Neurological Function In Rats Research Articles

Remifentanil protects neurological function of rats with cerebral ischemia-reperfusion injury via NR2B/CaMKIIα signaling pathway.

The effects of remifentanil were studied on cerebral ischemia-reperfusion injury (IRI) in rats, and its regulatory effect was explored on the N-methyl D-aspartate receptor subtype 2B (NR2B)/calcium/calmodulin-dependent protein kinase type II subunit alpha (CaMKII) signaling pathway in cerebral tissues. A total of 40 Sprague-Dawley rats were randomly assigned into sham group, model group, low-dose group (remifentanil injected into the caudal veins at 2 μg/kg) and high-dose group (remifentanil injected into the caudal veins at 10 μg/kg). Then, in the model, low-dose and high-dose groups, the rat model of cerebral IRI was established through middle cerebral artery occlusion (namely, ischemia for 1 h and reperfusion for 2 h), while no thread was inserted into the rats in the sham group. Neurological function of rats in each group was evaluated, and the cerebral infarct size was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Moreover, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed to determine the neuronal apoptosis level, and mitochondrial membrane potential was measured via JC-1 assay. Moreover, the reactive oxygen species (ROS) level in neurons and the malondialdehyde (MDA) content in cerebral tissues were determined using 2',7'-dichlorofluorescin diacetate (DCFH-DA) assay and enzyme-linked immunosorbent assay (ELISA), respectively. Finally, the expression levels of apoptosis-associated proteins and the NR2B/CaMKIIα signaling pathway-associated proteins in cerebral tissues were measured by Western blotting. Remifentanil preconditioning substantially decreased the neurological score of rats (p<0.01), cerebral infarct size (p<0.01), neuronal apoptosis level (p<0.01), ROS level in neurons (p<0.01), MDA content (p<0.01) and expression levels of cysteinyl aspartate specific proteinase-3 (Caspase-3), NR2B, phosphorylated CaMKIIα (p-CaMKIIα) and p-cAMP responsive element binding protein (p-CREB) (p<0.01), but it increased the mitochondrial membrane potential (p<0.01) and B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) ratio (p<0.01). Remifentanil can repress the NR2B/ CaMKIIα signaling pathway in the neurons of rats with cerebral IRI to decrease the p-CREB expression, ROS level and MDA content in neurons, neuronal apoptosis level and cerebral infarct size, and increase the mitochondrial membrane potential, thereby protecting the neurological function.

Protective effect of BMSCs-derived exosomes mediated by BDNF on TBI via miR-216a-5p.

BackgroundTransplantation of exosomes derived from mesenchymal stem cells (MSCs-Exo) can improve the recovery of neurological function in rats after traumatic brain injury (TBI). We tested a new hypothesis that brain-derived neurotrophic factor (BDNF)-induced MSCs-Exo can effectively promote functional recovery and neurogenesis in rats after TBI.Material/MethodsBM-MSCs of rats were extracted by whole bone marrow culture, BDNF was added to BM-MSCs as an intervention, supernatant was collected, and exosomes were separated and purified by ultracentrifugation. Exosomes were identified by Western blot (WB), transmission electron microscopy (TEM), and particle size analysis and were subsequently used in cell and animal experiments. The experimental animals were divided into a sham group, a PBS group, an MSCs-Exo group, and a BDNF-induced MSCs-Exo group (n=12). An electric cortical contusion impactor (eCCI) was used to cause TBI in all rats except the sham group. We investigated the recovery of sensorimotor function and spatial learning ability, inflammation inhibition, and neuron regeneration in rats after TBI.ResultsCompared with the MSCs-Exo group, the BDNF-induced MSCs-Exo group showed better effects in promoting the recovery of sensorimotor function and spatial learning ability. BDNF-induced MSCs-Exo successfully inhibited inflammation and promoted neuronal regeneration in vivo and in vitro. We further analyzed miRNAs in BDNF-induced MSCs-Exo and MSCs-Exo and found that the expression of miR-216a-5p in BDNF-induced MSCs-Exo was significantly higher than that in MSCs-Exo as determined by qRT-PCR. Rescue experiments indicated that miR-216a-5p had a similar function as BDNF-induced MSCs-Exo.ConclusionsWe found that BDNF-induced MSCs-Exo can improve cell migration and inhibit apoptosis better than MSCs-Exo in rats after TBI, and the mechanism may be related to the high expression of miR-216a-5p.

Overexpression of miR-124 Protects Against Neurological Dysfunction Induced by Neonatal Hypoxic-Ischemic Brain Injury.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a major cause of lifelong disabilities worldwide, without effective therapies and clear regulatory mechanisms. MicroRNAs (miRNAs) act as a significant regulator in neuroregeneration and neuronal apoptosis, thus holding great potential as therapeutic targets in HIE. In this study, we established the hypoxia-ischemia (HI) model in vivo and oxygen-glucose deprivation (OGD) model in vitro. Zea-longa score and magnetic resonance imaging were applied to verify HI-induced neuronal dysfunction and brain infarction. Subsequently, a miRNA microarray analysis was employed to profile miRNA transcriptomes. Down-regulated miR-124 was found 24h after HIE, which corresponded to the change in PC12, SHSY5Y, and neurons after OGD. To determine the function of miR-124, mimics and lentivirus-mediated overexpression were used to regulate miR-124 in vivo and in vitro, respectively. Our results showed that miR-124 overexpression obviously promoted cell survival and suppressed neuronal apoptosis. Further, the memory and neurological function of rats was also obviously improved at 1 and 2 months after HI, indicated by the neurological severity score, Y-maze test, open field test, and rotating rod test. Our findings showed that overexpression of miR-124 can be a promising new strategy for HIE therapy in future clinical practice.

Human Umbilical Cord–Derived Mesenchymal Stem Cell Therapy Effectively Protected the Brain Architecture and Neurological Function in Rat After Acute Traumatic Brain Injury

Intracranial hemorrhage from stroke and head trauma elicits a cascade of inflammatory and immune reactions detrimental to neurological integrity and function at cellular and molecular levels. This study tested the hypothesis that human umbilical cord–derived mesenchymal stem cell (HUCDMSC) therapy effectively protected the brain integrity and neurological function in rat after acute traumatic brain injury (TBI). Adult male Sprague-Dawley rats (n = 30) were equally divided into group 1 (sham-operated control), group 2 (TBI), and group 3 [TBI + HUCDMSC (1.2 × 106 cells/intravenous injection at 3 h after TBI)] and euthanized by day 28 after TBI procedure. The results of corner test and inclined plane test showed the neurological function was significantly progressively improved from days 3, 7, 14, and 28 in groups 1 and 3 than in group 2, and group 1 than in group 3 (all P < 0.001). By day 28, brain magnetic resonance imaging brain ischemic volume was significantly increased in group 2 than in group 3 (P < 0.001). The protein expressions of apoptosis [mitochondrial-bax positive cells (Bax)/cleaved-caspase3/cleaved-poly(adenosine diphosphate (ADP)-ribose) polymerase], fibrosis (Smad3 positive cells (Smad3)/transforming growth factor-β), oxidative stress (NADPH Oxidase 1 (NOX-1)/NADPH Oxidase 2 (NOX-2)/oxidized-protein/cytochrome b-245 alpha chain (p22phox)), and brain-edema/deoxyribonucleic acid (DNA)–damaged biomarkers (Aquaporin-4/gamma H2A histone family member X ( (γ-H2AX)) displayed an identical pattern to neurological function among the three groups (all P < 0.0001), whereas the protein expressions of angiogenesis biomarkers (vascular endothelial growth factor/stromal cell–derived factor-1α/C-X-C chemokine receptor type 4 (CXCR4)) significantly increased from groups 1 to 3 (all P < 0.0001). The cellular expressions of inflammatory biomarkers (cluster of differentiation 14 (+) cells (CD14+)/glial fibrillary acidic protein positive cells (GFAP+)/ a member of a new family of EGF-TM7 molecules positive cells (F4/80+)) and DNA-damaged parameter (γ-H2AX) exhibited an identical pattern, whereas cellular expressions of neural integrity (hexaribonucleotide Binding Protein-3 positive cells (NeuN+)/nestin+/doublecortin+) exhibited an opposite pattern of neurological function among the three groups (all P < 0.0001). Xenogeneic HUCDMSC therapy was safe and it significantly preserved neurological function and brain architecture in rat after TBI.

Improvement of Neurological Function in Rats with Ischemic Stroke by Adipose-derived Pericytes

Pericytes possess high multipotent features and cell plasticity, and produce angiogenic and neurotrophic factors that indicate their high regenerative potential. The aim of this study was to investigate whether transplantation of adipose-derived pericytes can improve functional recovery and neurovascular plasticity after ischemic stroke in rats. Rat adipose-derived pericytes were isolated from subcutaneous adipose tissue by fluorescence-activated cell sorting. Adult male Wistar rats were subjected to 90 min of middle cerebral artery occlusion followed by intravenous injection of rat adipose-derived pericytes 24 h later. Functional recovery evaluations were performed at 1, 7, 14, and 28 days after injection of rat adipose-derived pericytes. Angiogenesis and neurogenesis were examined in rat brains using immunohistochemistry. It was observed that intravenous injection of adipose-derived pericytes significantly improved recovery of neurological function in rats with stroke compared to phosphate-buffered saline–treated controls. Immunohistochemical analysis revealed that the number of blood capillaries was significantly increased along the ischemic boundary zone of the cortex and striatum in stroke rats treated with adipose-derived pericytes. In addition, treatment with adipose-derived pericytes increased the number of doublecortin positive neuroblasts. Our data suggest that transplantation of adipose-derived pericytes can significantly improve the neurologic status and contribute to neurovascular remodeling in rats after ischemic stroke. These data provide a new insight for future cell therapies that aim to treat ischemic stroke patients.

Analysis of Antiapoptosis Effect of Netrin-1 on Ischemic Stroke and Its Molecular Mechanism under Deleted in Colon Cancer/Extracellular Signal-Regulated Kinase Signaling Pathway.

To analyze the regulatory effect of Netrin-1 in ischemic stroke and its influence on Deleted in Colon Cancer (DCC)/Extracellular Signal-regulated Kinase (ERK) signaling pathway, 20 male rats were selected to construct the rat model of middle cerebral artery occlusion (MCAO), 10 normal rats were selected as healthy controls (Normal Saline (NS)), and they were divided into the MCAO+Netrin-1 group, MCAO group, and NS group according to different treatment schemes. The positive expression of Netrin-1 was detected by immunostaining, magnetic resonance imaging (MRI) was adopted to detect the percentage of rat cerebral infarct volume in the cerebral hemispheres, and Modified Neurological Severity Score (mNSS) was adopted to evaluate postoperative neurological function in rats. Besides, a tunnel staining experiment was applied to detect the apoptosis rate of rat neurons, the sticker removal test was applied to evaluate the postoperative sensory function of rats, and fluorescence staining was adopted to detect the expression of DCC and ERK in rats. The results showed that the percentage of cerebral infarction volume in the cerebral hemispheres of the MCAO+Netrin-1 group was higher than that of the MCAO and NS groups (P < 0.05); in the MCAO+Netrin-1 group, the MCAO mNSS scoring and the time spent in the sticker removal test were lower than the MCAO group (P < 0.05); the apoptosis rate of rats in the MCAO+Netrin-1 group was lower than that in the MCAO group (P < 0.05); the average fluorescence intensity of DCC and p-ERK in the MCAO+Netrin-1 group was higher than that in the MCAO group (P < 0.05); the average fluorescence intensity of p-ERK in the MCAO+Netrin-1 group was higher than that in the MCAO group (P < 0.05). In short, Netrin-1 can effectively reduce the brain tissue damage in rats with ischemic stroke, improve the nerve function and sensory function of rats, and inhibit neuronal cell apoptosis. Netrin-1 can promote DCC expression and ERK phosphorylation, and the EPK signaling pathway may be involved in the antiapoptotic effect of Netrin-1.

Cerebral ischemia-reperfusion aggravated cerebral infarction injury and possible differential genes identified by RNA-Seq in rats

Numerous studies have shown that local excessive inflammatory response in brain tissue was an important pathogenesis of secondary injury following cerebral ischemia-reperfusion (I/R). However, the inflammatory-related targets and pathways after cerebral I/R injury are still unclear. This study was to investigate possible targets and mechanisms after cerebral I/R injury. Rats were subjected to transient or permanent middle cerebral artery occlusion (MCAO). Neurological deficit scores test was used to evaluate neurological function. Cerebral infarction was evaluated by MRI, TTC staining and Nissl staining. Microglia activation was detected by immunofluorescence using Iba-1 antibody. Inflammatory factors were detected by ELISA assay. RNA-sequencing transcriptome analysis was processed and the differential genes were verified by real-time quantitative PCR (qPCR) and western blotting. The results showed that neurological function of rats in I/R group was more severe than that in I group on the 7th after cerebral I/R. Therefore, the differences between cerebral ischemia and cerebral I/R for 7 days were studied in further study. The results showed that the levels of pro-inflammatory factors in I/R group were higher and the levels of anti-inflammatory factors were lower than those in I group. KEGG pathway and gene network enrichment analysis revealed that some common differential up- and down-regulated genes were involved in most of significant pathways. These common differential up-regulated genes belonged to TLR4/MYD88 inflammatory signaling pathway and common differential down-regulated genes belonged to HRAS/RAF1 neurotrophic signaling pathway. Interestingly, according to the genetic interaction analysis of string database, these up-regulated differential genes might promote the development of inflammation, while the down-regulated differential genes might inhibit the development of inflammation. Furthermore, qPCR and WB results verified that these pro-inflammatory genes in the I/R group were higher than those in the I group, while possible anti-inflammatory genes in the I/R group were lower than those in the I group. It is concluded that TLR4/MYD88 inflammatory signaling pathway and HRAS/RAF1 neurotrophic signaling pathway may play different roles after cerebral I or I/R and may be therapeutic targets for stroke recovery.

Effects of lncRNA gm4419 on rats with hypertensive cerebral atherosclerosis through NF-κB pathway.

To explore the effects of long non-coding ribonucleic acid (lncRNA) Gm4419 on rats with hypertensive cerebral atherosclerosis through the nuclear factor-kappa B (NF-κB) pathway. Healthy male rats were selected and randomly divided into control group, model group (hypertensive cerebral atherosclerosis model), and lncRNA group (hypertensive cerebral atherosclerosis model + lncRNA injection). Neurological deficit scoring criteria, flow cytometry, Western blotting, and staining method were adopted to measure the differences in the neurological function score, NF-κB activity, and chemerin level of rats in the three groups. The neurological scores revealed that the neurological function of rats was not damaged in control group, while it was severely damaged in model group. However, the neurological function of rats was more severely damaged in lncRNA group than that in control group and model group, while the neurological function deficits were slighter in model group. In terms of NF-κB expression activity in mononuclear cells, the serum activity of NF-κB in control group appeared the lowest among the three groups and was significantly higher in lncRNA group than in model group. The serum chemerin level was evidently increased in model group compared with control group, while it was significantly decreased in lncRNA group compared with model group and control group. Moreover, the levels of NF-κB and chemerin were most evidently influenced in lncRNA group. Activating the NF-κB signal, lncRNA Gm4419 promotes the expression of chemerin signal, accelerates the apoptosis of nerve cells, and motivates the deterioration of hypertensive cerebral arteriosclerosis.

Edaravone combined with cinepazide maleate on neurocyte autophagy and neurological function in rats with subarachnoid hemorrhage.

Effects of edaravone combined with cinepazide maleate on neurocyte autophagy and neurological function in rats with subarachnoid hemorrhage were investigated. Eighty SD rats were selected to establish subarachnoid hemorrhage (SAH) rat models, which were divided into sham operation group, SAH group, MCI group and combined group. Hippocampal tissue of each group was taken to observe the number of neurocytes. The expression levels of Beclin-1 and (light chain LC3)-II of rats in each group were detected by ELISA. Pearson's correlation factors were used to analyze the correlation between Beclin-1 and LC3-11, and neurological function tests were carried out on rats of each group 14 and 28 days after administration. The morphological and structural damage of nerve cells in the combined group was further alleviated, and the survival rate of neurons significantly increased at all time points (P<0.05). The expression levels of Beclin-1 and LC3-11 in combined group was significantly higher than those in SAH group and CMI group (P<0.05), and Beclin-1 was positively correlated with LC3-11 (r=0.9454). Longa score of the combined group was significantly lower than that of the other two groups, and muscle strength score was significantly higher than that of the other two groups (P<0.05). Edaravone combined with cinepazide maleate can effectively increase the survival rate of brain cells and promote the volatilization of neurological function in the treatment of hemorrhage in the subretinal space of the omentum, which is worthy of popularization and application.

Platelet-Rich Plasma Combined with Low-Dose Ultrashort Wave Therapy Accelerates Peripheral Nerve Regeneration.

Finding treatments that accelerate peripheral nerve regeneration, prolongation, and functional recovery remains a challenging task. Platelet-rich plasma (PRP) contains numerous growth factors and active proteins, and low-dose ultrashort waves (USWs) stimulate the formation of nerve-nourishing vessels, which are powerful for nerve regeneration. The goal of this study was to evaluate the synergistic effects of serial ultrasound-guided PRP injections combined with low-dose USWs radiation on peripheral nerve regeneration in a crush injury model. Fifty rabbits were equally and randomly divided into normal control, model, USW, PRP, and PRP+USW groups. The neurological function, electrophysiological recovery, and histological and morphological evaluation of regenerated nerves, as well as a targeted muscle recovery assessment, were performed to investigate the regenerative effect of PRP combined with USW therapy. Our results showed that the PRP+USW group had the better early axonal regeneration and displayed the earliest positive compound muscle action potential among the treatment groups. At postintervention week 12, a histological evaluation showed similar expression of the S-100 protein in the PRP+USW and normal control groups. Moreover, the morphological assessment revealed a significant increase in the myelinated nerve fiber density and diameter and myelin sheath thickness compared with the USW and PRP groups. The morphometry of the target muscles indicated the lowest reduction in the percent volume in the PRP+USW group, and an ultrasound examination of the targeted muscle showed the best improvement in stiffness and perfusion parameters at 12 weeks after crush injury. Thus, serial ultrasound-guided PRP injections combined with low-dose USW radiation exert a synergistic effect on accelerating functional axon recovery and decreasing atrophy of the target muscles in a crush injury model. Impact Statement This research describes that the application of platelet-rich plasma combined with low-dose ultrashort waves treatment exert a synergistic effect on accelerating peripheral nerve regeneration. With the extensive use of platelet-rich plasma and physical factors in regenerative medicine or clinical rehabilitation medicine, our findings may help establish effective strategies for repairing peripheral nerve injury.

Protective effect of propofol on neurological function in rats after traumatic brain injury and related mechanism

Objective To investigate the protective effect of propofol on neurological function in rats after traumatic brain injury (TBI) and its possible mechanism. Methods A total of 96 SD rats were randomly divided into sham operation group, sham operation+ propofol group, TBI group and TBI + propofol group, with 24 rats in each group. The TBI model was prepared by modified Feeney method. The sham operation+ propofol group and the TBI+ propofol group were given 50 mg/kg of propofol once daily. The sham operation group and the TBI group were injected with the same amount of normal saline. Modified neurobehavioral functional scores (mNSS) were evaluated at 1, 3, 7 and 14 days after injury; dry-wet specific gravity method was used to detect brain water content in injured area; TUNEL staining was used to detect neuronal apoptosis; chemiluminescence was used to detect activity of Oxygen cluster (ROS) content; Western blot was used to determine the expressions of inositol requirement enzyme 1 (IRE-1), enhancer binding protein homolog protein (CHOP), heme oxygenase 1 (HO-1), quinone oxidoreductase 1 (NQO1) and nuclear factor E2 related factor 2 (Nrf2) protein. Results Compared with the sham operation group and the sham operation + propofol group, the mNSS, brain tissue water content, apoptosis number and ROS increased at 1, 3, 7 and 14 days after TBI in the TBI group and TBI + propofol group (P<0.05). Compared with TBI group, mNSS in TBI+ propofol group decreased significantly [(9.3±1.4)points ∶(10.9±1.2)points] 7 days after injury (P<0.05); the brain tissue water content decreased significantly [(81.0±0.8)%∶(82.1±0.8)%] 3 days after injury (P<0.05); the number of apoptotic cells decreased significantly 7 days after injury[(14.1±1.4)%∶(15.6±1.6)%], with the most significant decrease at 14 days after injury [( 10.4±1.5)%∶(13.2±1.4)% (P<0.05); and ROS decreased significantly 7 days after injury [(61.5±4.0)RFU∶(77.3±5.5)RFU](P<0.05). Compared with the sham operation group and the sham operation+ propofol group, the expressions of IRE-1 and CHOP were significantly up-regulated in the TBI group and the TBI+ propofol group (P<0.05); the expressions of HO-1, NQO1 and Nrf2 in the TBI group were significantly decreased (P<0.05); the expressions of HO-1 and NQO1 in TBI+ propofol group were increased (P<0.05) while the expression of Nrf2 were decreased slightly (P<0.05). Compared with the TBI group, the expressions of IRE-1 and CHOP in TBI+ propofol group were decreased (P<0.05), while the expressions of HO-1, NQO1 and Nrf2 were significantly increased (P<0.05). Conclusion After TBI in rats, propofol can reduce oxidative stress by activating the Nrf2-antioxidant element (ARE) pathway, reduce brain edema, and inhibit neuronal apoptosis, thus playing a neuro-protective role. Key words: Brain injuries; Oxidative stress; Nuclear factor-E2-related factor 2

P5746Human induced pluripotent stem cell-derived mesenchymal stem cell therapy effectively reduced brain infarct volume and preserved neurological function in rat after acute intracranial hemorrhage

Abstract Intracerebral hemorrhage (ICH) causes 10%-20% of all strokes and results in higher morbidity compared to other subtypes of cerebral stroke. Although early surgical intervention can clear the expanding hematoma, clinical outcomes following ICH have not significantly improved over the decades. Since ICH elicits neuroinflammation to exacerbate brain edema, damage the blood-brain barrier (BBB), lead to secondary neuronal injury, anti-inflammation may be a critical therapeutic strategy. Mesenchymal stem cell (MSC) therapy processes anti-inflammatory, immunomodulatory and tissue regenerative properties, suggesting that MSC therapy could be an effective therapy for ICH. Therefore, this study tested the hypothesis that human induced pluripotent stem cell-derived mesenchymal stem cell (iPSC-MSC) therapy could effectively reduce brain-infract volume (BIV) and improve neurological function in rat after acute ICH induced by a weight-drop device. Adult-male SD rats (n=40) were equally divided into group 1 (sham-operated control), group 2 (ICH), group 3 (ICH + hyaluronic acid (HA)/intracranial injection/3h after ICH), group 4 [ICH + HA + iPSC-MSC (1.2x106 cells/intracranial injection/3h after ICH)] and euthanized by day 28 after ICH procedure. In vitro study showed that hemorrhagic-brain tissue augmented protein expressions of inflammation (HMGB1/MyD88/TLR-4/TLR-2/NF-κB/TNF-α/iNOS/IL-1β) in cultured neurons that were significantly inhibited by iPSC-MSC treatment (all p&lt;0.001). By days 7/14 after ICH procedure, circulating inflammatory levels of TNF-α/IL-6/MPO expressed were lowest in group 1, highest in group 2 and significantly lower in group 4 than in group 3 (all p&lt;0.0001). By day 14 after ICH procedure, neurological function and BIV expressed an opposite pattern, whereas protein expressions of inflammation (HMGB1/MyD88/TLR-4/TLR-2/NF-κB/I-kB/TNF-α/iNOS/IL-1β/MMP-9), oxidative stress (NOX-1/NOX-2/oxidized protein) and apoptosis (mitochondrial-Bax/cleaved-caspase-2/PARP) in brain exhibited an identical pattern to circulating inflammation among the four groups (all p&lt;0.001). Microscopy demonstrated that the number of vascular remodeling/GFAP+/53BP1+/γ-H2AX+ cells displayed an identical pattern of inflammation, whereas the NeuN+ cells displayed an opposite pattern of inflammation among the four groups (all p&lt;0.001). In conclusion, iPSC-MSC therapy markedly reduced BIV and preserved neurological function mainly by inhibiting inflammatory/oxidative-stress generation. Acknowledgement/Funding Kaohsiung Chang Gung Memorial Hospital, Taiwan Society of Stem Cell Research

Cyclosporine A Plus Ischemic Postconditioning Improves Neurological Function in Rats After Cardiac Resuscitation.

Attenuation of neuronal apoptosis helps maintain neurological function in patients after cardiac arrest. After ischemia-reperfusion, both cyclosporin A (CsA) and ischemic postconditioning independently protect mitochondria and thus reduce nerve injury. This study employed a rat model to evaluate the neuroprotective effect of combining ischemic postconditioning with CsA after cardiopulmonary resuscitation (CPR). Rats were apportioned equally to model control, postconditioned, CsA-treated, or CsA + postconditioned groups. Asphyxial cardiac arrest was imposed using modified Utstein-style guidelines. In the appropriate groups, postconditioning was implemented by ischemia and reperfusion (clamping and loosening the left femoral artery); CsA treatment was delivered with a single intravenous dose. Neurological deficits were scored at different times after CPR. Histological evaluation and electron microscopy were used to evaluate tissue damage, and TUNEL and flow cytometry were used to measure the apoptotic rate of hippocampal neurons and size of the mitochondrial permeability transition pore (mPTP) opening. The apoptotic rate was significantly lower in the postconditioned and CsA-treated groups compared with the model control and lowest in the CsA + postconditioned group. By histological evaluation and electron microscopy, the least damage was observed in the CsA + postconditioned group. The neurological deficit score of the CsA + postconditioned group was significantly higher than that of the CsA-treated group, but the size of the mPTP openings of these two groups was comparable. Ischemic postconditioning combined with CsA exerted a better neuroprotective effect after CPR than did either postconditioning or CsA alone. Inhibiting the opening of the mPTP is not the only neuroprotective mechanism.

Granulocyte colony-stimulating factor and stromal cell-derived factor-1 combination therapy: A more effective treatment for cerebral ischemic stroke.

Drugs that promote angiogenesis include statins, recombinant human granulocyte colony-stimulating factor, and stromal cell-derived factor-1. Low doses of atorvastatin could significantly increase the vascular expressions of endothelial growth factor, and the number of peripheral blood endothelial progenitor cells (EPCs), thus improving angiogenesis and local blood flow. G-CSF is an EPC-mobilization agent used in ischemia studies for targeting angiogenesis after cerebral ischemia via EPCs. In previous clinical trials, consistent conclusions have not been reached about the effectiveness of G-CSF on ischemic stroke. Therefore, the therapeutic effect of G-CSF and its combination with other medicines need further experimental verification. It is known that atorvastatin, rhG-CSF, and SDF-1 are considered the most promising neuroprotective candidates, but a comprehensive comparison of their effects is lacking. To compare the effects of atorvastatin, stromal cell-derived factor-1, and recombinant human granulocyte colony-stimulating factor on ischemic stroke. Adult male Sprague-Dawley rats were randomly allocated to three groups: normal, sham-operated, and middle cerebral artery occlusion operated. Middle cerebral artery occlusion operated rats were further allocated into saline, atorvastatin, recombinant human granulocyte colony-stimulating factor, and recombinant human granulocyte colony-stimulating factor + stromal cell-derived factor-1 groups. Neurological function evaluation, cerebral infarction and the blood-brain barrier integrity analysis, identification of angiogenic factors, assessment of angiogenesis, expression of growth-associated protein-43, neuroglobin, glial cell-derived neurotrophic factor, and cleaved caspase 3, were performed. Compared with atorvastatin or recombinant human granulocyte colony-stimulating factor alone, recombinant human granulocyte colony-stimulating factor + stromal cell-derived factor-1 treatment improved neurological performance, reduced cerebral infarction and blood-brain barrier disruption after stroke, and increased the content of stromal cell-derived factor-1, vascular endothelial growth factor, monocyte chemotactic protein 1, and basic fibroblast growth factor in peripheral blood. In addition, recombinant human granulocyte colony-stimulating factor + stromal cell-derived factor-1 promoted greater angiogenesis than atorvastatin or recombinant human granulocyte colony-stimulating factor alone and increased the expression of growth-associated protein-43, neuroglobin, and glial cell-derived neurotrophic factor, while decreasing the levels of cleaved caspase 3 in the brain after ischemic stroke. Combination therapy with recombinant human granulocyte colony-stimulating factor and stromal cell-derived factor-1 is more effective than atorvastatin or recombinant human granulocyte colony-stimulating factor alone in protecting against stroke-induced damage and could be an optimal therapeutic strategy for stroke.

P.279Pragmatic cognitive and psychosocial health monitoring in clinical practice in Duchenne muscular dystrophy

We performed a prospective, cross-sectional cognitive assessment in subjects with Duchenne muscular dystrophy (DMD). Thirty subjects (29 boys and 1 girl) with out-of-frame mutations in dystrophin (DMD) gene were assessed using the National institutes of health toolbox cognition battery (NIHTB-CB). The NIH toolbox is a scientifically rigorous, multi-dimensional measure designed to bring uniformity to evaluation of neurological function across the life span. The NIHTB-CB tests have previously been evaluated for their reliability, validity, and other psychometric properties, including among individuals with several different neurodevelopmental disorders. A caregiver completed the Behavior Rating Inventory of Executive Functioning (BRIEF), a standardized rating scale of executive functioning, for their child. Overall, the children with DMD scored one standard deviation (SD) below age-corrected norms on the NIHTB-CB total cognition score. They scored 1.5 SD below age-corrected norms in fluid cognition, which evaluates the cognitive domains of executive function, working memory, episodic memory, attention, and processing speed. In crystallized cognition score, which evaluates vocabulary and reading, their performance was consistent with age expectations (within 1 SD below age-corrected norms). Children with DMD had higher T-scores in several domains of BRIEF, demonstrating greater difficulty in executive functioning. The NIHTB-CB sensitively identifies core deficits in the specific domains of cognition in DMD, and can be easily incorporated in a clinic setting to assess cognitive needs and psychosocial health in DMD.

SOD3 overexpression alleviates cerebral ischemia-reperfusion injury in rats.

BackgroundIschemic stroke is a deadly disease that poses a serious threat to human life. Superoxide dismutase 3 (SOD3, ECSOD) is the main antioxidant enzyme that removes superoxide anions from cells. This study aimed to investigate the effect of SOD3 overexpression on cerebral ischemia‐reperfusion injury in rats.MethodsGV230‐EGFP‐ECSOD, the recombinant SOD3‐overexpressed vector, was constructed by genetic engineering technology, and mesenchymal stem cells (MSCs) were infected with lentiviral packaging. In animal experiment, cerebral ischemia‐reperfusion injury model rats were successfully established. ECSOD‐MSCs are the MSCs that successfully transfected with SOD3 overexpression vector. The animals were injected with ECSOD‐MSCs (ECSOD‐MSC group), normal MSCs (MSCs group), PBS (PBS group), and not do any processing (Model group) via the tail vein. Then MRI was used to detect the infarct volume of rats, modified Neurological Severity Scores (mNSS), and immunohistochemistry were used to evaluate the expression of neurological function and apoptosis‐related genes in rats.ResultsWestern blot analysis revealed that the SOD3 was highly expressed in MSCs. Animal experiments showed that the transplantation of ECSOD‐MSCs significantly reduced the infarct volume of ischemic stroke rats (p < 0.05), significantly improved neurological function in rats (p < 0.05), and found proapoptotic gene, Bax, expression was significantly decreased (p < 0.05), the expression of anti‐apoptotic gene, Bcl‐2, was significantly increased (p < 0.05). The highly expressed SOD3 has no correction with brain infarct volume, and the highly expressed SOD3 has a positive correlation with cell apoptosis. It is speculated that overexpression of SOD3 affects the expression of Bax and Bcl‐2, and improves apoptosis to alleviate ischemic stroke.ConclusionOur results indicated that MSCs transfected with SOD3 can effectively alleviate cerebral ischemia‐reperfusion injury in rats.

Effects of ISO-Alpha-Acids on the Hematoma Volume, Inflammation and Neurological Functions in Intracerebral Hemorrhage Rats and its Potential Mechanism

Abstract INTRODUCTION ISO-alpha-acids (IAA) was reported to be useful for the prevention of dementia as a PPAR-gamma agonist in rats, but the application of IAA in intracerebral hemorrhage (ICH) was still limited. METHODS ICH models were built in Vivo and 90 SD rats were randomized to 5 groups: ICH + IAA intraperitoneal injection (IAA), ICH + normal saline intraperitoneal injection (control), ICH without any intraperitoneal injections (vehicle), Sham, and Mock. Neurological functions of rats, hematoma volume, and brain water content were evaluated 1-d, 3-d, and 7-d postoperation. Microglia of SD rats were sub cultured in Vitro. According to different interventions, 4 groups were set, including IAA, PPAR-gamma antagonist, normal saline (control), and normal cultured (vehicle). Western blotting, immunofluorescence, flow cytometry, and RT-PCR were used to detect the expression of molecular markers, proteins and mRNAs. RESULTS In ICH rats, intraperitoneal injection of IAA was able to promote hematoma clearance, to relieve brain water content and improve neurological functions. Detected by western blot, immunofluorescence, flow cytometry, and reverse transcription polymerase chain reaction (RT-PCR), IAA was able to significantly increase the expression of CD36, CD11b, and CD206, and inhibit the expression of CD80, CD86, TLR4, NF-kappaB, TNF-alpha, and IL-1beta comparing with other groups (P &lt; .05) in Vivo and Vitro. IAA was able to significantly increase both in CD11b and CD206 double-positive anti-inflammatory type microglia and in microglia phagocytosis in the brain (P &lt; .05). CONCLUSION IAA induces microglia to M2 type, characterized by phagocytosis and anti-inflammation, via activating PPAR-gamma signaling pathway in Vivo and Vitro. In ICH rats, administration of IAA is able to facilitate intracerebral hematoma absorption, alleviate brain edema, suppress inflammatory reactions, protect neurological functions, and finally improve rat's outcome.

Effect of miR-124 on neuronal apoptosis in rats with cerebral infarction through Wnt/β-catenin signaling pathway.

The aim of this study was to observe the influence of micro ribonucleic acid (miR)-124 on neuronal apoptosis in rats with cerebral infarction (CI), and to further investigate the underlying mechanism of miR-124 in CI occurrence and development. A total of 60 adult male Wistar rats were randomly divided into the Sham group, the CI group and the CI + miR-124 mimics group using a random number table. The focal CI model was established using the suture-occluded method. After successful modeling, miR-124 mimics were stereotactically injected into the lateral ventricle of rats. 24 h after operation, the neurological function of rats in each group was scored using modified neurological severity score (mNSS). Meanwhile, the infarction area of brain tissues was evaluated by the triphenyl tetrazolium chloride (TTC) method. The protein expression levels of apoptosis-related genes, including B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), C-Caspase and T-Caspase, were detected via Western blotting. The expression and location of caspase-3 in brain tissues were detected via immunofluorescence staining. Moreover, the level of apoptosis in each group was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In addition, the expression levels of the Wnt/β-catenin signaling pathway-related proteins were detected via Western blotting. Polymerase Chain Reaction (PCR) results revealed that the expression level of miR-124 in the CI group was significantly decreased when compared with that of the Sham group (p<0.05). The mNSS and TTC staining results manifested that injection of miR-124 mimics could significantly reduce the CI-induced neurological deficits and CI area (p<0.05). At the same time, the levels of Bax and C-caspase/T-Caspase were significantly decreased, whereas the expression of Bcl-2 was remarkably increased after the injection of miR-124 mimics (p<0.05). Besides, the number of apoptotic cells in the CI + miR-124 mimic group was remarkably decreased (p<0.05). In addition, miR-124 mimics significantly activated the expression levels of Wnt and β-catenin (p<0.05). The inhibitory effect of miR-124 on neuronal apoptosis in CI rats is probably related to the activation of the Wnt/β-catenin signaling pathway. Furthermore, miR-124 is expected to be a target drug for the clinical treatment of CI.

14-3-3ζ protein protects against brain ischemia/reperfusion injury and induces BDNF transcription after MCAO in rat.

Brain ischemia is a leading cause of death and disability worldwide that occurs when blood supply of the brain is disrupted. Brain-derived neurotrophic factor (BDNF) is a protective factor in neurodegenerative conditions. Nevertheless, there are some problems when exogenous BDNF is to be used in the clinic. 14-3-3ζ is a pro-survival highly-expressed protein in the brain that protects neurons against death. This study evaluates 14-3-3ζ effects on BDNF transcription at early time point after ischemia and its possible protective effects against ischemia damage. Human 14-3-3ζ protein was purified after expression. Rats were assigned into four groups, including sham, ischemia, and two treatment groups. Stereotaxic cannula implantation was carried out in the right cerebral ventricle. After one week, rats underwent middle cerebral artery occlusion (MCAO) surgery and received 14-3-3ζ (produced in our laboratory or standard form as control) in the middle of ischemia time. At 6 h of reperfusion after ischemia, brain parts containing the hippocampus, the cortex, the piriform cortex-amygdala and the striatum were collected for real time PCR analysis. At 24 h of reperfusion after ischemia, neurological function evaluation and infarction volume measurement were performed. The present study showed that 14-3-3ζ could up-regulate BDNF mRNA at early time point after ischemia in the hippocampus, in the cortex and in the piriform cortex-amygdala and could also improve neurological outcome and reduce infarct volume. It seems that 14-3-3ζ could be a candidate factor for increasing endogenous BDNF in the brain and a potential therapeutic factor against brain ischemia.

Hypothermic properties of dexmedetomidine provide neuroprotection in rats following cerebral ischemia-reperfusion injury.

Dexmedetomidine (Dex) is a sedative and analgesic agent that is widely administered to patients admitted to the intensive care unit, and has been demonstrated to result in hypothermia. Many patients have been revealed to benefit from therapeutic hypothermia, which can mitigate cerebral ischemia/reperfusion (I/R) injury following successful cardiopulmonary resuscitation. However, studies investigating the efficacy of Dex in I/R treatment is lacking. The present study aimed to investigate the efficacy of Dex in mitigating neuronal damage, and to determine the possible mechanism of its effects in a rat model of cardiac arrest (CA). CA was induced in Sprague-Dawley rats by asphyxiation for 5 min. Following successful resuscitation, the surviving rats were randomly divided into two treatment groups; one group was intraperitoneally administered with Dex (D group), whereas the control group was treated with normal saline (N group). Critical parameters, including core temperature and blood pressure were monitored following return of spontaneous circulation (ROSC). Arterial blood samples were collected at 10 min after surgery (baseline) 30 and 120 min post-ROSC; and neurological deficit scores (NDS) of the rats were taken 12 or 24 h after ROSC prior to euthanasia. The hippocampal tissue was then removed for analysis by histology, electron microscopy and western blotting. Rats in the D group exhibited a lower core temperature and higher NDS scores compared with the N group (P<0.05). In addition, Dex injection resulted in reduced expression of apoptotic and autophagy-associated factors in the hippocampus (P<0.05). Dex treatment induced hypothermia and improved neurological function in rats after ROSC following resuscitation from CA by inhibiting neuronal apoptosis and reducing autophagy, which suggested that Dex may be a potential therapy option for patients with CA.