Melanocortin Derivatives Induced Vascularization and Neuroglial Proliferation in the Rat Brain under Conditions of Cerebral Ischemia.

Stroke remains the second leading cause of death worldwide. The development of new therapeutic agents focused on restoring vascular function and neuroprotection of viable tissues is required. In this study the neuroprotective activity of melanocortin-like ACTH(4-7)PGP and ACTH(6-9)PGP peptides was investigated in rat brain at 24 h after transient middle cerebral artery occlusion (tMCAO). The severity of ischemic damage, changes in the proliferative activity of neuroglial cells and vascularization of rat brain tissue were analyzed. The administration of peptides resulted in a significant increase in the volume density of neurons in the perifocal zone of infarction compared to rats subjected to ischemia and receiving saline. Immunohistochemical analysis of the proliferative activity of neuroglia cells using PCNA antibodies showed a significant increase in the number of proliferating cells in the penumbra and in the intact cerebral cortex of rats receiving peptide treatment. The effect of peptides on vascularization was examined using CD31 antibodies under tMCAO conditions, revealing a significant increase in the volume density of vessels and their sizes in the penumbra after administration of ACTH(4-7)PGP and ACTH(6-9)PGP. These findings confirm the neuroprotective effect of peptides due to the activation of neuroglia proliferation and the enhancement of collateral blood flow.

Synthetic Adrenocorticotropic Peptides Modulate the Expression Pattern of Immune Genes in Rat Brain following the Early Post-Stroke Period.

Ischemic stroke is an acute local decrease in cerebral blood flow due to a thrombus or embolus. Of particular importance is the study of the genetic systems that determine the mechanisms underlying the formation and maintenance of a therapeutic window (a time interval of up to 6 h after a stroke) when effective treatment can be provided. Here, we used a transient middle cerebral artery occlusion (tMCAO) model in rats to study two synthetic derivatives of adrenocorticotropic hormone (ACTH). The first was ACTH(4-7)PGP, which is known as Semax. It is actively used as a neuroprotective drug. The second was the ACTH(6-9)PGP peptide, which is elucidated as a prospective agent only. Using RNA-Seq analysis, we revealed hundreds of ischemia-related differentially expressed genes (DEGs), as well as 131 and 322 DEGs related to the first and second peptide at 4.5 h after tMCAO, respectively, in dorsolateral areas of the frontal cortex of rats. Furthermore, we showed that both Semax and ACTH(6-9)PGP can partially prevent changes in the immune- and neurosignaling-related gene expression profiles disturbed by the action of ischemia at 4.5 h after tMCAO. However, their different actions with regard to predominantly immune-related genes were also revealed. This study gives insight into how the transcriptome depends on the variation in the structure of the related peptides, and it is valuable from the standpoint of the development of measures for early post-stroke therapy.

Isoflurane Anesthesia's Impact on Gene Expression Patterns of Rat Brains in an Ischemic Stroke Model.

BACKGROUND: Ischemic stroke (IS) is one of the most severe brain diseases. Animal models with anesthesia are actively used to study stroke genomics and pathogenesis. However, the anesthesia-related gene expression patterns of ischemic rat brains remain poorly understood. In this study, we sought to elucidate the impact of isoflurane (ISO) anesthesia on the extent of ischemic brain damage and gene expression changes associated with stroke. METHODS: We used the transient middle cerebral artery occlusion (tMCAO) model under long-term and short-term ISO anesthesia, magnetic resonance imaging (MRI), RNA sequencing, and bioinformatics. RESULTS: We revealed that the volume of cerebral damage at 24 h after tMCAO was inversely proportional to the duration of ISO anesthesia. Then, we revealed hundreds of overlapping ischemia-related differentially expressed genes (DEGs) with a cutoff of >1.5; Padj < 0.05, and 694 and 1557 DEGs only under long-term and short-term anesthesia, respectively, using sham-operated controls. Concomitantly, unique DEGs identified under short-term anesthesia were mainly associated with neurosignaling systems, whereas unique DEGs identified under long-term anesthesia were predominantly related to the inflammatory response. CONCLUSIONS: We were able to determine the effects of the duration of anesthesia using isoflurane on the transcriptomes in the brains of rats at 24 h after tMCAO. Thus, specific genome responses may be useful in developing potential approaches to reduce damaged areas after cerebral ischemia and neuroprotection.

Dynamic MRI of the Mesenchymal Stem Cells Distribution during Intravenous Transplantation in a Rat Model of Ischemic Stroke.

Systemic transplantation of mesenchymal stem cells (MSCs) is a promising approach for the treatment of ischemia-associated disorders, including stroke. However, exact mechanisms underlying its beneficial effects are still debated. In this respect, studies of the transplanted cells distribution and homing are indispensable. We proposed an MRI protocol which allowed us to estimate the dynamic distribution of single superparamagnetic iron oxide labeled MSCs in live ischemic rat brain during intravenous transplantation after the transient middle cerebral artery occlusion. Additionally, we evaluated therapeutic efficacy of cell therapy in this rat stroke model. According to the dynamic MRI data, limited numbers of MSCs accumulated diffusely in the brain vessels starting at the 7th minute from the onset of infusion, reached its maximum by 29 min, and gradually eliminated from cerebral circulation during 24 h. Despite low numbers of cells entering brain blood flow and their short-term engraftment, MSCs transplantation induced long lasting improvement of the neurological deficit, but without acceleration of the stroke volume reduction compared to the control animals during 14 post-transplantation days. Taken together, these findings indicate that MSCs convey their positive action by triggering certain paracrine mechanisms or cell-cell interactions or invoking direct long-lasting effects on brain vessels.

Differential gene expression in the contralateral hemisphere of the rat brain after focal ischemia.

Ischemic stroke is one of the most severe polygenic brain diseases. Here, we performed further functional genetic analysis of the processes occurring in the contralateral hemisphere (CH) after ischemia-reperfusion injury in rat brain. Comparison of RNA sequencing data for subcortical samples from the ipsilateral hemisphere (IH) and CH after 90 min of transient middle cerebral artery occlusion (tMCAO) and corresponding sham-operated (SO) controls showed four groups of genes that were associated with ischemic processes in rat brain at 24 h after tMCAO. Among them, 2672 genes were differentially expressed genes (DEGs) for IH but non-DEGs for CH, 34 genes were DEGs for CH but non-DEGs for IH, and 114 genes had codirected changes in expression in both hemispheres. The remaining 16 genes exhibited opposite changes at the mRNA level in the two brain hemispheres after tMCAO. These findings suggest that the ischemic process caused by a focal ischemia induces complex bilateral reactions at the transcriptome level in the rat brain. We believe that specific genome responses in the CH and IH may provide a useful model for the study of the potential for brain repair after stroke.

Insight into Glyproline Peptides' Activity through the Modulation of the Inflammatory and Neurosignaling Genetic Response Following Cerebral Ischemia-Reperfusion.

Glyprolines are Gly-Pro (GP)- or Pro-Gly (PG)-containing biogenic peptides. These peptides can act as neutrophil chemoattractants, or atheroprotective, anticoagulant, and neuroprotective agents. The Pro-Gly-Pro (PGP) tripeptide is an active factor of resistance to the biodegradation of peptide drugs. The synthetic Semax peptide, which includes Met-Glu-His-Phe (MEHF) fragments of adrenocorticotropic hormone and the C-terminal tripeptide PGP, serves as a neuroprotective drug for the treatment of ischemic stroke. Previously, we revealed that Semax mostly prevented the disruption of the gene expression pattern 24 h after a transient middle cerebral artery occlusion (tMCAO) in a rat brain model. The genes of this pattern were grouped into an inflammatory cluster (IC) and a neurotransmitter cluster (NC). Here, using real-time RT-PCR, the effect of other PGP-containing peptides, PGP and Pro-Gly-Pro-Leu (PGPL), on the expression of a number of genes in the IC and NC was studied 24 h after tMCAO. Both the PGP and PGPL peptides showed Semax-unlike effects, predominantly without changing gene expression 24 h after tMCAO. Moreover, there were IC genes (iL1b, iL6, and Socs3) for PGP, as well as IC (iL6, Ccl3, Socs3, and Fos) and NC genes (Cplx2, Neurod6, and Ptk2b) for PGPL, that significantly changed in expression levels after peptide administration compared to Semax treatment under tMCAO conditions. Furthermore, gene enrichment analysis was carried out, and a regulatory gene network was constructed. Thus, the spectra of the common and unique effects of the PGP, PGPL, and Semax peptides under ischemia-reperfusion were distinguished.

Comparative Use of Contralateral and Sham-Operated Controls Reveals Traces of a Bilateral Genetic Response in the Rat Brain after Focal Stroke.

Ischemic stroke is a multifactorial disease with a complex etiology and global consequences. Model animals are widely used in stroke studies. Various controls, either brain samples from sham-operated (SO) animals or symmetrically located brain samples from the opposite (contralateral) hemisphere (CH), are often used to analyze the processes in the damaged (ipsilateral) hemisphere (IH) after focal stroke. However, previously, it was shown that focal ischemia can lead to metabolic and transcriptomic changes not only in the IH but also in the CH. Here, using a transient middle cerebral artery occlusion (tMCAO) model and genome-wide RNA sequencing, we identified 1941 overlapping differentially expressed genes (DEGs) with a cutoff value >1.5 and Padj < 0.05 that reflected the general transcriptome response of IH subcortical cells at 24 h after tMCAO using both SO and CH controls. Concomitantly, 861 genes were differentially expressed in IH vs. SO, whereas they were not vs. the CH control. Furthermore, they were associated with apoptosis, the cell cycle, and neurotransmitter responses. In turn, we identified 221 DEGs in IH vs. CH, which were non-DEGs vs. the SO control. Moreover, they were predominantly associated with immune-related response. We believe that both sets of non-overlapping genes recorded transcriptome changes in IH cells associated with transhemispheric differences after focal cerebral ischemia. Thus, the specific response of the CH transcriptome should be considered when using it as a control in studies of target brain regions in diseases that induce a global bilateral genetic response, such as stroke.

The Impact of Cerebral Perfusion on Mesenchymal Stem Cells Distribution after Intra-Arterial Transplantation: A Quantitative MR Study.

Intra-arterial (IA) mesenchymal stem cells (MSCs) transplantation providing targeted cell delivery to brain tissue is a promising approach to the treatment of neurological disorders, including stroke. Factors determining cell distribution after IA administration have not been fully elucidated. Their decoding may contribute to the improvement of a transplantation technique and facilitate translation of stroke cell therapy into clinical practice. The goal of this work was to quantitatively assess the impact of brain tissue perfusion on the distribution of IA transplanted MSCs in rat brains. We performed a selective MR-perfusion study with bolus IA injection of gadolinium-based contrast agent and subsequent IA transplantation of MSCs in intact rats and rats with experimental stroke and evaluated the correlation between different perfusion parameters and cell distribution estimated by susceptibility weighted imaging (SWI) immediately after cell transplantation. The obtained results revealed a certain correlation between the distribution of IA transplanted MSCs and brain perfusion in both intact rats and rats with experimental stroke with the coefficient of determination up to 30%. It can be concluded that the distribution of MSCs after IA injection can be partially predicted based on cerebral perfusion data, but other factors requiring further investigation also have a significant impact on the fate of transplanted cells.

Genome-Wide RNA-Sequencing Reveals Massive Circular RNA Expression Changes of the Neurotransmission Genes in the Rat Brain after Ischemia-Reperfusion.

Ischemic brain stroke is one of the most serious and socially significant diseases. In addition to messenger RNAs (mRNAs), encoding protein, the study of regulatory RNAs in ischemic has exceptional importance for the development of new strategies for neuroprotection. Circular RNAs (circRNAs) have a closed structure, predominantly brain-specific expression, and remain highly promising targets of research. They can interact with microRNAs (miRNAs), diminish their activity and thereby inhibit miRNA-mediated repression of mRNA. Genome-wide RNA-Seq analysis of the subcortical structures of the rat brain containing an ischemic damage focus and penumbra area revealed 395 circRNAs changed their expression significantly at 24 h after transient middle cerebral artery occlusion model (tMCAO) conditions. Furthermore, functional annotation revealed their association with neuroactive signaling pathways. It was found that about a third of the differentially expressed circRNAs (DECs) originate from genes whose mRNA levels also changed at 24 h after tMCAO. The other DECs originate from genes encoding non-regulated mRNAs under tMCAO conditions. In addition, bioinformatic analysis predicted a circRNA-miRNA-mRNA network which was associated with the neurotransmission signaling regulation. Our results show that such circRNAs can persist as potential miRNA sponges for the protection of mRNAs of neurotransmitter genes. The results expanded our views about the neurotransmission regulation in the rat brain after ischemia-reperfusion with circRNA action.

A Novel Phenylpyrrolidine Derivative: Synthesis and Effect on Cognitive Functions in Rats with Experimental Ishemic Stroke.

We performed an in silico, in vitro, and in vivo assessment of a potassium 2-[2-(2-oxo-4-phenylpyrrolidin-1-yl) acetamido]ethanesulfonate (compound 1) as a potential prodrug for cognitive function improvement in ischemic brain injury. Using in silico methods, we predicted the pharmacological efficacy and possible safety in rat models. In addition, in silico data showed neuroprotective features of compound 1, which were further supported by in vitro experiments in a glutamate excitotoxicity-induced model in newborn rat cortical neuron cultures. Next, we checked whether compound 1 is capable of crossing the blood-brain barrier in intact and ischemic animals. Compound 1 improved animal behavior both in intact and ischemic rats and, even though the concentration in intact brains was low, we still observed a significant anxiety reduction and activity escalation. We used molecular docking and molecular dynamics to support our hypothesis that compound 1 could affect the AMPA receptor function. In a rat model of acute focal cerebral ischemia, we studied the effects of compound 1 on the behavior and neurological deficit. An in vivo experiment demonstrated that compound 1 significantly reduced the neurological deficit and improved neurological symptom regression, exploratory behavior, and anxiety. Thus, here, for the first time, we show that compound 1 can be considered as an agent for restoring cognitive functions.

Brain Protein Expression Profile Confirms the Protective Effect of the ACTH(4-7)PGP Peptide (Semax) in a Rat Model of Cerebral Ischemia-Reperfusion.

The Semax (Met-Glu-His-Phe-Pro-Gly-Pro) peptide is a synthetic melanocortin derivative that is used in the treatment of ischemic stroke. Previously, studies of the molecular mechanisms underlying the actions of Semax using models of cerebral ischemia in rats showed that the peptide enhanced the transcription of neurotrophins and their receptors and modulated the expression of genes involved in the immune response. A genome-wide RNA-Seq analysis revealed that, in the rat transient middle cerebral artery occlusion (tMCAO) model, Semax suppressed the expression of inflammatory genes and activated the expression of neurotransmitter genes. Here, we aimed to evaluate the effect of Semax in this model via the brain expression profiling of key proteins involved in inflammation and cell death processes (MMP-9, c-Fos, and JNK), as well as neuroprotection and recovery (CREB) in stroke. At 24 h after tMCAO, we observed the upregulation of active CREB in subcortical structures, including the focus of the ischemic damage; downregulation of MMP-9 and c-Fos in the adjacent frontoparietal cortex; and downregulation of active JNK in both tissues under the action of Semax. Moreover, a regulatory network was constructed. In conclusion, the suppression of inflammatory and cell death processes and the activation of recovery may contribute to the neuroprotective action of Semax at both the transcriptome and protein levels.

Intra-Arterial Stem Cell Transplantation in Experimental Stroke in Rats: Real-Time MR Visualization of Transplanted Cells Starting With Their First Pass Through the Brain With Regard to the Therapeutic Action.

Cell therapy is an emerging approach to stroke treatment with a potential to limit brain damage and enhance its restoration after the acute phase of the disease. In this study we tested directly reprogrammed neural precursor cells (drNPC) derived from adult human bone marrow cells in the rat middle cerebral artery occlusion (MCAO) model of acute ischemic stroke using human placenta mesenchymal stem cells (pMSC) as a positive control with previously confirmed efficacy. Cells were infused into the ipsilateral (right) internal carotid artery of male Wistar rats 24 h after MCAO. The main goal of this work was to evaluate real-time distribution and subsequent homing of transplanted cells in the brain. This was achieved by performing intra-arterial infusion directly inside the MRI scanner and allowed transplanted cells tracing starting from their first pass through the brain vessels. Immediately after transplantation, cells were observed in the periphery of the infarct zone and in the brain stem, 15 min later small numbers of cells could be discovered deep in the infarct core and in the contralateral hemisphere, where drNPC were seen earlier and in greater numbers than pMSC. Transplanted cells in both groups could no longer be detected in the rat brain 48-72 h after infusion. Histological and histochemical analysis demonstrated that both the drNPC and pMSC were localized inside blood vessels in close contact with the vascular wall. No passage of labeled cells through the blood brain barrier was observed. Additionally, the therapeutic effects of drNPC and pMSC were compared. Both drNPC and pMSC induced substantial attenuation of neurological deficits evaluated at the 7th and 14th day after transplantation using the modified neurological severity score (mNSS). Some of the effects of drNPC and pMSC, such as the influence on the infarct volume and the survival rate of animals, differed. The results suggest a paracrine mechanism of the positive therapeutic effects of IA drNPC and pMSC infusion, potentially enhanced by the cell-cell interactions. Our data also indicate that the long-term homing of transplanted cells in the brain is not necessary for the brain's functional recovery.

Combined Cell Therapy in the Treatment of Neurological Disorders.

Cell therapy of neurological diseases is gaining momentum. Various types of stem/progenitor cells and their derivatives have shown positive therapeutic results in animal models of neurological disorders and in clinical trials. Each tested cell type proved to have its advantages and flaws and unique cellular and molecular mechanism of action, prompting the idea to test combined transplantation of two or more types of cells (combined cell therapy). This review summarizes the results of combined cell therapy of neurological pathologies reported up to this point. The number of papers describing experimental studies or clinical trials addressing this subject is still limited. However, its successful application to the treatment of neurological pathologies including stroke, spinal cord injury, neurodegenerative diseases, Duchenne muscular dystrophy, and retinal degeneration has been reported in both experimental and clinical studies. The advantages of combined cell therapy can be realized by simple summation of beneficial effects of different cells. Alternatively, one kind of cells can support the survival and functioning of the other by enhancing the formation of optimum environment or immunomodulation. No significant adverse events were reported. Combined cell therapy is a promising approach for the treatment of neurological disorders, but further research needs to be conducted.

Novel Insights into the Protective Properties of ACTH(4-7)PGP (Semax) Peptide at the Transcriptome Level Following Cerebral Ischaemia-Reperfusion in Rats.

Cerebral ischaemia is the most common cause of impaired brain function. Biologically active peptides represent potential drugs for reducing the damage that occurs after ischaemia. The synthetic melanocortin derivative, ACTH(4-7)PGP (Semax), has been used successfully in the treatment of patients with severe impairment of cerebral blood circulation. However, its molecular mechanisms of action within the brain are not yet fully understood. Previously, we used the transient middle cerebral artery occlusion (tMCAO) model to study the damaging effects of ischaemia-reperfusion on the brain transcriptome in rats. Here, using RNA-Seq analysis, we investigated the protective properties of the Semax peptide at the transcriptome level under tMCAO conditions. We have identified 394 differentially expressed genes (DEGs) (>1.5-fold change) in the brains of rats at 24 h after tMCAO treated with Semax relative to saline. Following tMCAO, we found that Semax suppressed the expression of genes related to inflammatory processes and activated the expression of genes related to neurotransmission. In contrast, ischaemia-reperfusion alone activated the expression of inflammation-related genes and suppressed the expression of neurotransmission-related genes. Therefore, the neuroprotective action of Semax may be associated with a compensation of mRNA expression patterns that are disrupted during ischaemia-reperfusion conditions.

Genome-wide transcriptome analysis using RNA-Seq reveals a large number of differentially expressed genes in a transient MCAO rat model.

BACKGROUND: The transient middle cerebral artery occlusion (tMCAO) model is used for studying the molecular mechanisms of ischemic damage and neuroprotection. Numerous studies have demonstrated the role of individual genes and associated signaling pathways in the pathogenesis of ischemic stroke. Here, the tMCAO model was used to investigate the genome-wide response of the transcriptome of rat brain tissues to the damaging effect of ischemia and subsequent reperfusion. RESULTS: Magnetic resonance imaging and histological examination showed that the model of focal ischemia based on endovascular occlusion of the right middle cerebral artery for 90 min using a monofilament, followed by restoration of the blood flow, led to reproducible localization of ischemic damage in the subcortical structures of the brain. High-throughput RNA sequencing (RNA-Seq) revealed the presence of differentially expressed genes (DEGs) in subcortical structures of rat brains resulting from hemisphere damage by ischemia after tMCAO, as well as in the corresponding parts of the brains of sham-operated animals. Real-time reverse transcription polymerase chain reaction expression analysis of 20 genes confirmed the RNA-Seq results. We identified 469 and 1939 genes that exhibited changes in expression of > 1.5-fold at 4.5 and 24 h after tMCAO, respectively. Interestingly, we found 2741 and 752 DEGs under ischemia-reperfusion and sham-operation conditions at 24 h vs. 4.5 h after tMCAO, respectively. The activation of a large number of genes involved in inflammatory, immune and stress responses, apoptosis, ribosome function, DNA replication and other processes was observed in ischemia-reperfusion conditions. Simultaneously, massive down-regulation of the mRNA levels of genes involved in the functioning of neurotransmitter systems was recorded. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that dozens of signaling pathways were associated with DEGs in ischemia-reperfusion conditions. CONCLUSIONS: The data obtained revealed a global profile of gene expression in the rat brain sub-cortex under tMCAO conditions that can be used to identify potential therapeutic targets in the development of new strategies for the prevention and treatment of ischemic stroke.

MRI Guiding of the Middle Cerebral Artery Occlusion in Rats Aimed to Improve Stroke Modeling.

The middle cerebral artery occlusion (MCAO) model in rats closely imitates ischemic stroke and is widely used. Existing instrumental methods provide a certain level of MCAO guidance, but monitoring of the MCA-occluding intraluminal filament position and possible complications can be improved. The goal of this study was to develop a MRI-based method of simultaneous control of the filament position, blood flow in the intracranial vessels, and hemorrhagic complications. Rats were subjected to either MRI-guided MCAO (group 1, n = 51) or MCAO without MRI control (group 2, n = 38). After operation, group 1 rats were transferred into a MRI scanner for the control of the filament position and possible complications. Ninety minutes after the onset of MCAO, the filament was removed in rats of both groups and MRI control of the infarct volume and hemorrhagic complications performed. High-resolution T1- and T2-weighted imaging performed immediately after filament insertion provided visualization of the filament position, blood flow in brain arteries, and complications related to inappropriate filament insertion. It permitted replacement of wrongly positioned filaments and exclusion of animals with complications from the experiment. MRI-based MCAO guiding provided real-time intra-operational monitoring of crucial parameters determining MCAO suitability for stroke modeling, including better assessment of the operation outcomes in individual animals and significant enhancement of the model success rate. The possibility of simultaneous visualization of the filament, blood flow in the arteries, brain tissue, and hemorrhagic complications is the principal advantage of the proposed method over other instrumental methods of MCAO quality control. Graphical Abstract MRI-guided middle cerebral artery occlusion technique permits intra-operational monitoring via direct non-invasive simultaneous visualization of the filament, blood flow in the arteries, brain tissue, and hemorrhagic complications. It provides better assessment of MCAO outcomes in individual animals and significant enhancement of MCAO success rate.

Magnetic resonance spectroscopy of the ischemic brain under lithium treatment. Link to mitochondrial disorders under stroke.

Recent evidence suggests that mitochondria are one of the main factors in the pathogenesis in different organs including brain. The pathogenesis after brain damage is caused not only by the change in bioenergetics, but also involves impairment of alternative functions of mitochondria, particularly those related to the control of cell death. In this study we evaluated partial metabolic pathways under the simulation of a stroke by using the occlusion of the middle cerebral artery in rats. The analysis shows that the induced switch to a non-oxidative energy metabolism (glycolysis) due to the block of tissue oxygen supply does not ensure the adequate supply of the tissue with ATP. Moreover, the well-known acidification of the ischemic tissue is not associated with the so-called traditionally and incorrectly considered "lactic acidosis" (the generation of lactate from glucose by itself does not lead to excessive generation of protons), but occurs because of the consumption of tissue ATP under its reduced resynthesis. Incubation of mitochondria isolated from normal rat brain at neutral and slightly acidic pH, mimicking the intracellular pH of normal and ischemic tissues correspondingly, revealed serious changes in mitochondrial bioenergetics, partially reflected in the magnitude of respiratory control and the basal and maximally stimulated respiration rates. Measurement of available metabolites by (1)H MR spectra of normal and ischemia-damaged brains showed a significant increase in lactate and myo-inositol and a moderate decrease in N-acetylaspartate 24h after reperfusion. Remarkably, the administration of lithium chloride in the reperfusion phase normalized the levels of metabolites. Moreover, the introduction of lithium salts (chloride or succinate) in the bloodstream, restored after ischemia, reduced both the size of the ischemia-induced brain damage and the degree of brain swelling. Besides, post-ischemic introduction of lithium salts largely restored the neurological status of the animal.