Brain and blood microRNA expression profiling of ischemic stroke, intracerebral hemorrhage, and kainate seizures

MicroRNAs (miRNAs) regulate gene expression and have a critical role in many biologic and pathologic processes. We hypothesized that miRNA expression profiles in injured brain (hippocampus) would show common as well as unique profiles when compared with those of blood. Adult, untouched, control rats were compared with rats with sham surgeries, ischemic strokes, brain hemorrhage (lysed blood, fresh blood, or thrombin), and kainate-induced seizures. Brain and whole-blood miRNA expression profiles were assessed 24 h later using TaqMan rodent miRNA arrays. MicroRNA response profiles were different for each condition. Many miRNAs changed more than 1.5-fold in brain and blood after each experimental manipulation, and several miRNAs were upregulated or downregulated in both brain and blood after a given injury. A few miRNAs (e.g., miR-298, miR-155, and miR-362-3p) were upregulated or downregulated more than twofold in both brain and blood after several different injuries. The results show the possible use of blood miRNAs as biomarkers for brain injury; that selected blood miRNAs may correlate with miRNA changes in the brain; and that many of the mRNAs, previously shown to be regulated in brain and blood after brain injury, are likely accounted for by changes in miRNA expression.     

Ontogeny of adrenomedullary responses to hypoxia and hypoglycemia: Role of splanchnic innervation

Adrenals of neonatal rats were denervated at 3 days of age (just before functional neuronal connections are ordinarily made). At 14 days of age, rats with denervated adrenals did not secrete catecholamines in response to a neurogenic reflex stimulus (insulin-induced hypoglycemia), confirming that innervation had failed to develop; sham-operated littermates were fully responsive to insulin. In contrast, hypoxia was still able to cause depletion of adrenal catecholamines in the denervated group, indicating the persistence of a non-neurogenic secretory mechanism well past the age at which it should have disappeared (8 days). Prolonged deprivation of neural input in adult rats also led to the re-emergence of non-neurogenic capabilities. Twenty-one days after surgery in adulthood, adrenal responses to insulin were still not present but those to hypoxia were. Thus, the development of neural stimulation itself is responsible for the ontogenetic loss of the immature type of adrenal catecholamine release mechanism and does so by suppressing the non-neurogenic component; consequently the mechanism can reappear when nerve traffic is interrupted for extended periods.     

Blood genomic responses differ after stroke, seizures, hypoglycemia, and hypoxia: blood genomic fingerprints of disease.

Using microarray technology, we investigated whether the gene expression profile in white blood cells could be used as a fingerprint of different disease states. Adult rats were subjected to ischemic strokes, hemorrhagic strokes, sham surgeries, kainate-induced seizures, hypoxia, or insulin-induced hypoglycemia, and compared with controls. The white blood cell RNA expression patterns were assessed 24 hours later using oligonucleotide microarrays. Results showed that many genes were upregulated or downregulated at least twofold in white blood cells after each experimental condition. Blood genomic response patterns were different for each condition. These results demonstrate the potential of blood gene expression profiling for diagnostic, mechanistic, and therapeutic assessment of a wide variety of disease states.     

Relationships between brain and body temperature,clinical and imaging outcomes after ischemic stroke

Pyrexia soon after stroke is associated with severe stroke and poor functional outcome. Few studies have assessed brain temperature after stroke in patients, so little is known of its associations with body temperature, stroke severity, or outcome. We measured temperatures in ischemic and normal-appearing brain using ¹H-magnetic resonance spectroscopy and its correlations with body (tympanic) temperature measured four-hourly, infarct growth by 5 days, early neurologic (National Institute of Health Stroke Scale, NIHSS) and late functional outcome (death or dependency). Among 40 patients (mean age 73 years, median NIHSS 7, imaged at median 17 hours), temperature in ischemic brain was higher than in normal-appearing brain on admission (38.6°C-core, 37.9°C-contralateral hemisphere, P=0.03) but both were equally elevated by 5 days; both were higher than tympanic temperature. Ischemic lesion temperature was not associated with NIHSS or 3-month functional outcome; in contrast, higher contralateral normal-appearing brain temperature was associated with worse NIHSS, infarct expansion and poor functional outcome, similar to associations for tympanic temperature. We conclude that brain temperature is higher than body temperature; that elevated temperature in ischemic brain reflects a local tissue response to ischemia, whereas pyrexia reflects the systemic response to stroke, occurs later, and is associated with adverse outcomes.     

Acute ischemic stroke treatment and the occurrence of seizures

Background and objectives

Early intravenous treatment with recombinant tissue plasminogen activator (rt-PA) improves the outcome of patients with an acute ischemic stroke. This retrospective observational study analyses whether rt-PA treatment also prevents the occurrence of early- and late-onset seizures.

Patients and methods

Thirty-eight patients treated with rt-PA were compared to 269 receiving anticoagulants (ACs) and 769 on antithrombotics (ATs) for an acute cardiac- or thrombo-embolic stroke. The epidemiological and clinical data, and the vascular risk factors were determined in the three groups. The incidence, onset and types of seizures were compared.

Results

The patients treated with rt-PA had more severe stroke signs on admission and remained more dependent than those treated with ACs and ATs. The appearance of early-onset seizures was related to the severity of the stroke. The incidence of the late-onset ones tended to be low in the rt-PA group. None of the patients developed status epilepticus or epilepsy.

Conclusion

The occurrence of early-onset seizures in the rt-PA treated group is related to the severity of the stroke and not to the treatment modality. Thrombolysis prevents partly the occurrence of late-onset seizures, probably, by a better reperfusion of the ischemic brain regions.     

Differential neuronal fates in the CA1 hippocampus after hypoxia in newborn and 7-day-old rats: effects of pre-treatment with MK-801

The brain displays an age-dependent sensitivity to ischemic insults. However, the consequences of oxygen deprivation per se in the developing brain remain unclear, and the role of glutamate excitotoxicity via N-methyl-D-aspartate (NMDA) receptors is controversial. To gain a better understanding of the mechanisms involved in the cerebral response to severe hypoxia, cell damage was temporally monitored in the CA1 hippocampus of rat pups transiently exposed to in vivo hypoxia (100% N2) at either 24 h or 7 days of age. Also, the influence of a pre-treatment with the NMDA receptor antagonist MK-801 (5 mg/kg, i.p.) was examined. At both ages, morphometric analyses and cell counts showed hypoxia-induced significant neuronal loss (30-35%) in the pyramidal layer, with injury appearing more rapidly in rats exposed at 7 days. Morphological alterations of 4,6-diamidino-2-phenylindole (DAPI)-labeled nuclei, DNA fragmentation patterns on agarose gels, as well as expression profiles of the apoptosis-related regulatory proteins Bax and Bcl-2 showed that apoptosis was prevalent in younger animals, whereas only necrosis was detected in hippocampi of rats treated at 7 days. Moreover, pre-treatment with MK-801 was ineffective in protecting hippocampal neurons from hypoxic injury in newborn rats, but significantly reduced necrosis in older subjects. These data confirm that hypoxia alone may trigger neuronal death in vivo, and the type of cell death is strongly influenced by the degree of brain maturity. Finally, NMDA receptors are not involved in the apoptotic consequences of hypoxia in the newborn rat brain, but they were found to mediate necrosis at 7 days of age.     

Adenosine kinase determines the degree of brain injury after ischemic stroke in mice

Adenosine kinase (ADK) is the major negative metabolic regulator of the endogenous neuroprotectant and homeostatic bioenergetic network regulator adenosine. We used three independent experimental approaches to determine the role of ADK as a molecular target for predicting the brain''s susceptibility to ischemic stroke. First, when subjected to a middle cerebral artery occlusion model of focal cerebral ischemia, transgenic fb-Adk-def mice, which have increased ADK expression in striatum (164%) and reduced ADK expression in cortical forebrain (65%), demonstrate increased striatal infarct volume (126%) but almost complete protection of cortex (27%) compared with wild-type (WT) controls, indicating that cerebral injury levels directly correlate to levels of ADK in the CNS. Second, we demonstrate abrogation of lipopolysaccharide (LPS)-induced ischemic preconditioning in transgenic mice with brain-wide ADK overexpression (Adk-tg), indicating that ADK activity negatively regulates LPS-induced tolerance to stroke. Third, using adeno-associated virus-based vectors that carry Adk-sense or -antisense constructs to overexpress or knockdown ADK in vivo, we demonstrate increased (126%) or decreased (51%) infarct volume, respectively, 4 weeks after injection into the striatum of WT mice. Together, our data define ADK as a possible therapeutic target for modulating the degree of stroke-induced brain injury.     

Interleukin-10 overexpression does not synergize with the neuroprotective action of RGD-containing vectors after postnatal brain excitotoxicity but modulates the main inflammatory cell responses

Antiinflammatory cytokines such as interleukin-10 (IL-10) have been used to modulate and terminate inflammation and provide neuroprotection. Recently, we reported that the modular recombinant transfection vector NLSCt is an efficient tool for transgene overexpression in vivo, which induces neuroprotection as a result of its RGD-mediated integrin-interacting capacity. We here sought to evaluate the putative synergic neuroprotective action exerted by IL-10 overexpression using NLSCt as a transfection vector after an excitotoxic injury to the postnatal rat brain. For this purpose, lesion volume, neurodegeneration, astroglial and microglial responses, neutrophil infiltration, and proinflammatory cytokine production were analyzed at several survival times after intracortical NMDA injection in postnatal day 9 rats, followed by injection of NLSCt combined with the IL-10 gene, a control transgene, or saline vehicle solution. Our results show no combined neuroprotective effect between RGD-interacting vectors and IL-10 gene therapy; instead, IL-10 overexpression using NLSCt as transfection vector increased lesion volume and neuronal degeneration at 12 hr and 3 days postlesion. In parallel, NLSCt/IL-10 treated animals displayed increased density of neutrophils and microglia/macrophages, and a reduced astroglial content of GFAP and vimentin. Moreover, NLSCt/IL-10 treated animals did not show any variation in interleukin-1β or tumor necrosis factor-α expression but a slight increase in interleukin-6 content at 7 days postlesion. In conclusion, overexpression of IL-10 by using NLSCt transfection vector did not synergistically neuroprotect the excitotoxically damaged postnatal rat brain but induced changes in the astroglial and microglial and inflammatory cell response.     

Antioxidant effect of acetylsalicylic and salicylic acid in rat brain slices subjected to hypoxia

Acetylsalicylic acid (ASA) reduces the incidence of ischemic stroke mainly through its antithrombotic action; however, it also has a direct neuroprotective effect. The present study was designed to evaluate the effect of ASA on oxidative stress and the activity of nitric oxide synthase (NOS) in an in vitro model of hypoxia in rat brain slices. Rat brain slices were perfused with nitrogen (hypoxia) for a maximum of 120 min, after which we measured lipid peroxidation, glutathione levels, glutathione-related enzyme activities, and constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) activities. In brain tissue subjected to hypoxia, ASA reduced oxidative stress and iNOS activity (all increased by hypoxia), but only when used at higher concentrations. The effects of salicylic acid (SA) were similar but more intense than were those of ASA. After oral administration, the effect of SA was much greater than that of ASA, and the decrease in cell death with SA was seen much more clearly. In view of the greater effect of SA compared to ASA on changes in oxidative stress parameters in a model of hypoxia, and higher brain concentrations of SA when it is administered alone than when ASA is given (undetectable levels), we conclude that SA plays an important role in the cytoprotective effect in brain tissue after ASA administration.     

Remote ischemic conditioning enhances oxygen supply to ischemic brain tissue in a mouse model of stroke: Role of elevated 2,3-biphosphoglycerate in erythrocytes

Oxygen supply for ischemic brain tissue during stroke is critical to neuroprotection. Remote ischemic conditioning (RIC) treatment is effective for stroke. However, it is not known whether RIC can improve brain tissue oxygen supply. In current study, we employed a mouse model of stroke created by middle cerebral artery occlusion (MCAO) to investigate the effect of RIC on oxygen supply to the ischemic brain tissue using a hypoxyprobe system. Erythrocyte oxygen-carrying capacity and tissue oxygen exchange were assessed by measuring oxygenated hemoglobin and oxygen dissociation curve. We found that RIC significantly mitigated hypoxic signals and decreased neural cell death, thereby preserving neurological functions. The tissue oxygen exchange was markedly enhanced, along with the elevated hemoglobin P50 and right-shifted oxygen dissociation curve. Intriguingly, RIC markedly elevated 2,3-biphosphoglycerate (2,3-BPG) levels in erythrocyte, and the erythrocyte 2,3-BPG levels were highly negatively correlated with the hypoxia in the ischemic brain tissue. Further, adoptive transfusion of 2,3-BPG-rich erythrocytes prepared from RIC-treated mice significantly enhanced the oxygen supply to the ischemic tissue in MCAO mouse model. Collectively, RIC protects against ischemic stroke through improving oxygen supply to the ischemic brain tissue where the enhanced tissue oxygen delivery and exchange by RIC-induced 2,3-BPG-rich erythrocytes may play a role.     

Differential sensitivity of medium- and large-sized striatal neurons to NMDA but not kainate receptor activation in the rat

Infrared videomicroscopy and differential interference contrast optics were used to identify medium- and large-sized neurons in striatal slices from young rats. Whole-cell patch-clamp recordings were obtained to compare membrane currents evoked by application of N-methyl-d-aspartate (NMDA) and kainate. Inward currents and current densities induced by NMDA were significantly smaller in large- than in medium-sized striatal neurons. The negative slope conductance for NMDA currents was greater in medium- than in large-sized neurons and more depolarization was required to remove the Mg2+ blockade. In contrast, currents induced by kainate were significantly greater in large-sized neurons whilst current densities were approximately equal in both cell types. Spontaneous excitatory postsynaptic currents occurred frequently in medium-sized neurons but were relatively infrequent in large-sized neurons. Excitatory postsynaptic currents evoked by electrical stimulation were smaller in large- than in medium-sized neurons. A final set of experiments assessed a functional consequence of the differential sensitivity of medium- and large-sized neurons to NMDA. Cell swelling was used to examine changes in somatic area in both neuronal types after prolonged application of NMDA or kainate. NMDA produced a time-dependent increase in somatic area in medium-sized neurons whilst it produced only minimal changes in large interneurons. In contrast, application of kainate produced significant swelling in both medium- and large-sized cells. We hypothesize that reduced sensitivity to NMDA may be due to variations in receptor subunit composition and/or the relative density of receptors in the two cell types. These findings help define the conditions that put neurons at risk for excitotoxic damage in neurological disorders.     

Loss of miR-29b following acute ischemic stroke contributes to neural cell death and infarct size

Glutathione depletion and 12-lipoxygenase-dependent metabolism of arachidonic acid are known to be implicated in neurodegeneration associated with acute ischemic stroke. The objective of this study was to investigate the significance of miR-29 in neurodegeneration associated with acute ischemic stroke. Neural cell death caused by arachidonic acid insult of glutathione-deficient cells was preceded by a 12-lipoxygenase-dependent loss of miR-29b. Delivery of miR-29b mimic to blunt such loss was neuroprotective. miR-29b inhibition potentiated such neural cell death. 12-Lipoxygenase knockdown and inhibitors attenuated the loss of miR-29b in challenged cells. In vivo, stroke caused by middle-cerebral artery occlusion was followed by higher 12-lipoxygenase activity and loss of miR-29b as detected in laser-captured infarct site tissue. 12-Lipoxygenase knockout mice demonstrated protection against such miR loss. miR-29b gene delivery markedly attenuated stroke-induced brain lesion. Oral supplementation of α-tocotrienol, a vitamin E 12-lipoxygenase inhibitor, rescued stroke-induced loss of miR-29b and minimized lesion size. This work provides the first evidence demonstrating that loss of miR-29b at the infarct site is a key contributor to stroke lesion. Such loss is contributed by activity of the 12-lipoxygenase pathway providing maiden evidence linking arachidonic acid metabolism to miR-dependent mechanisms in stroke.     

Fibrinolysis and aspiration of experimental intracerebral hematoma reduces the volume of ischemic brain in rats

Abstract

The hypothesis was tested in rats that brain ischemia by an intracerebral hematoma can be ameliorated by fibrinolysis and aspiration of the hematoma. Intraparenchymal blood clots were generated by the injection of 50μI of autologous blood into the right caudate nucleus in two portions seven minutes apart. Thirty or 120 min later 12 fil recombinant tissue plasminogen activator (rtPA) or 0.9% NaCI were injected and after 30 min the resolved hematoma was aspirated. Six hours later cerebral blood flow (CBF) was determined by 14C-iodoantipyrine autoradiography. Tissue volumes of CBF < 10 ml 1 00 g^–1 min-1 and CBF < 30 ml g"1 min’1 were determined. Clot and lesion volume were quantified histologically from serial sections stained for succinate-dehydrogenase (SDH) activity. In rtPA-treated rats the major part of the hematoma could be evacuated 30 min as well as 120 min after production of the clot. The volume of ischemic brain (CBF < 10) was significantly reduced fp<0.05) in the rtPA group compared to saline- treated and control groups irrespective of the time of treatment. In contrast, no difference was found between the control group and the experimental groups when the volumes of brain tissue surrounding the lesion were compared which had values of CBF<30 ml lOOgmin^–1. In a rat model of intracerebral hemorrhage, treatment by local fibrinolysis followed by aspiration of the hematoma is effective in reducing the volume of ischemic brain tissue and of the remaining clot volume. [Neurol Res 1999; 21: 517–523]     

急性期血浆脑钠肽水平与缺血性脑卒中近期预后关系分析

目的 探讨缺血性脑卒中患者急性期血浆BNP水平与近期预后的关系.方法 选取43例缺血性脑卒中急性期患者为研究对象,所有研究对象均在入院第2天清晨空腹抽血行BNP测定.将同期门诊40例中老年健康体检者为对照组.比较2组血浆BNP水平,同时比较不同预后缺血性脑卒中患者首次血浆BNP水平.结果 缺血性脑卒中患者组血浆BNP浓度高于对照组(P＜0.01);而近期预后不良的缺血性脑卒中患者急性期血浆BNP水平较近期预后良好的患者高(P＜0.01).结论 缺血性脑卒中患者急性期血浆BNP水平与缺血性卒中近期预后有密切关系.     

Downregulation of miR‐181b in mouse brain following ischemic stroke induces neuroprotection against ischemic injury through targeting heat shock protein A5 and ubiquitin carboxyl‐terminal hydrolase isozyme L1

Understanding the molecular mechanism of cerebral hypoxic preconditioning (HPC)‐induced endogenous neuroprotection may provide potential therapeutic targets for ischemic stroke. By using bioinformatics analysis, we found that miR‐181b, one of 19 differentially expressed miRNAs, may target aconitate hydratase (ACO2), heat shock protein A5 (HSPA5), and ubiquitin carboxyl‐terminal hydrolase isozyme L1 (UCHL1) among 26 changed protein kinase C isoform‐specific interacting proteins in HPC mouse brain. In this study, the role of miR‐181b in oxygen–glucose deprivation (OGD)‐induced N2A cell ischemic injury in vitro and mouse middle cerebral artery occlusion (MCAO)‐induced cerebral ischemic injury in vivo, and its regulation of ACO2, HSPA5, and UCHL1 were further determined. We found that miR‐181b expression levels significantly decreased in mouse brain following MCAO and in OGD‐treated N2A cells. Up‐ and downregulation of miR‐181b by transfection of pre‐ or anti‐miR‐181b could negatively regulate HSPA5 and UCHL1 (but not ACO2) protein levels as well as N2A cell death and programmed cell death in OGD‐treated N2A cells. By using a T7 promoter‐driven control dual luciferase assay, we confirmed that miR‐181b could bind to the 3′‐untranslated rergions of HSPA5 and UCHL1 mRNAs and repress their translations. miR‐181b antagomir reduced caspase‐3 cleavage and neural cell loss in cerebral ischemic cortex and improved neurological deficit of mice after MCAO. In addition, HSPA5 and UCHL1 short interfering RNAs (siRNAs) blocked anti‐miR‐181b‐mediated neuroprotection against OGD‐induced N2A cell injury in vitro. These results suggest that the downregulated miR‐181b induces neuroprotection against ischemic injury through negatively regulating HSPA5 and UCHL1 protein levels, providing a potential therapeutic target for ischemic stroke. © 2013 Wiley Periodicals, Inc.     

Effect of hypoxia on the incorporation of [2-3H]glycerol and [1-14C]-palmitate into lipids of various brain regions

The lipid metabolism in guinea pig brain after intermittent hypoxia, prolonged for 80 hrs, was markedly impaired. The in vivo incorporation of [2-³H]glycerol and [1-¹⁴C]palmitate into lipids of microsomes, mitochondria, myelin, and synaptosomes, purified from cerebral hemispheres, was significantly lower in the hypoxic animals than in the controls. The same effect was observed on the incorporation of labeled precursors into lipids of mitochondria purified from cerebellum and brainstem. In particular, the labeling of the major phospholipids present – ie, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) – in the mitochondria of the three brain regions examined decreased after hypoxic treatment.     

Computed tomographic changes of the brain and clinical outcome of patients with seizures and epilepsy after an ischaemic hemispheric stroke.

It is not well established whether seizures and epilepsy after an ischaemic stroke increase the disability of patients. Seventy-two patients with delayed seizures after a hemispheric infarct (37 with a single seizure and 35 with epilepsy) were included in the study. The modified Rankin scale was used to compare disability of the patients at 1 month after stroke and at 2 weeks after single or the last seizure, in case of epilepsy. The size of the X-ray hypoattenuation zone was compared on computed tomographic (CT) scans, performed in the weeks after the stroke and 1 week after single or repeated seizures. Lesion size was determined by superimposing the CT slices on digital cerebral vascular maps, on which the contours of the infarct area were delineated. The extent of the infarcts was expressed as the percentage fraction of the total surface area of the cerebral hemisphere. Groups with a single seizure and with epilepsy were mutually compared. Infarcts predominated in the parieto-temporal cortical regions. In the overall group the median Rankin score worsened significantly after seizures. The average size of the X-ray hypoattenuation zone was also significantly increased on the CT scans after the seizures, compared with those after stroke, without clear evidence of recent infarction. Mutual comparison of patients with a single seizure episode and of those with epilepsy showed only a trend of more severe disability and of increase in lesion size in the post-stroke epilepsy group. Delayed seizures and epilepsy after ischaemic stroke are accompanied by an increase in lesion size on CT and by worsening of the disability of the patients. This study does not allow to determine whether this is due to stroke recurrence or due to additional damage as a result of the seizures themselves.