Bioinformatic Analysis of Exosomal MicroRNAs of Cerebrospinal Fluid in Ischemic Stroke Rats After Physical Exercise.

Physical exercise is beneficial to the structural and functional recovery of post-ischemic stroke, but its molecular mechanism remains obscure. Herein, we aimed to explore the underlying mechanism of exercise-induced neuroprotection from the perspective of microRNAs (miRNAs). Adult male Sprague-Dawley (SD) rats were randomly distributed into 4 groups, i.e., the physical exercise group with the transient middle cerebral artery occlusion (tMCAO) surgery (PE-IS, n = 28); the physical exercise group without tMCAO surgery (PE, n = 6); the sedentary group with tMCAO surgery (Sed-IS, n = 28); and the sedentary group without tMCAO surgery (Sed, n = 6). Notably, rats in the PE-IS and PE groups were subjected to a running exercise for 28 days while rats in the Sed-IS and Sed groups received no exercise training. After long-term exercise, exosomal miRNAs of cerebrospinal fluid (CSF) were analyzed using high-throughput sequencing. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were employed for the differentially expressed miRNAs. Physical exercise improved the neurological function and attenuated the lesion expansion after stroke. In total, 41 differentially expressed miRNAs were screened for the GO and KEGG analysis. GO enriched terms were associated with the central nervous system, including cellular response to retinoic acid, vagus nerve morphogenesis, cellular response to hypoxia, dendritic cell chemotaxis, cell differentiation, and regulation of neuron death. Besides, these differentially expressed miRNAs were linked to the pathophysiological process of stroke, including axon guidance, NF-kappa B signaling pathway, thiamine metabolism, and MAPK signaling pathway according to KEGG analysis. In summary, exercise training significantly alleviated the neurological damage at both functional and structural levels. Moreover, the differentially expressed miRNAs regulating multiple signal pathways were potentially involved in the neuroprotective effects of physical exercise. Therefore, these miRNAs altered by physical exercise might represent the therapeutic strategy for cerebral ischemia.

Plasma Exosomal miR-450b-5p as a Possible Biomarker and Therapeutic Target for Transient Ischaemic Attacks in Rats.

Transient ischaemic attack (TIA) and cerebral infarction are difficult to identify within the thrombolytic time window. Blood markers are efficient, economical and noninvasive and can be beneficial in the diagnosis of many diseases. Plasma exosomal biomarkers are rarely reported in TIA. Exosomal microRNAs (miRNAs) were extracted from plasma and cerebrospinal fluid after middle cerebral artery occlusion (MCAo) in rats (0 min, 5 min, 10 min, 2 h). Deep sequencing was used to detect exosomal miRNAs in rat plasma and confirm significant differentially expressed miRNAs. Polymerase chain reaction (PCR) was used to detect the differentially expressed miRNAs in plasma and cerebrospinal fluid. Exosomal miRNAs with the same expression trends in plasma and cerebrospinal fluid were selected, and bioinformatics analysis was then carried out. Finally, the area under the curve (AUC) of the receiver operating characteristic (ROC) curve was determined to assess the diagnostic accuracy of miRNAs for TIA in rats. First, high-throughput sequencing was used to detect the expression level of plasma exosome miRNA, and rno-miR-450b-5p with a decreasing expression level was screened. Second, the expression levels of exosomal miRNAs were verified in cerebrospinal fluid and plasma samples by PCR, and the results indicated that exosomal rno-miR-450b-5p was similarly expressed in cerebrospinal fluid and plasma. ROC analysis showed high AUC values for rno-miR-450b-5p (0.880) in the 10 min ischaemia rats compared with the control rats. Finally, bioinformatic analysis indicated that exosomal rno-miR-450b-5p may be involved in cerebral ischaemia. Plasma exosomal rno-miR-450b-5p has a high diagnostic value and may become a therapeutic target for rat TIA.

JM-20 Treatment After MCAO Reduced Astrocyte Reactivity and Neuronal Death on Peri-infarct Regions of the Rat Brain.

Stroke is frequently associated with severe neurological decline and mortality, and its incidence is expected to increase due to aging population. The only available pharmacological treatment for cerebral ischemia is thrombolysis, with narrow therapeutic windows. Efforts aimed to identify new therapeutics are crucial. In this study, we look into plausible molecular and cellular targets for JM-20, a new hybrid molecule, against ischemic stroke in vivo. Male Wistar rats were subjected to 90 min middle cerebral artery occlusion (MCAO) following 23 h of reperfusion. Animals treated with 8 mg/kg JM-20 (p.o., 1 h after reperfusion) showed minimal neurological impairment and lower GABA and IL-1ß levels in CSF when compared to damaged rats that received vehicle. Immunocontent of pro-survival, phosphorylated Akt protein decreased in the cortex after 24 h as result of the ischemic insult, accompanied by decreased number of NeuN+ cells in the peri-infarct cortex, cornu ammonis 1 (CA1) and dentate gyrus (DG) areas. Widespread reactive astrogliosis in both cortex and hippocampus (CA1, CA3, and DG areas) was observed 24 h post-ischemia. JM-20 prevented the activated Akt reduction, neuronal death, and astrocytes reactivity throughout the brain. Overall, the results reinforce the pharmacological potential of JM-20 as neuroprotective agent and provide important evidences about its molecular and cellular targets in this model of cerebral ischemia.

A Case of a 4-Year-Old Female with a Primary Spinal Malignancy Presenting with Froin's Syndrome.

We report the case of a 4-year-old female with a primary extradural intramedullary atypical teratoid/rhabdoid tumor (AT/RT) leading to a middle cerebral artery (MCA) infarct and Froin's syndrome. She presented with a 6-pound weight loss over the previous week, as well as a decreased urinary output and an altered mental status. She underwent a brain MRI that revealed a left MCA infarct, mild ventriculomegaly, and bilateral internal carotid artery, M1, and A1 stenosis. An external ventricular drain (EVD) was placed due to increased intracranial pressure. Cerebrospinal fluid (CSF) was analyzed via lumbar puncture that revealed extremely elevated protein. However, CSF sampled from the EVD was completely normal, a phenomenon called Froin's syndrome. The following day, she developed a right MCA infarct. Her grim prognosis was discussed with her family and care was eventually withdrawn. The patient underwent an autopsy which confirmed a spinal AT/RT. To our knowledge, this is the first reported case of stroke and Froin's syndrome as the initial manifestations of a primary spinal AT/RT with a late onset of spinal cord compression due to tumor obstruction.

Emergency LP in a patient receiving dabigatran after antagonization with idarucizumab.

Idarucizumab is an antibody fragment which is used to reverse the anticoagulant effects of dabigatran. We report on the first successful use of idarucizumab before performing an emergency lumbar puncture in a patient on effective anticoagulation with dabigatran thought to have infective cerebral disease (such as temporal encephalitis).

(1)H NMR-based metabolomics exploring biomarkers in rat cerebrospinal fluid after cerebral ischemia/reperfusion.

In our study, metabolomics was used to investigate biochemical changes in the early stages of rats focal cerebral ischemia/reperfusion (I/R) injury. Cerebrospinal fluid (CSF) samples at 0, 0.5, 1, 3, and 6 h of reperfusion (n = 10), based on (1)H NMR spectroscopy and multivariate data analyses, were tested to analyze the changing of metabolites during the early disease process. Partial least squares-discriminant analysis scores plots of the (1)H NMR data revealed clear differences among the experiment groups. Combining the results of the loading plot and t-test, we found that twenty-seven metabolites were changed significantly (p < 0.05) in the CSF samples among the different groups. Among that, the potential biomarkers in CSF of ischemic rats were: acetic acid, 3-hydroxyisovaleric acid, 3-hydroxybutyric acid, choline, l-alanine, creatine, creatinine, glycine, pyruvic acid, glycerol, glutamic acid, d-fructose, l-lactic acid and acetone. These findings help us understand the biochemical metabolite changes in CSF of I/R rats in early stages. What's more, metabolomics may, therefore, have the potential to be developed into a clinically useful diagnostic tool of ischemic brain injury.

Insulin and glucagon share the same mechanism of neuroprotection in diabetic rats: role of glutamate.

In patients with acute ischemic stroke, diabetes and hyperglycemia are associated with increased infarct size, more profound neurologic deficits and higher mortality. Notwithstanding extensive clinical and experimental data, treatment of stroke-associated hyperglycemia with insulin is controversial. In addition to hyperglycemia, diabetes and even early prediabetic insulin resistance are associated with increased levels of amino acids, including the neurotoxic glutamate, in the circulation. The pleiotropic metabolic effects of insulin include a reduction in the concentration of amino acids in the circulation. In this article, we show that in diabetic rats exposed to transient middle cerebral artery occlusion, a decrease of plasma glutamate by insulin or glucagon reduces CSF glutamate, improves brain histology, and preserves neurologic function. The neuroprotective effect of insulin and glucagon was similar, notwithstanding their opposite effects on blood glucose. The therapeutic window of both hormones overlapped with the short duration (~30 min) of elevated brain glutamate following brain trauma in rodents. Similar neuroprotective effects were found after administration of the glutamate scavenger oxaloacetate, which does not affect glucose metabolism. These data indicate that insulin and glucagon exert a neuroprotective effect within a very brief therapeutic window that correlates with their capacity to reduce glutamate, rather than by modifying glucose levels.

Ubiquitin C-terminal hydrolase-L1 as a biomarker for ischemic and traumatic brain injury in rats.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1), also called neuronal-specific protein gene product 9.5, is a highly abundant protein in the neuronal cell body and has been identified as a possible biomarker on the basis of a recent proteomic study. In this study, we examined whether UCH-L1 was significantly elevated in cerebrospinal fluid (CSF) following controlled cortical impact (CCI) and middle cerebral artery occlusion (MCAO; model of ischemic stroke) in rats. Quantitative immunoblots of rat CSF revealed a dramatic elevation of UCH-L1 protein 48 h after severe CCI and as early as 6 h after mild (30 min) and severe (2 h) MCAO. A sandwich enzyme-linked immunosorbent assay constructed to measure UCH-L1 sensitively and quantitatively showed that CSF UCH-L1 levels were significantly elevated as early as 2 h and up to 48 h after CCI. Similarly, UCH-L1 levels were also significantly elevated in CSF from 6 to 72 h after 30 min of MCAO and from 6 to 120 h after 2 h of MCAO. These data are comparable to the profile of the calpain-produced alphaII-spectrin breakdown product of 145 kDa biomarker. Importantly, serum UCH-L1 biomarker levels were also significantly elevated after CCI. Similarly, serum UCH-L1 levels in the 2-h MCAO group were significantly higher than those in the 30-min group. Taken together, these data from two rat models of acute brain injury strongly suggest that UCH-L1 is a candidate brain injury biomarker detectable in biofluid compartments (CSF and serum).

Accumulation of calpain and caspase-3 proteolytic fragments of brain-derived alphaII-spectrin in cerebral spinal fluid after middle cerebral artery occlusion in rats.

Preclinical studies have identified numerous neuroprotective drugs that attenuate brain damage and improve functional outcome after cerebral ischemia. Despite this success in animal models, neuroprotective therapies in the clinical setting have been unsuccessful. Identification of biochemical markers common to preclinical and clinical cerebral ischemia will provide a more sensitive and objective measure of injury severity and outcome to facilitate clinical management and treatment. However, there are currently no effective biomarkers available for assessment of stroke. Nonerythroid alphaII-spectrin is a cytoskeletal protein that is cleaved by calpain and caspase-3 proteases to signature alphaII-spectrin breakdown products (alphaII-SBDPs) after cerebral ischemia in rodents. This investigation examined accumulation of calpain- and caspase-3-cleaved alphaII-SBDPs in cerebrospinal fluid (CSF) of rodents subjected to 2 hours of transient focal cerebral ischemia produced by middle cerebral artery occlusion (MCAO) followed by reperfusion. After MCAO injury, full-length alphaII-spectrin protein was decreased in brain tissue and increased in CSF from 24 to 72 hours after injury. Whereas alphaII-SBDPs were undetectable in sham-injured control animals, calpain but not caspase-3 specific alphaII-SBDPs were significantly increased in CSF after injury. However, caspase-3 alphaII-SBDPS were observed in CSF of some injured animals. These results indicate that alphaII-SBDPs detected in CSF after injury, particularly those mediated by calpain, may be useful diagnostic indicators of cerebral infarction that can provide important information about specific neurochemical events that have occurred in the brain after acute stroke.

Nitric oxide and subarachnoid hemorrhage: elevated level in cerebrospinal fluid and their implications.

OBJECTIVE: Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury after brain ischemia, and decreased levels of NO have been implicated in the pathogenesis of vasospasm after subarachnoid hemorrhage (SAH). In this study, we measured the ventricular cerebrospinal fluid (CSF) NO levels in patients with SAH and correlated the levels with clinical grade and middle cerebral artery velocities measured with transcranial Doppler ultrasound. METHODS: All patients with spontaneous SAH documented on computed tomography and with an external ventricular drain inserted within 24 hours of hemorrhage were included in the study. A total of 16 patients were studied between August 1999 and August 2000. CSF was collected serially at the time of surgery and subsequently at daily intervals. It was collected during the time that the external ventricular drain remained patent and in situ. NO levels were measured by photometric analysis by using a nitrite/nitrate assay kit (Cayman Chemical, Ann Arbor, MI). RESULTS: The peak NO level in patients with SAH ranged from 9.96 to 168.16 micromol, with a median of 36.93 micromol. The levels were significantly elevated as compared with the control group (5.16 micromol, P < 0.05). The median NO level in patients with poor-grade SAH was 67.14 micromol as compared with 27.42 micromol in patients with good-grade hemorrhage (P < 0.05). No correlation was seen between CSF NO levels and middle cerebral artery velocities. The median NO level was 33.2 micromol in patients with a poor outcome as compared with 30.25 micromol in patients with a good outcome (P > 0.05). CONCLUSION: This study showed that NO levels are elevated after spontaneous SAH, and the degree of elevation is higher in patients with poor-grade SAH.