Angiogenesis, the formation of new blood vessels from preexisting endothelium, is a process that involves a series of interassociated and mutually interactive pathophysiological processes. It is accepted that microRNAs (miRNAs) regulate endothelial cell behavior, including their involvement in angiogenesis. However, it remains unclear whether miRNAs are involved in the regulation of angiogenesis following cerebral ischemia. Therefore, the present study aimed to investigate the role of miRNAs in angiogenesis and the underlying mechanism following cerebral ischemia. Expression profiles of miRNAs in rat brain samples following middle cerebral artery occlusion (MCAO) were investigated using a miRNA microarray. The expression of candidate miRNA, miR‑195 was further validated using reverse transcription‑quantitative polymerase chain reaction. Then, the effects of miR‑195 on cell migration and tube formation of human umbilical vein vascular endothelial cells (HUVECs) were investigated following miR‑195 silencing, and overexpression. The specific target genes of miR‑195 were predicted using microRNA prediction bioinformatics software (http://www.microrna.org/microrna/home.do), and then confirmed using a dual‑luciferase reporter assay and rescue experiment. It was demonstrated that miR‑195 was significantly downregulated in the brains of rats following MCAO and in hypoxia‑induced HUVECs. Furthermore, it was revealed that miR‑195 overexpression inhibited the invasion ability and tube formation of HUVECs in vitro, while miR‑195 silencing enhanced these functions. In addition, vascular endothelial growth factor A (VEGFA) was identified as a direct target of miR‑195 and was negatively correlated with miR‑195 expression. In addition, the rescue experiment revealed that overexpression of VEGFA reversed the inhibitory effects of miR‑195 overexpression on the invasion ability and tube formation of HUVECs. The present study has provided a novel insight into the promoting roles of miR‑195 downregulation on angiogenesis following cerebral infarction and suggests that the miR‑195/VEGFA signaling pathway is a putative therapeutic target in cerebral ischemia.

In the progression of ischemia, pH is important and is essential in elucidating the association between metabolic disruption, lactate formation, acidosis and tissue damage. Chemical exchange‑dependent saturation transfer (CEST) imaging can be used to detect tissue pH and, in particular, a specific form of CEST magnetic resonance imaging (MRI), termed amide proton transfer (APT) MRI, which is sensitive to pH and can detect ischemic lesions, even prior to diffusion abnormalities. The critical parameter governing the ability of CEST to detect pH is the sequence. In the present study, a novel strategy was used, based on the gradient echo sequence (GRE), which involved the insertion of a magnetization transfer pulse in each repetition time (TR) and minimizing the TR for in vivo APT imaging. The proposed GRE‑APT MRI method was initially verified using a tissue‑like pH phantom and optimized MRI parameters for APT imaging. In order to assess the range of acute cerebral infarction, rats (n=4) were subjected to middle cerebral artery occlusion (MCAO) and MRI scanning at 7 telsa (T). Hyperacute ischemic tissue damage was characterized using multiparametric imaging techniques, including diffusion, APT and T2‑Weighted MRI. By using a magnetization transfer pulse and minimizing TR, GRE‑APT provided high spatial resolution and a homogeneous signal, with clearly distinguished cerebral anatomy. The GRE‑APT and diffusion MRI were significantly correlated with lactate content and the area of cerebral infarction in the APT and apparent diffusion coefficient (ADC) maps matched consistently during the hyperacute period. In addition, compared with the infarction area observed on the ADC MRI map, the APT map contained tissue, which had not yet been irreversibly damaged. Therefore, GRE‑APT MRI waa able to detect ischemic lactic acidosis with sensitivity and spatiotemporal resolution, suggesting the potential use of pH MRI as a surrogate imaging marker of impaired tissue metabolism for the diagnosis and prognosis of hyperacute stroke.

Physical exercise is beneficial for the functional recovery of neurons after stroke. It has been suggested that exercise regulates proliferation and differentiation of endogenous neural stem cells (NSCs); however, the underlying molecular mechanisms are still largely unknown. In the present study, the aim was to investigate whether physical exercise activates the extracellular signal‑regulated kinase (ERK) signaling pathway to promote proliferation and differentiation of NSCs in rats with cerebral infarction, thereby improving neurological function. Following middle cerebral artery occlusion, rats underwent physical exercise and neurological behavior was analyzed at various time points. Immunofluorescence staining was performed to detect proliferation and differentiation of NSCs, and western blotting was used to analyze cyclin‑dependent kinase 4 (CDK4), Cyclin D1, retinoblastoma protein (p‑Rb), P‑16, phosphorylated (p)‑ERK1/2 and c‑Fos expression. The results indicated that physical exercise promoted proliferation and differentiation of NSCs, and led to improved neural function. In addition, the expression levels of CDK4, Cyclin D1, p‑Rb, p‑ERK1/2 and c‑Fos were upregulated, whereas the expression of P‑16 was downregulated following exercise. U0126, an inhibitor of ERK signaling, reversed the beneficial effects of exercise. Therefore, it may be hypothesized that physical exercise enhances proliferation and differentiation of endogenous NSCs in the hippocampus of rats with cerebral infarction via the ERK signaling pathway.

The present study aimed to investigate the effects of sevoflurane post‑conditioning in a rat brain cerebral ischemia‑reperfusion (I/R) model and examine its possible mechanism. Rats were randomly divided into six groups: Sham control group (Sham), I/R group, sevoflurane group (Se), Toll‑like receptor‑4 (TLR4) inhibitor group (Tak‑242), nuclear factor (NF)‑κB inhibitor group (QNZ) and Sevoflurane post‑conditioning combined with TLR4‑NF‑κB signaling pathway inhibitor group (Se + Tak‑242). Morris water maze test and tetrazolium chloride staining were used to investigate the I/R injury. The nerve cell apoptosis and autophagy in cortical tissue were detected by TUNEL and transmission electron microscopy, respectively. The expression of TLR4 protein in cortical tissue was observed by immunohistochemical staining. The expression of autophagy and apoptotic associated proteins in cortical tissues and the activity of TLR4‑NF‑κB signaling pathway were assayed by western blot analysis. Sevoflurane post‑conditioning improved the learning and memory dysfunction caused by cerebral I/R injury. The cerebral infarction area, nerve cell apoptosis and formation of autophagic vacuoles were reduced after sevoflurane administration. The expression of light chain 3II/I, Beclin‑1, Bad and Cleaved‑Caspase‑3 proteins were inhibited and the expression of Bcl‑2 protein was upregulated after sevoflurane administration. Sevoflurane post‑conditioning also inhibited the TLR4 protein and NF‑κB phosphorylation, and increased inhibitor of kBα phosphorylation. The treatment effect of Tak‑242 and QNZ groups were not significantly different compared with the Se group (P>0.05), and the Se + Tak‑242 group had the best results. The present study demonstrated that sevoflurane post‑conditioning could protect middle cerebral artery occlusion‑induced brain injury rats by inhibiting autophagy and apoptosis, and that its mechanism is related to the TLR4‑NF‑κB signaling pathway.

Mitochondrial autophagy serves a key role in clearing damaged mitochondria. P‑hydroxybenzyl alcohol (pHBA) can improve neuronal injury induced by cerebral ischemia‑reperfusion (I/R). However, the mechanism of pHBA improving I/R damage through the mitochondrial pathway remains unclear. A rat model of middle cerebral artery occlusion and reperfusion (MCAO/R) was used in the present study. The rats were treated with sirtuin 1 (SIRT1) inhibitor EX527 and pHBA for 7 days, followed by reperfusion. At 24 h after reperfusion, the infarct size was calculated and the severity of nerve damage was evaluated. Hematoxylin and eosin and Nissl staining revealed cellular changes in the ischemic penumbra. Changes in mitochondrial structure were observed using electron microscopy. Mitochondrial function was evaluated by detecting mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (mPTP) and ATP levels using commercially available kits. In addition, the ischemic penumbra tissues were used for immunofluorescence staining for p62 and LC3 proteins. The expression of SIRT1 and mitochondrial autophagy‑related proteins, PTEN‑induced kinase 1 (PINK1) and Parkin, were detected by western blotting. Finally, apoptosis was analyzed by TUNEL staining and the expression of apoptosis‑related proteins (Bax, Bcl‑2 and Caspase‑3) by western blotting. The results suggested that postoperative pHBA treatment may reduce the size of cerebral infarction and damage to the nervous system, and may improve cell damage in the ischemic penumbra of MCAO/R rats. Compared with rats in the untreated MCAO/R group, the mitochondrial structure of the pHBA‑treated group was improved, the levels of MMP and ATP were increased, and the degree of opening of mPTP was decreased. Simultaneously, immunofluorescence and western blotting results showed that compared with the MCAO/R group, the number of LC3‑ and TUNEL‑positive cells increased, the number of p62‑positive cells decreased, SIRT1 and autophagy protein (PINK1, Parkin and LC3 II/I) expression levels increased and p62 expression decreased in the pHBA group. However, these improvements were blocked by treatment with EX527. In summary, results from the present study suggested that pHBA may improve neuronal injury in the ischemic penumbra of MCAO/R rats through SIRT1‑activated mitochondrial autophagy and mitochondrial‑mediated neuronal apoptosis.

There is evidence that the transplantation of mesenchymal stem cells into rat models of cerebral ischemia reduces ischemic damage; however, the mechanism remains to be elucidated. The present study aimed to assess the effect of transplantation of human bone marrow stromal cells (hBMSCs) on neurologic function and the expression of vascular endothelial growth factor (VEGF) in a rat model of focal cerebral ischemia. The left middle cerebral artery of adult Wistar rats was occluded for 90 min using a nylon thread, followed by reperfusion for 1 h. hBMSCs labeled with 5-bromo-2-deoxyuridine (BrdU) were stereotaxically injected into the ischemic boundary zone. Behavioral analysis using the Neurological Severity Score (NSS) was conducted on days 1, 3, 7 and 28, and a histologic evaluation was performed simultaneously. VEGF was detected by immunofluorescence staining and western blot analysis. Fifty rats were divided equally into five groups: Normal control, sham‑operated, operated (no transplantation), Dulbecco's medium Eagle's medium (DMEM)-injected (received only serum-free DMEM), and hBMSC-transplanted. The hBMSC-transplanted group showed significantly improved behavioral recovery compared with the operated and DMEM-transplanted groups on days 3, 7 and 28. Histological examination showed that transplanted cells migrated from the injection site into nearby areas including the cortex. Expression of VEGF was significantly greater in the hBMSC group compared with the other four groups on each assessment day. The expression of VEGF was found to be beneficial for functional recovery following cerebral ischemic injury and hBMSC transplantation stimulated the expression of VEGF. Transplantation of BMSCs may be a promising therapeutic strategy for treating cerebral infarction.

Gastrodia elata Blume has been widely used to treat various central and peripheral nerve diseases, and Para‑hydroxybenzaldehyde (PHBA) is one of the indicated components suggested to provide a neuroprotective effect. In our previous, it was shown that PHBA protected mitochondria against cerebral ischemia‑reperfusion (I/R) injury in rats. In the present study, how PHBA regulated the metabolic mechanism in blood following cerebral I/R was assessed to identify an effective therapeutic target for the prevention and treatment of ischemic stroke (IS). First, a rat model of cerebral ischemia‑reperfusion injury was established via middle cerebral artery occlusion/reperfusion (MCAO/R). The therapeutic effect of PHBA on brain I/R was evaluated by assessing the neurological function score, triphenyl tetrazolium chloride, hematoxylin and eosin, and Nissl staining. Next, a non‑targeted metabolomic based on high‑performance liquid chromatography quadrupole time‑of‑flight mass spectrometry was established to identify differential metabolites. Finally, a targeted metabolic spectrum was analyzed and the potential therapeutic targets were verified by Western blotting. The results showed that the neurological function score, cerebral infarction area, hippocampal morphology, and the number of neurons in the PHBA group were significantly improved compared with the model group. Metabonomic analysis showed that 13 different metabolites were identified between the model and PHBA group, which may be involved in the 'tricarboxylic acid cycle', 'glutathione metabolism', and 'mutual transformation of pentose and glucuronates', amongst others. Among these, the levels of the most significant differential metabolite, dGMP, decreased significantly following PHBA treatment. Western blotting was used to verify the expression of membrane‑associated guanosine kinase PSD‑95 and the subunit of glutamate AMPA receptor GluA1, which significantly increased after PHBA treatment. In addition, it was also found that PHBA increased the expression of the light chain‑3 protein and autophagy effector protein 1, whilst the expression of sequestosome‑1 decreased, indicating that PHBA promoted autophagy. Similarly, in TUNEL staining and detection of apoptosis‑related proteins, it was found that MCAO/R upregulated the expression of Bax and cleaved‑caspase‑3 whilst downregulating the expression of Bcl‑2 and increasing the apoptosis of hippocampal neurons; PHBA reversed this situation. These results suggest that cerebral I/R causes postsynaptic dysfunction by disrupting the interaction between PSD‑95 and AMPARs, and the inhibition of the autophagy system eventually leads to the apoptosis of hippocampal neurons.

Iridoid glycosides of Radix Scrophulariae (IGRS) are a group of the major bioactive components from Radix Scrophulariae with extensive pharmacological activities. The present study investigated the effects of IGRS on cerebral ischemia‑reperfusion injury (CIRI) and explored its potential mechanisms of action. A CIRI model in rats was established by occlusion of the right middle cerebral artery for 90 min, followed by 24 h of reperfusion. Prior to surgery, 30, 60 or 120 mg/kg IGRS was administered to the rats once a day for 7 days. Then, the neurological scores, brain edema and volume of the cerebral infarction were measured. The apoptosis index was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The effects of IGRS on the histopathology of the cortex in brain tissues and the endoplasmic reticulum ultrastructure in the hippocampus were analyzed. Finally, the expression of endoplasmic reticulum stress (ERS)‑regulating mediators, endoplasmic reticulum chaperone BiP (GRP78), DNA damage‑inducible transcript 3 protein (CHOP) and caspase‑12, were detected by reverse transcription quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis. The volume of cerebral infarction and brain water content in the IGRS‑treated groups treated at doses of 60 and 120 mg/kg were decreased significantly compared with the Model group. The neurological scores were also significantly decreased in the IGRS‑treated groups. IGRS treatment effectively decreased neuronal apoptosis resulting from CIRI‑induced neuron injury. In addition, the histopathological damage and the endoplasmic reticulum ultrastructure injury were partially improved in CIRI rats following IGRS treatment. RT‑qPCR and western blot analysis data indicated that IGRS significantly decreased the expression levels of GRP78, CHOP and caspase‑12 at both mRNA and protein levels. The results of the present study demonstrated that IGRS exerted a protective effect against CIRI in brain tissue via the inhibition of apoptosis and ERS.

Vascular endothelial growth factor (VEGF) inhibition has been demonstrated to be an effective strategy in preserving the integrity of the blood-brain barrier (BBB) in patients with acute ischemic stroke. Loss of the BBB is the key event associated with morbidity and mortality in these patients. However, the underlying mechanisms remain poorly understood. In the present study, the effects of VEGF inhibition and the possible mechanism that underlies acute cerebral ischemia in rats was investigated. Following the induction of transient middle cerebral artery occlusion for a 90‑min period, either an anti‑VEGF neutralizing antibody (RB‑222; 5 or 10 µg), or IgG (control), was administered by intracerebroventricular injection at 1 h following reperfusion. Functional outcomes, BBB leakage, brain edema, microvessel numbers and the relative protein levels of VEGF, matrix metalloproteinase (MMP)-2, MMP-9, occludin and collagen-IV were then determined using neurological assessments, Evans Blue staining, brain water content, CD31 staining and western blotting. Treatment with RB‑222 at a dose of 5 and 10 µg significantly improved neurological functional outcomes and diminished infarct size, BBB leakage and brain edema compared with the MCAO and IgG groups at 24 h following reperfusion; 10 µg RB‑222 was more effective than a 5 µg dose of the antibody. In addition, RB‑222 reduced the number of immature microvessels, which subsequently attenuated BBB permeability. RB‑222 significantly repressed VEGF expression as well as decreased MMP‑2 and MMP‑9 expression. However, it enhanced occludin and collagen‑IV levels in the ischemic rat brain compared with the MCAO and IgG groups. Taken together, the results indicate that early inhibition of VEGF may have significant potential against cerebral ischemia, partly by regulating the expression of MMPs.

Reperfusion is the only approved therapy for acute ischemic stroke; however, it can cause excessive inflammation responses and aggravate brain damage. Therefore, supplementary treatment against inflammation caused by reperfusion is required. In a previous study from our group, curcumin was demonstrated to decrease infarction volume, brain edema and blood‑brain barrier (BBB) disruption against cerebral ischemia/reperfusion (I/R) injury. However, the underlying mechanisms remain unclear. The present study was conducted to understand whether curcumin protects against cerebral I/R injury through anti‑inflammatory and antiapoptotic properties. Ischemia for 1 h was induced in vivo in Wistar rats by middle cerebral artery occlusion (MCAO), followed by reperfusion for 24 h, and curcumin was injected intraperitoneally at 30 min prior to reperfusion. Immunohistochemistry was performed to analyze the expression levels of nuclear factor (NF)‑κB, intercellular adhesion molecule (ICAM)‑1, matrix metalloproteinase (MMP)‑9 and caspase‑3. The findings revealed that inflammation (NF‑κB, ICAM‑1 and MMP‑9) and apoptosis (caspase‑3)‑related markers were significantly downregulated in the curcumin‑treated MCAO group compared with the vehicle‑treated MCAO group. Furthermore, brain infarction size, brain edema and neurological dysfunction were attenuated in the curcumin‑treated MCAO group compared with the vehicle‑treated MCAO group. Taken together, the present results provided evidence that the protective effect of curcumin against cerebral I/R injury might be mediated by anti‑inflammatory and anti‑apoptotic properties. Therefore, curcumin may be a promising supplementary agent against cerebral I/R injury in the future.

Despite improvements in imaging techniques, it remains challenging to quantitatively assess the time of ischemic onset of an acute ischemic stroke. It is crucial to evaluate the early signs of infarction, which are predictive of responses to recombinant tissue plasminogen activator within a treatment window of 4.5 h after stroke induction. The aim of the present study was to assess and quantify the onset time for hyperacute middle cerebral artery occlusion (MCAO) ischemic stroke by measuring the apparent diffusion coefficient (ADC) of diffusion‑weighted imaging (DWI) and 1H‑magnetic resonance spectroscopy (MRS) at 7.0 T. DWI, conventional T2‑weighted imaging (T2WI) and subsequent focal ADCs were employed to evaluate ischemic brain lesions in a rat model of MCAO (n=20) at different time‑points following a stroke. A quantitation of local changes in metabolite concentrations within the lesions was performed using MRS. Proton metabolites were quantified automatically using LCModel software. At 30 min after MCAO, intense signals were observed in the DWI spectra of all animals. No abnormal signal was observed within 3 h by T2WI. ADC images of the central area, peripheral striping and on the fringes of the infarction demonstrated a lower signal than that of the normal side. The ADC decreased significantly within 30 min after infarction, followed by a gradual elevation in volatility levels and then becoming relatively stable at a lower level 3 h later. MRS exhibited a consistent elevation of lactate and reduced N‑acetyl aspartic acid. Glutamate and taurine reached a maximum 2 h after MCAO and began to decrease 1 h later. In conclusion, the present study demonstrated that hyperacute ischemic stroke can be quantitatively detected with the application of ADC, DWI and MRS. These methods may also be used to quantitatively assess the ischemic onset time of a hyperacute stroke.