Stroke studies in large animals: Prospects of mitochondrial transplantation and enhancing efficiency using hydrogels and nanoparticle-assisted delivery.

One of the most frequent reasons for mortality and disability today is acute ischemic stroke, which occurs by an abrupt disruption of cerebral circulation. The intricate damage mechanism involves several factors, such as inflammatory response, disturbance of ion balance, loss of energy production, excessive reactive oxygen species and glutamate release, and finally, neuronal death. Stroke research is now carried out using several experimental models and potential therapeutics. Furthermore, studies are being conducted to address the shortcomings of clinical care. A great deal of research is being done on novel pharmacological drugs, mitochondria targeting compounds, and different approaches including brain cooling and new technologies. Still, there are many unanswered questions about disease modeling and treatment strategies. Before these new approaches may be used in therapeutic settings, they must first be tested on large animals, as most of them have been done on rodents. However, there are several limitations to large animal stroke models used for research. In this review, the damage mechanisms in acute ischemic stroke and experimental acute ischemic stroke models are addressed. The current treatment approaches and promising experimental methods such as mitochondrial transplantation, hydrogel-based interventions, and strategies like mitochondria encapsulation and chemical modification, are also examined in this work.

Transient Intraluminal Filament Middle Cerebral Artery Occlusion Stroke Model in Rats: A Step-by-Step Guide and Technical Considerations.

OBJECTIVE: Stroke is a leading cause of disability and mortality worldwide. Related research, although already providing significant insights regarding the underlying pathophysiology and potential treatment strategies, has been far from conclusive. Stroke models have been proved of extreme significance for laboratories around the world. In the present report, we have described in detail the most popular to date focal stroke model, the transient intraluminal filament middle cerebral artery occlusion (tifMCAO) model in rats. This model reliably mimics stroke in humans and also approximates endovascular thrombectomy. METHODS: The tifMCAO model was performed using Wistar rats weighing 300-400 g. We have described the surgical technique in a stepwise manner, with figures and/or high-definition video provided for each step. We have also introduced the use of complete arteriotomy of the external carotid artery stump during the procedure. RESULTS: We performed tifMCAO in 65 rats (male and female) involved in various experimental protocols. Although the initial mortality was 48%, practice reduced the rate to 10%. The mean procedural time was 53 minutes (range, 38-85 minutes). In a group of 8 rats ischemia was confirmed in 7 of them, with the stroke induction rate being 87.5%. CONCLUSIONS: The tifMCAO stroke model in rats is the most often used experimental model of focal ischemia because of its clinical relevance. We revisited the procedure and divided it, for instructional purposes, into 15 consecutive and distinct steps.

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.

Cell Therapy of Stroke: Do the Intra-Arterially Transplanted Mesenchymal Stem Cells Cross the Blood-Brain Barrier?

Animal model studies and first clinical trials have demonstrated the safety and efficacy of the mesenchymal stem cells' (MSCs) transplantation in stroke. Intra-arterial (IA) administration looks especially promising, since it provides targeted cell delivery to the ischemic brain, is highly effective, and can be safe as long as the infusion is conducted appropriately. However, wider clinical application of the IA MSCs transplantation will only be possible after a better understanding of the mechanism of their therapeutic action is achieved. On the way to achieve this goal, the study of transplanted cells' fate and their interactions with the blood-brain barrier (BBB) structures could be one of the key factors. In this review, we analyze the available data concerning one of the most important aspects of the transplanted MSCs' action-the ability of cells to cross the blood-brain barrier (BBB) in vitro and in vivo after IA administration into animals with experimental stroke. The collected data show that some of the transplanted MSCs temporarily attach to the walls of the cerebral vessels and then return to the bloodstream or penetrate the BBB and either undergo homing in the perivascular space or penetrate deeper into the parenchyma. Transmigration across the BBB is not necessary for the induction of therapeutic effects, which can be incited through a paracrine mechanism even by cells located inside the blood vessels.

Does the inability of CA1 area to respond to ischemia with early rapid adenosine release contribute to hippocampal vulnerability?: An Editorial Highlight for "Spontaneous, transient adenosine release is not enhanced in the CA1 region of hippocampus during severe ischemia models".

This Editorial highlights a remarkable study in the current issue of the Journal of Neurochemistry in which Ganesana & Venton (2021) report new data showing that brain ischemia does not elicit transient adenosine release in the CA1 hippocampal area. Using fast-scan cyclic voltammetry at a carbon-fiber microelectrode implanted in the CA1 subfield of the hippocampus, it was shown that none of three different ischemia/reperfusion models could increase spontaneous, transient adenosine release, and more severe models even suppressed this presumably neuroprotective release. Since the authors have previously shown that in the caudate putamen, ischemia increased the frequency of spontaneous adenosine release (Ganesana & Venton, 2018), the new data may disclose a mechanism underlying important regional differences in rapid neuroprotective adenosine signaling. The phenomenon of selective susceptibility of the hippocampus to ischemia/hypoxia is well-documented, and the reported failure of its CA1 area to respond to ischemia by rapid adenosine release may be indicative of an insufficiency of this neuroprotective mechanism contributing to hippocampal vulnerability.

Spontaneous, transient adenosine release is not enhanced in the CA1 region of hippocampus during severe ischemia models.

Ischemic stroke causes damage in the brain, and a slow buildup of adenosine is neuroprotective during ischemic injury. Spontaneous, transient adenosine signaling, lasting only 3 s per event, has been discovered that increases in frequency in the caudate-putamen during early stages of mild ischemia-reperfusion injury. However, spontaneous adenosine changes have not been studied in the hippocampus during ischemia, an area highly susceptible to stroke. Here, we investigated changes of spontaneous, transient adenosine in the CA1 region of rat hippocampus during three different models of the varied intensity of ischemia. During the early stages of the milder bilateral common carotid artery occlusion (BCCAO) model, there were fewer spontaneous, transient adenosine, but no change in the concentration of individual events. In contrast, during the moderate 2 vertebral artery occlusion (2VAO) and severe 4 vessel occlusion (4VO) models, both the frequency of spontaneous, transient adenosine and the average event adenosine concentration decreased. Blood flow measurements validate that the ischemia models decreased blood flow, and corresponding pathological changes were observed by transmission electron microscopy (TEM). 4VO occlusion showed the most severe damage in histology and BCCAO showed the least. Overall, our data suggest that there is no enhanced spontaneous adenosine release in the hippocampus during moderate and severe ischemia, which could be due to depletion of the rapidly releasable adenosine pool. Thus, during ischemic stroke, there are fewer spontaneous adenosine events that could inhibit neurotransmission, which might lead to more damage and less neuroprotection in the hippocampus CA1 region. Read the Editorial Highlight for this article on page 800.

Translational Block in Stroke: A Constructive and "Out-of-the-Box" Reappraisal.

Why can we still not translate preclinical research to clinical treatments for acute strokes? Despite > 1000 successful preclinical studies, drugs, and concepts for acute stroke, only two have reached clinical translation. This is the translational block. Yet, we continue to routinely model strokes using almost the same concepts we have used for over 30 years. Methodological improvements and criteria from the last decade have shed some light but have not solved the problem. In this conceptual analysis, we review the current status and reappraise it by thinking "out-of-the-box" and over the edges. As such, we query why other scientific fields have also faced the same translational failures, to find common denominators. In parallel, we query how migraine, multiple sclerosis, and hypothermia in hypoxic encephalopathy have achieved significant translation successes. Should we view ischemic stroke as a "chronic, relapsing, vascular" disease, then secondary prevention strategies are also a successful translation. Finally, based on the lessons learned, we propose how stroke should be modeled, and how preclinical and clinical scientists, editors, grant reviewers, and industry should reconsider their routine way of conducting research. Translational success for stroke treatments may eventually require a bold change with solutions that are outside of the box.

Effect of hemopexin treatment on outcome after intracerebral hemorrhage in mice.

Heme release from hemoglobin may contribute to secondary injury after intracerebral hemorrhage (ICH). The primary endogenous defense against heme toxicity is hemopexin, a 57 kDa glycoprotein that is depleted in the CNS after hemorrhagic stroke. We hypothesized that systemic administration of exogenous hemopexin would reduce perihematomal injury and improve outcome after experimental ICH. Intraperitoneal treatment with purified human plasma hemopexin beginning 2 h after striatal ICH induction and repeated daily for the following two days reduced blood-brain barrier disruption and cell death at 3 days. However, it had no effect on neurological deficits at 4 or 7 days or striatal cell viability at 8 days. Continuous daily hemopexin administration had no effect on striatal heme content at 3 or 7 days, and did not attenuate neurological deficits, inflammatory cell infiltration, or perihematomal cell viability at 8 days. These results suggest that systemic hemopexin treatment reduces early injury after ICH, but this effect is not sustained, perhaps due to an imbalance between striatal tissue heme and hemopexin content at later time points. Future studies should investigate its effect when administered by methods that more efficiently target CNS delivery.

Impact of aging and comorbidities on ischemic stroke outcomes in preclinical animal models: A translational perspective.

Ischemic stroke is a highly complex and devastating neurological disease. The sudden loss of blood flow to a brain region due to an ischemic insult leads to severe damage to that area resulting in the formation of an infarcted tissue, also known as the ischemic core. This is surrounded by the peri-infarct region or penumbra that denotes the functionally impaired but potentially salvageable tissue. Thus, the penumbral tissue is the main target for the development of neuroprotective strategies to minimize the extent of ischemic brain damage by timely therapeutic intervention. Given the limitations of reperfusion therapies with recombinant tissue plasminogen activator or mechanical thrombectomy, there is high enthusiasm to combine reperfusion therapy with neuroprotective strategies to further reduce the progression of ischemic brain injury. Till date, a large number of candidate neuroprotective drugs have been identified as potential therapies based on highly promising results from studies in rodent ischemic stroke models. However, none of these interventions have shown therapeutic benefits in stroke patients in clinical trials. In this review article, we discussed the urgent need to utilize preclinical models of ischemic stroke that more accurately mimic the clinical conditions in stroke patients by incorporating aged animals and animal stroke models with comorbidities. We also outlined the recent findings that highlight the significant differences in stroke outcome between young and aged animals, and how major comorbid conditions such as hypertension, diabetes, obesity and hyperlipidemia dramatically increase the vulnerability of the brain to ischemic damage that eventually results in worse functional outcomes. It is evident from these earlier studies that including animal models of aging and comorbidities during the early stages of drug development could facilitate the identification of neuroprotective strategies with high likelihood of success in stroke clinical trials.

Hypothesis and Theory: A Pathophysiological Concept of Stroke-Induced Acute Phase Response and Increased Intestinal Permeability Leading to Secondary Brain Damage.

Gut integrity impairment leading to increased intestinal permeability (IP) is hypothesized to be a trigger of critically illness. Approximately 15-20% of human ischemic stroke (IS) victims require intensive care, including patients with impaired level of consciousness or a high risk for developing life-threatening cerebral edema. Local and systemic inflammatory reactions are a major component of the IS pathophysiology and can significantly aggravate brain tissue damage. Intracerebral inflammatory processes following IS have been well studied. Until now, less is known about systemic inflammatory responses and IS consequences apart from a frequently observed post-IS immunosuppression. Here, we provide a hypothesis of a crosstalk between systemic acute phase response (APR), IP and potential secondary brain damage during acute and subacute IS stages supported by preliminary experimental data. Alterations of the acute phase proteins (APPs) C-reactive protein and lipopolysaccharide-binding protein and serum level changes of antibodies directed against Escherichia coli-cell extract antigen (IgA-, IgM-, and IgG-anti-E. coli) were investigated at 1, 2, and 7 days following IS in ten male sheep. We found an increase of both APPs as well as a decrease of all anti-E. coli antibodies within 48 h following IS. This may indicate an early systemic APR and increased IP, and underlines the importance of the increasingly recognized gut-brain axis and of intestinal antigen release for systemic immune responses in acute and subacute stroke stages.

Click by Click Microporous Annealed Particle (MAP) Scaffolds.

Macroporous scaffolds are being increasingly used in regenerative medicine and tissue repair. While the recently developed microporous annealed particle (MAP) scaffolds have overcome issues with injectability and in situ hydrogel formation, limitations with respect to tunability to be able to manipulate hydrogel strength and rigidity for broad applications still exist. To address these key issues, here hydrogel microparticles (HMPs) of hyaluronic acid (HA) are synthesized using the thiol-norbornene click reaction and then HMPs are subsequently annealed into a porous scaffold using the tetrazine-norbornene click reaction. This assembly method allows for straightforward tuning of bulk scaffold rigidity by varying the tetrazine to norbornene ratio, with increasing tetrazine resulting in increasing scaffold storage modulus, Young's modulus, and maximum stress. These changes are independent of void fraction. Further incorporation of human dermal fibroblasts throughout the porous scaffold reveals the biocompatibility of this annealing strategy as well as differences in proliferation and cell-occupied volume. Finally, injection of porous HA-Tet MAP scaffolds into an ischemic stroke model shows this chemistry is biocompatible in vivo with reduced levels of inflammation and astrogliosis as previously demonstrated for other crosslinking chemistries.

Short-Chain Fatty Acids Improve Poststroke Recovery via Immunological Mechanisms.

Recovery after stroke is a multicellular process encompassing neurons, resident immune cells, and brain-invading cells. Stroke alters the gut microbiome, which in turn has considerable impact on stroke outcome. However, the mechanisms underlying gut-brain interaction and implications for long-term recovery are largely elusive. Here, we tested the hypothesis that short-chain fatty acids (SCFAs), key bioactive microbial metabolites, are the missing link along the gut-brain axis and might be able to modulate recovery after experimental stroke. SCFA supplementation in the drinking water of male mice significantly improved recovery of affected limb motor function. Using in vivo wide-field calcium imaging, we observed that SCFAs induced altered contralesional cortex connectivity. This was associated with SCFA-dependent changes in spine and synapse densities. RNA sequencing of the forebrain cortex indicated a potential involvement of microglial cells in contributing to the structural and functional remodeling. Further analyses confirmed a substantial impact of SCFAs on microglial activation, which depended on the recruitment of T cells to the infarcted brain. Our findings identified that microbiota-derived SCFAs modulate poststroke recovery via effects on systemic and brain resident immune cells.SIGNIFICANCE STATEMENT Previous studies have shown a bidirectional communication along the gut-brain axis after stroke. Stroke alters the gut microbiota composition, and in turn, microbiota dysbiosis has a substantial impact on stroke outcome by modulating the immune response. However, until now, the mediators derived from the gut microbiome affecting the gut-immune-brain axis and the molecular mechanisms involved in this process were unknown. Here, we demonstrate that short-chain fatty acids, fermentation products of the gut microbiome, are potent and proregenerative modulators of poststroke neuronal plasticity at various structural levels. We identified that this effect was mediated via circulating lymphocytes on microglial activation. These results identify short-chain fatty acids as a missing link along the gut-brain axis and as a potential therapeutic to improve recovery after stroke.

Rapid loss of perihematomal cell viability in the collagenase intracerebral hemorrhage model.

The effective time window of any therapeutic in an experimental stroke model is limited by the rate of injury progression. Intracerebral hemorrhage in rodents is commonly induced by striatal injection of either autologous blood or bacterial collagenase, which digests local blood vessels. During time window studies of the heme oxygenase-1 inducer hemin, which is protective when administered within 1-3 h in both models, the rate of perihematomal injury was directly compared after striatal blood or collagenase injection. Surprisingly, about 80% of the loss of perihematomal cell viability as measured by MTT reduction assay occurred within 6 h of collagenase injection. In contrast, significant viability loss was not observed at this time point after autologous blood injection, but rather it progressed over the subsequent four days to a level similar to that produced by collagenase. Consistent with these observations, systemic hemin therapy reduced blood-brain barrier disruption and perihematomal cell injury when initiated at 6 h after striatal injection of blood but not collagenase. These results indicate that the rate of early cell injury differs markedly in the collagenase and blood injection ICH models, which may contribute to inconsistent results in time window studies. The blood injection model may be more appropriate for prolonged time window studies of a neuroprotective agent.

Modulator of apoptosis-1 is a potential therapeutic target in acute ischemic injury.

Modulator of apoptosis 1 (MOAP-1) is a Bax-associating protein highly enriched in the brain. In this study, we examined the role of MOAP-1 in promoting ischemic injuries following a stroke by investigating the consequences of MOAP-1 overexpression or deficiency in in vitro and in vivo models of ischemic stroke. MOAP-1 overexpressing SH-SY5Y cells showed significantly lower cell viability following oxygen and glucose deprivation (OGD) treatment when compared to control cells. Consistently, MOAP-1-/- primary cortical neurons were observed to be more resistant against OGD treatment than the MOAP-1+/+ primary neurons. In the mouse transient middle cerebral artery occlusion (tMCAO) model, ischemia triggered MOAP-1/Bax association, suggested activation of the MOAP-1-dependent apoptotic cascade. MOAP-1-/- mice were found to exhibit reduced neuronal loss and smaller infarct volume 24 h after tMCAO when compared to MOAP-1+/+ mice. Correspondingly, MOAP-1-/- mice also showed better integrity of neurological functions as demonstrated by their performance in the rotarod test. Therefore, both in vitro and in vivo data presented strongly support the conclusion that MOAP-1 is an important apoptotic modulator in ischemic injury. These results may suggest that a reduction of MOAP-1 function in the brain could be a potential therapeutic approach in the treatment of acute stroke.

Sensory stimulation in acute stroke therapy.

The beneficial effects of cortical activation for functional recovery after ischemic stroke have been well described. However, little is known about the role of early sensory stimulation, i.e. stimulation during first 6 h after stroke onset even during acute treatment. In recent years, various preclinical studies reported significant effects of acute sensory stimulation that range from entire neuroprotection to increased infarct volumes by 30-50%. Systematic knowledge about the effect of acute sensory stimulation on stroke outcome is highly relevant as stroke patients are subject to uncontrolled sensory stimulation during transport, acute treatment, and critical care. This article discusses the current stage of knowledge about acute sensory stimulation and provides directions for future experimental and clinical trials.

Anti-inflammatory treatments for stroke: from bench to bedside.

So far, intravenous tissue-type plasminogen activator (tPA) and mechanical removal of arterial blood clot (thrombectomy) are the only available treatments for acute ischemic stroke. However, the short therapeutic window and the lack of specialized stroke unit care make the overall availability of both treatments limited. Additional agents to combine with tPA administration or thrombectomy to enhance efficacy and improve outcomes associated with stroke are needed. Stroke-induced inflammatory processes are a response to the tissue damage due to the absence of blood supply but have been proposed also as key contributors to all the stages of the ischemic stroke pathophysiology. Despite promising results in experimental studies, inflammation-modulating treatments have not yet been translated successfully into the clinical setting. This review will (a) describe the timing of the stroke immune pathophysiology; (b) detail the immune responses to stroke sift-through cell type; and

The Intersection of Central Dopamine System and Stroke: Potential Avenues Aiming at Enhancement of Motor Recovery.

Dopamine, a major neurotransmitter, plays a role in a wide range of brain sensorimotor functions. Parkinson's disease and schizophrenia are two major human neuropsychiatric disorders typically associated with dysfunctional dopamine activity levels, which can be alleviated through the druggability of the dopaminergic systems. Meanwhile, several studies suggest that optimal brain dopamine activity levels are also significantly impacted in other serious neurological conditions, notably stroke, but this has yet to be fully appreciated at both basic and clinical research levels. This is of utmost importance as there is a need for better treatments to improve recovery from stroke. Here, we discuss the state of knowledge regarding the modulation of dopaminergic systems following stroke, and the use of dopamine boosting therapies in animal stroke models to improve stroke recovery. Indeed, studies in animals and humans show stroke leads to changes in dopamine functioning. Moreover, evidence from animal stroke models suggests stimulation of dopamine receptors may be a promising therapeutic approach for enhancing motor recovery from stroke. With respect to the latter, we discuss the evidence for several possible receptor-linked mechanisms by which improved motor recovery may be mediated. One avenue of particular promise is the subtype-selective stimulation of dopamine receptors in conjunction with physical therapy. However, results from clinical trials so far have been more mixed due to a number of potential reasons including, targeting of the wrong patient populations and use of drugs which modulate a wide array of receptors. Notwithstanding these issues, it is hoped that future research endeavors will assist in the development of more refined dopaminergic therapeutic approaches to enhance stroke recovery.

Inhibiting 12/15-lipoxygenase to treat acute stroke in permanent and tPA induced thrombolysis models.

12/15-Lipoxygenase (12/15-LOX) contributes to the brain damage after middle cerebral artery occlusion (MCAO) in the acute phase of stroke. The aim of this study was to investigate the effects of a 12/15-LOX inhibitor, LOXBlock-1(LB1), in mice using a FeCl3-induced permanent distal MCAO model and FeCl3-induced ischemia/thrombolysis with tPA. In order to induce permanent distal MCAO, 30% FeCl3 was used in C57BL6 mice. LB1 or DMSO treatments were applied intraperitoneally 2 h following MCAO. For FeCl3-induced ischemia/thrombolysis experiments, 10% FeCl3 was preferred so as to obtain reperfusion with tPA in CD1 mice. 4 h following ischemia either LB1 or DMSO and iv tPA was administered. Outcomes were NSS, weight loss, infarct volume, hemorrhage area and reperfusion rate. FeCl3-induced distal MCAO caused an increase in 12/15-LOX signal in the ischemic cortex with an increase in MDA2 and AIF immunoreactivity. LB1 treatment, applied 2 h after ischemia, significantly decreased the infarct volume at 24 h of permanent distal MCAO. Weight loss was also significantly reduced in LB1 treated group. Distal MCAO and tPA application with LB1 or DMSO showed that treatment significantly decreased the infarct volume and the hemorrhage area. The reperfusion rate in the LB1-treated group was surprisingly higher than in the DMSO group and NSS results were significantly improved. These data suggest that LB1 can be used as an adjuvant agent to tPA. This study not only shows the effects of LB1 treatment in distal MCAO but also confirms that FeCl3-induced MCAO model can be a useful tool to screen novel treatment options in stroke.

Cerebral collateral circulation in experimental ischemic stroke.

Cerebral collateral circulation is a subsidiary vascular network, which is dynamically recruited after arterial occlusion, and represents a powerful determinant of ischemic stroke outcome. Although several methods may be used for assessing cerebral collaterals in the acute phase of ischemic stroke in humans and rodents, they are generally underutilized. Experimental stroke models may play a unique role in understanding the adaptive response of cerebral collaterals during ischemia and their potential for therapeutic modulation. The systematic assessment of collateral perfusion in experimental stroke models may be used as a "stratification factor" in multiple regression analysis of neuroprotection studies, in order to control the within-group variability. Exploring the modulatory mechanisms of cerebral collaterals in stroke models may promote the translational development of therapeutic strategies for increasing collateral flow and directly compare them in term of efficacy, safety and feasibility. Collateral therapeutics may have a role in the hyperacute (even pre-hospital) phase of ischemic stroke, prior to recanalization therapies.

[General anesthetics as a factor of effective neuroprotection in ischemic stroke models].

Stroke is the second leading cause of death in the world. Unfortunately, only a few drugs have been proved in clinical trials. Drug development of the last decade has been focused substantially on a promising and heterogeneous group of neuroprotective drugs. Hundreds of compounds were suggested as new putative neuroprotectors, which effectiveness was confirmed in preclinical trials only. At the present time discrepancy between results of preclinical studies and clinical trials requires careful analysis. One of the least evaluated and probably the most noticeable reasons is general anesthesia--an obligatory component of an overwhelming majority of existing animal stroke models. The aim of the review is to describe known mechanisms of common general anesthetics influence on ionotropic and metabotropic plasma membrane receptors, and key signal pathways involved in neuronal hypoxic-ischemic injury and survival.