprisingly, field potentials (fEPSPs) recorded during redox modulation in brain slices taken from TRPM2(-/-) mice revealed increased excitability, a phenomenon normally associated with ischemic vulnerability, whereas wild-type fEPSPs were unaffected. The upregulation in fEPSP in TRPM2(-/-) neurons was blocked selectively by a GluN2A antagonist. This increase in excitability of TRPM2(-/-) fEPSPs during redox modulation depended on the upregulation and downregulation of GluN2A- and GluN2B-containing NMDARs, respectively, and on augmented prosurvival signaling via Akt and ERK pathways culminating in the inhibition of the proapoptotic factor GSK3ß. Our results suggest that TRPM2 plays a role in downregulating prosurvival signals in central neurons and that TRPM2 channels may comprise a therapeutic target for preventing ischemic damage.

A translational paradigm for the preclinical evaluation of the stroke neuroprotectant Tat-NR2B9c in gyrencephalic nonhuman primates.

Over decades, all attempts to translate acute stroke neuroprotectants from discovery in lower-order species to human clinical use have failed. This raised concerns about the predictive validity of preclinical studies in animals for outcomes in human stroke trials. To bridge this translational gap, we used high-order gyrencephalic nonhuman primates subjected to an experimental protocol that mimicked that of a corresponding, separately reported, clinical trial in which the human subjects underwent endovascular cerebral aneurysm repair. Both placebo-controlled studies tested neuroprotection by Tat-NR2B9c, a prospective therapeutic compound, in anesthetized subjects. Embolic strokes were produced by small intra-arterial emboli caused by the endovascular procedure. We show that primates treated with Tat-NR2B9c after the onset of embolic strokes exhibited significantly reduced numbers and volumes of strokes, as visualized by diffusion- and T2-weighted magnetic resonance imaging. These results correctly anticipated the outcome of the corresponding human trial, thus validating this study design as a predictor of neuroprotective efficacy in humans. This strategy may facilitate the evaluation of promising neuroprotectants before undertaking similar studies in human subjects.

Treatment of stroke with a PSD-95 inhibitor in the gyrencephalic primate brain.

All attempts at treating strokes by pharmacologically reducing the human brain's vulnerability to ischaemia have failed, leaving stroke as a leading cause of death, disability and massive socioeconomic loss worldwide. Over decades, research has failed to translate over 1,000 experimental treatments from discovery in cells and rodents to use in humans, a scientific crisis that gave rise to the prevailing belief that pharmacological neuroprotection is not feasible or practicable in higher-order brains. To provide a strategy for advancing stroke therapy, we used higher-order gyrencephalic non-human primates, which bear genetic, anatomical and behavioural similarities to humans and tested neuroprotection by PSD-95 inhibitors--promising compounds that uncouple postsynaptic density protein PSD-95 from neurotoxic signalling pathways. Here we show that stroke damage can be prevented in non-human primates in which a PSD-95 inhibitor is administered after stroke onset in clinically relevant situations. This treatment reduced infarct volumes as gauged by magnetic resonance imaging and histology, preserved the capacity of ischaemic cells to maintain gene transcription in genome-wide screens of ischaemic brain tissue, and significantly preserved neurological function in neurobehavioural assays. The degree of tissue neuroprotection by magnetic resonance imaging corresponded strongly to the preservation of neurological function, supporting the intuitive but unproven dictum that integrity of brain tissue can reflect functional outcome. Our findings establish that tissue neuroprotection and improved functional outcome after stroke is unequivocally achievable in gyrencephalic non-human primates treated with PSD-95 inhibitors. Efforts must ensue to translate these findings to humans.

Suppression of hippocampal TRPM7 protein prevents delayed neuronal death in brain ischemia.

Cardiac arrest victims may experience transient brain hypoperfusion leading to delayed death of hippocampal CA1 neurons and cognitive impairment. We prevented this in adult rats by inhibiting the expression of transient receptor potential melastatin 7 (TRPM7), a transient receptor potential channel that is essential for embryonic development, is necessary for cell survival and trace ion homeostasis in vitro, and whose global deletion in mice is lethal. TRPM7 was suppressed in CA1 neurons by intrahippocampal injections of viral vectors bearing shRNA specific for TRPM7. This had no ill effect on animal survival, neuronal and dendritic morphology, neuronal excitability, or synaptic plasticity, as exemplified by robust long-term potentiation (LTP). However, TRPM7 suppression made neurons resistant to ischemic death after brain ischemia and preserved neuronal morphology and function. Also, it prevented ischemia-induced deficits in LTP and preserved performance in fear-associated and spatial-navigational memory tasks. Thus, regional suppression of TRPM7 is feasible, well tolerated and inhibits delayed neuronal death in vivo.

Effectiveness of PSD95 inhibitors in permanent and transient focal ischemia in the rat.

BACKGROUND AND PURPOSE: Postsynaptic density-95 inhibitors reduce ischemic brain damage without inhibiting excitatory neurotransmission, circumventing the negative consequences of glutamatergic inhibition. However, their efficacy in permanent ischemia and in providing permanent neuroprotection and neurobehavioral improvement in a practical therapeutic window is unproven. These were tested here under conditions that included fever, which is a common occurrence in clinical stroke. METHODS: Six studies were performed in unfasted Sprague-Dawley rats. Two involved permanent pial vessel occlusion in male and female rats. Two involved permanent middle cerebral artery occlusion, which induced severe hyperthermia, and 2 involved transient middle cerebral artery occlusion. Animals were treated with a single intravenous injection of postsynaptic density-95 inhibitors (Tat-NR2B9c([SDV]) or Tat-NR2B9c([TDV])) 1 hour or 3 hours after stroke. Infarct volumes and neurobehavior were assessed in a blinded manner at 24 hours (pial vessel occlusion and permanent middle cerebral artery occlusion) or at 62 days (transient middle cerebral artery occlusion). RESULTS: Postsynaptic density-95 inhibitors dramatically reduced infarct size in male and female animals exposed to pial vessel occlusion (>50%), in hyperthermic animals with fever exceeding 39 degrees C exposed to permanent middle cerebral artery occlusion (approximately 50%), and at 62 days poststroke in animals exposed to transient middle cerebral artery occlusion (approximately 80%). Effectiveness of postsynaptic density-95 inhibitors was achieved without the drugs affecting body temperature. In transient middle cerebral artery occlusion, a single dose of postsynaptic density-95 inhibitor given 3 hours after stroke onset permanently maintained reduced infarct size and improved neurobehavior. CONCLUSIONS: Postsynaptic density-95 inhibitors administrated 3 hours after stroke onset reduced infarct volumes and improved long-term neurobehavioral functions in a wide therapeutic window. This raises the possibility that they may have future clinical usefulness.

Ischemia and status epilepitcus result in enhanced phosphorylation of calcium and calmodulin-stimulated protein kinase II on threonine 253.

Ca2+-stimulated protein kinase II (CaMKII) is critically involved in the regulation of synaptic function and is implicated in the neuropathology associated with ischemia and status epilepticus (SE). The activity and localization of CaMKII is regulated by multi-site phosphorylation. In the present study we investigated the effects of global ischemia followed by reperfusion and of SE on the phosphorylation of CaMKII on T253 in rat forebrains and compared this to the phosphorylation of T286. Both ischemia and SE resulted in marked increases in the phosphorylation of T253, and this was particularly marked in the postsynaptic density (PSD). Phosphorylation of T286 decreased rapidly towards basal levels following ischemia whereas phosphorylation of T253 remained elevated for between 1 and 6 h before decreasing to control values. Following SE, phosphorylation of T253 remained elevated for between 1 and 3 h before decreasing to control levels. In contrast, phosphorylation of T286 remained elevated for at least 24 h following the termination of SE. Total CaMKII associated with PSDs transiently increased 10 min following ischemia, but only several hours following SE. The results demonstrate that phoshorylation of CaMKII on T253 is enhanced following both ischemia/reperfusion and SE and indicate that the phosphorylation of T253 and T286 are differentially regulated.

Increased phosphorylation and redistribution of NMDA receptors between synaptic lipid rafts and post-synaptic densities following transient global ischemia in the rat brain.

Ischemia results in increased phosphorylation of NMDA receptors. To investigate the possible role of lipid rafts in this increase, lipid rafts and post-synaptic densities (PSDs) were isolated by the extraction of rat brain synaptosomes with Triton X-100 followed by sucrose density gradient centrifugation. Lipid rafts accounted for the majority of PSD-95, whereas SAP102 was predominantly located in PSDs. Between 50 and 60% of NMDA receptors were associated with lipid rafts. Greater than 85-90% of Src and Fyn were present in lipid rafts, whereas Pyk2 was mainly associated with PSDs. Lipid rafts and PSDs were isolated from animals subjected to 15 min of global ischemia followed by 6 h of recovery. Ischemia did not affect the yield, density, flotillin-1 or cholesterol content of lipid rafts. Following ischemia, the phosphorylation of NR1 by protein kinase C and tyrosine phosphorylation of NR2A and NR2B was increased in both lipid rafts and PSDs, with a greater increase in tyrosine phosphorylation occurring in the raft fraction. Following ischemia, NR1, NR2A and NR2B levels were elevated in PSDs and reduced in lipid rafts. The findings are consistent with a model involving close interaction between lipid rafts and PSDs and a role for lipid rafts in ischemia-induced signaling pathways.

Inhibition of protein kinase C reduces ischemia-induced tyrosine phosphorylation of the N-methyl-d-aspartate receptor.

The role of protein kinase C (PKC) in tyrosine phosphorylation of the N-methyl-d-aspartate receptor (NMDAR) following transient cerebral ischemia was investigated. Transient (15 min) cerebral ischemia was produced in adult rats by four-vessel occlusion and animals allowed to recover for 15 or 45 min. Following ischemia, tyrosine phosphorylation of NR2A and NR2B and activated Src-family kinases (SFKs) and Pyk2 were increased in post-synaptic densities (PSDs). Phosphorylation of NR2B on Y1472 by PSDs isolated from post-ischemic forebrains was inhibited by the SFK specific inhibitor PP2, and by the PKC inhibitors GF109203X (GF), Gö6976 and calphostin C. Intravenous injection of GF immediately following the ischemic challenge resulted in decreased phosphorylation of NR1 on PKC phosphorylation sites and reduced ischemia-induced increases in tyrosine phosphorylation of NR2A and NR2B without affecting the increase in total tyrosine phosphorylation of hippocampal proteins. Ischemia-induced increases in activated Pyk2 and SFKs in PSDs, but not the translocation of PKC, Pyk2 or Src to the PSD, were also inhibited by GF. The inactive homologue of GF, bisindolylmaleimide V, had no effect on these parameters. The results are consistent with a role for PKC in the ischemia-induced increase in tyrosine phosphorylation of the NMDAR, via a pathway involving Pyk2 and Src-family kinases.