Development Of Ischemic Brain Injury Research Articles

Acute Cerebral Ischemia Increases a Set of Brain-Specific miRNAs in Serum Small Extracellular Vesicles.

Small extracellular vesicles (sEVs) miRNAs are promising diagnosis and prognosis biomarkers for ischemic stroke (IS). This study aimed to determine the impact of IS on the serum sEVs miRNA profile of IS patients and a transient middle cerebral artery occlusion (tMCAO) mouse model. Small RNAseq was used to define the serum sEVs miRNA profile in IS patients and healthy controls (HC), and tMCAO mice and sham controls. Among the 1,444 and 1,373 miRNAs identified in human and mouse serum sEVs, the expression of 424 and 37 miRNAs was significantly altered in the IS patients and tMCAO mice, respectively (| Log2FC| ≥ 1, p < 0.01). Notably, five of the top 25 upregulated miRNAs in IS patients were brain-specific or enriched, including hsa-miR-9-3p, hsa-miR-124-3p, hsa-miR-143-3p, hsa-miR-98-5p, and hsa-miR-93-5p. Upregulation of these four miRNAs was further validated by qPCR. Nine of the 20 upregulated miRNAs in tMCAO mice were also brain-specific or enriched miRNAs. Temporal analysis indicated that the dynamics of mmu-miR-9-5p, mmu-miR-124-3p, mmu-miR-129-5p, and mmu-miR-433-3p were closely correlated with the evolution of ischemic brain injury, as their expression increased at 0.5 days after the onset of ischemia, peaked at day 1 or 3, and returned to normal levels at day 7 and 14. Notably, with the exceptions of mmu-miR-128-3p, the expression of the other eight miRNAs in the mouse serum sEVs was unaffected in the lipopolysaccharide (LPS)-induced neuroinflammation model. Together, in this study, we provided a comprehensive view of the influences of IS on the serum sEVs miRNA profile of IS patients and tMCAO mice and demonstrated the increment of a set of brain-specific miRNAs in serum sEVs after acute cerebral ischemia, which could be promising candidates directly reflecting the ischemic brain injury.

New PAR1 Agonist Peptide Demonstrates Protective Action in a Mouse Model of Photothrombosis-Induced Brain Ischemia.

Protease-activated receptors (PARs) are involved not only in hemostasis but also in the development of ischemic brain injury. In the present work, we examined in vivo effects of a new peptide (AP9) composing Asn47-Phen55 of PAR1 “tethered ligand” generated by activated protein C. We chose a mouse model of photothrombosis (PT)-induced ischemia to assess AP9 effects in vivo. To reveal the molecular mechanism of AP9 action, mice lacking β-arrestin-2 were used. AP9 was injected intravenously once 10 min before PT at doses of 0.2, 2, or 20 mg/kg, or twice, that is, 10 min before and 1 h after PT at a dose of 20 mg/kg. Lesion volume was measured by magnetic resonance imaging and staining of brain sections with tetrazolium salt. Neurologic deficit was estimated using the cylinder and the grid-walk tests. Blood–brain barrier (BBB) disruption was assessed by Evans blue dye extraction. Eosin-hematoxylin staining and immunohistochemical staining were applied to evaluate the number of undamaged neurons and activated glial cells in the penumbra. A single administration of AP9 (20 mg/kg), as well as its two injections (20 mg/kg), decreased brain lesion volume. A double administration of AP9 also reduced BBB disruption and neurological deficit in mice. We did not observe the protective effect of AP9 in mice lacking β-arrestin-2 after PT. Thus, we demonstrated for the first time protective properties of a PAR1 agonist peptide, AP9, in vivo. β-Arrestin-2 was required for the protective action of AP9 in PT-induced brain ischemia.

Treatment duration affects cytoprotective efficacy of positive allosteric modulation of α7 nAChRs after focal ischemia in rats.

To minimize irreversible brain injury after acute ischemic stroke (AIS), the time to treatment (i.e., treatment delay) should be minimized. However, thus far, all cytoprotective clinical trials have failed. Analysis of literature identified short treatment durations (≤72 h) as a common motif among completed cytoprotective clinical trials. Here, we argue that short cytoprotective regimens even if given early after AIS may only slow down the evolution of ischemic brain injury and fail to deliver sustained long-term solutions leading to relapses that may be misinterpreted for conceptual failure of cytoprotection. In this randomized blinded study, we used young adult male rats subjected to transient 90 min suture middle cerebral artery occlusion (MCAO) and treated with acute vs. sub-chronic regimens of PNU120596, a prototypical positive allosteric modulator of α7 nicotinic acetylcholine receptors with anti-inflammatory cytoprotective properties to test the hypothesis that insufficient treatment durations may reduce therapeutic benefits of otherwise efficacious cytoprotectants after AIS. A single acute treatment 90 min after MCAO significantly reduced brain injury and neurological deficits 24 h later, but these effects vanished 72 h after MCAO. These relapses were avoided by utilizing sub-chronic treatments. Thus, extending treatment duration augments therapeutic efficacy of PNU120596 after MCAO. Furthermore, sub-chronic treatments could offset the negative effects of prolonged treatment delays in cases where the acute treatment window after MCAO was left unexploited. We conclude that a combination of short treatment delays and prolonged treatment durations may be required to maximize therapeutic effects of PNU120596, reduce relapses and ensure sustained therapeutic efficacy after AIS. Similar concepts may hold for other cytoprotectants including those that failed in clinical trials.

Neuroprotective effects of amiodarone in a mouse model of ischemic stroke

BackgroundIon channels play a crucial role in the development of ischemic brain injury. Recent studies have reported that the blockade of various types of ion channels improves outcomes in experimental stroke models. Amiodarone, one of the most effective drugs for life-threatening arrhythmia, works as a multiple channel blocker and its characteristics cover all four Vaughan-Williams classes. Although it is known that amiodarone indirectly contributes to preventing ischemic stroke by maintaining sinus rhythm in patients with atrial fibrillation, the direct neuroprotective effect of amiodarone has not been clarified. The purpose of this study was to investigate the direct effect of amiodarone on ischemic stroke in mice.MethodsFocal cerebral ischemia was induced via distal permanent middle cerebral artery occlusion (MCAO) in adult male mice. The amiodarone pre-treatment group received 50 mg/kg of amiodarone 1 h before MCAO; the amiodarone post-treatment groups received 50 mg/kg of amiodarone immediately after MCAO; the control group received vehicle only. In addition, the sodium channel opener veratrine and selective beta-adrenergic agonist isoprotelenol were used to elucidate the targeted pathway. Heart rate and blood pressure were monitored perioperatively. Infarct volume analysis was conducted 48 h after MCAO. The body asymmetry test and the corner test were used for neurological evaluation.ResultsAmiodarone pre-treatment and post-treatment reduced the heart rate but did not affect the blood pressure. No mice showed arrhythmia. Compared with the control group, the amiodarone pre-treatment group had smaller infarct volumes (8.9 ± 2.1% hemisphere [mean ± SD] vs. 11.2 ± 1.4%; P < 0.05) and improved functional outcomes: lower asymmetric body swing rates (52 ± 17% vs. 65 ± 18%; P < 0.05) and fewer left turns (7.1 ± 1.2 vs. 8.3 ± 1.2; P < 0.05). In contrast, amiodarone post-treatment did not improve the outcomes after MCAO. The neuroprotective effect of amiodarone pre-treatment was abolished by co-administration of veratrine but not by isoproterenol.ConclusionsAmiodarone pre-treatment attenuated ischemic brain injury and improved functional outcomes without affecting heart rhythm and blood pressure. The present results showed that amiodarone pre-treatment has neuroprotective effects, at least in part, via blocking the sodium channels.

Abstract W P100: Alterations of γδT Cell Subsets in Stroke Patients with White Matter Disease

Background: γδT cells have been shown in experimental models to play a role in the evolution of ischemic brain injury by increasing cerebral infarct volumes and neurological deficits. We have previously reported decreased levels of γδT cells in the subacute phase of human stroke. In this study we aimed to determine the functional significance of alterations in levels of γδT cells and other T cell subsets on the extent of ischemic brain injury. Methods: Blood was drawn from ischemic stroke patients within 24 hours of recruitment and stained with CD3 (PE-TR), γδTCR (PE), CD4 (FITC) and CD8 (PE-Cy5) monoclonal antibodies. γδT cells were also divided into functional subsets - CD4+ γδT, CD8+ γδT and null γδT cells. Percentages of circulating lymphocytes were measured by a fluorescent activated cell counter. The extent of acute ischemic injury was measured using lesion volume on diffusion weighted images by Image Processing Analysis and Visualization (MIPAV), while the extent of chronic ischemic injury which is manifested as white matter changes(WMC) was assessed using the Age Related White Matter Changes (ARWMC) rating score. Independent T-tests and non-parametric tests were used for between group comparisons. Results: Of the 41 patients recruited, the mean age was 66.8±13 years and 18 (43.9%) were men. Blood was drawn at a mean time of 47 ±30 hours after symptom onset. Compared to patients with absent-mild WMC (ARWMC score of 0-1), patients with moderate-severe WMC (ARWMC of 2-3) had higher percentages of CD8+ γδT cells (6.6% vs. 12.0%, p=0.005) and CD8+ T cells (22.2% vs. 28.5% p=0.036), and lower percentages of CD4+ T cells (71.0% vs. 63.5%, p=0.048) and null γδT cells (72.7% vs. 59.6%, p=0.03). However γδT percentages did not differ significantly. The volumes of acute ischemic stroke lesions did not correlate with γδT levels or with other T cell subsets. Conclusions: Alterations of T cell subsets, most notably, CD8+ γδT and CD8+ T cells were present in stroke patients with cerebral white matter disease. Their role in the formation of cerebral small vessel disease needs to be further studied.

Endovascular acute ischemic stroke therapy

The interface between basic science and clinical medicine is a key to an enhanced understanding of disease pathophysiology and for developing novel therapies. The term translational research is applied to this interface and provides a framework for the interaction between basic scientists and clinicians. Ischemic stroke is a medical disorder for which translational research is particularly important, because of rapidly developing advances in understanding the cellular consequences of focal brain ischemia, the contributions of the neurovascular unit to disease pathophysiology, and the role of blood vessel abnormalities. These and other factors contribute to the development and evolution of ischemic brain injury when a thrombus impairs blood flow to a brain region. Reperfusion of these ischemic brain regions with thrombolytic drugs such as tissue plasminogen activator (tPA) or by deploying a mechanical device to remove the occluding thrombus is a logical approach to ischemic stroke therapy, because each method restores the availability of oxygen and glucose-rich blood flow that potentiate salvage of ischemic tissue destined for infarction. Currently, only administration of IV tPA within 3 hours of ischemic stroke onset (likely to be extended to 4.5 hours soon) is approved by regulatory authorities, and 2 devices, the MERCI retriever …

Toll-like receptor 2 deficiency leads to delayed exacerbation of ischemic injury

BackgroundUsing a live imaging approach, we have previously shown that microglia activation after stroke is characterized by marked and long-term induction of the Toll-like receptor (TLR) 2 biophotonic signals. However, the role of TLR2 (and potentially other TLRs) beyond the acute innate immune response and as early neuroprotection against ischemic injury is not well understood.MethodsTLR2−/− mice were subjected to transient middle cerebral artery occlusion followed by different reperfusion times. Analyses assessing microglial activation profile/innate immune response were performed using in situ hybridization, immunohistochemistry analysis, flow cytometry and inflammatory cytokine array. The effects of the TLR2 deficiency on the evolution of ischemic brain injury were analyzed using a cresyl violet staining of brain sections with appropriate lesion size estimation.ResultsHere we report that TLR2 deficiency markedly affects post-stroke immune response resulting in delayed exacerbation of the ischemic injury. The temporal analysis of the microglia/macrophage activation profiles in TLR2−/− mice and age-matched controls revealed reduced microglia/macrophage activation after stroke, reduced capacity of resident microglia to proliferate as well as decreased levels of monocyte chemotactic protein-1 (MCP-1) and consequently lower levels of CD45high/CD11b+ expressing cells as shown by flow cytometry analysis. Importantly, although acute ischemic lesions (24 to 72 h) were smaller in TLR2−/− mice, the observed alterations in innate immune response were more pronounced at later time points (at day 7) after initial stroke, which finally resulted in delayed exacerbation of ischemic lesion leading to larger chronic infarctions as compared with wild-type mice. Moreover, our results revealed that TLR2 deficiency is associated with significant decrease in the levels of neurotrophic/anti-apoptotic factor Insulin-like growth factor-1 (IGF-1), expressed by microglia in the areas both in and around ischemic lesion.ConclusionOur results clearly suggest that optimal and timely microglial activation/innate immune response is needed to limit neuronal damage after stroke.

Differential Modulation of Estrogen Receptors (ERs) in Ischemic Brain Injury: A Role for ERα in Estradiol-Mediated Protection against Delayed Cell Death

Estradiol enhances plasticity and survival of the injured brain. Our previous work demonstrates that physiological levels of estradiol protect against cerebral ischemia in the young and aging brain through actions involving estrogen receptors (ERs) and alterations in gene expression. The major goal of this study was to establish mechanisms of neuroprotective actions induced by low levels of estradiol. We first examined effects of estradiol on the time-dependent evolution of ischemic brain injury. Because estradiol is known to influence apoptosis, we hypothesized that it acts to decrease the delayed phase of cell death observed after middle cerebral artery occlusion (MCAO). Furthermore, because ERs are pivotal to neuroprotection, we examined the temporal expression profiles of both ER subtypes, ERalpha and ERbeta, after MCAO and delineated potential roles for each receptor in estradiol-mediated neuroprotection. We quantified cell death in brains at various times after MCAO and analyzed ER expression by RT-PCR, in situ hybridization, and immunohistochemistry. We found that during the first 24 h, the mechanisms of estradiol-induced neuroprotection after MCAO are limited to attenuation of delayed cell death and do not influence immediate cell death. Furthermore, we discovered that ERs exhibit distinctly divergent profiles of expression over the evolution of injury, with ERalpha induction occurring early and ERbeta modulation occurring later. Finally, we provide evidence for a new and functional role for ERalpha in estradiol-mediated protection of the injured brain. These findings indicate that physiological levels of estradiol protect against delayed cell death after stroke-like injury through mechanisms requiring ERalpha.

Estrogen and progestagens differentially modulate vascular proinflammatory factors

The potential benefit of ovarian hormone replacement therapy in cerebrovascular disease is well supported by experimental observations but not by recent large, randomized clinical trials. This discrepancy points out the need for better understanding of the vascular actions of ovarian hormones as well as medroxyprogesterone acetate (MPA), a synthetic analog of progesterone (P) widely prescribed in combination with estrogens. Therefore, we investigated whether in vivo exposure to 17beta-estradiol (E) and/or P or MPA modifies inflammation in the cerebral vasculature, a key process in the evolution of ischemic brain injury. Female rats were injected (ip) with LPS to induce inflammation, and 6 h later brains were taken for blood vessel isolation and Western blot analysis of the inflammatory enzymes inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). In ovariectomized (O) females, LPS induced cerebrovascular iNOS and COX-2; however, this effect was significantly decreased when O animals were treated for 3 wk with E. In contrast, treatment of O females with either MPA or P exacerbated the cerebrovascular inflammatory response to LPS. In intact females, LPS induction of iNOS and COX-2 in cerebral vessels was found to vary with the stage of the estrous cycle: LPS had the greatest effect during estrus, when circulating estrogen is low and progesterone is high. Thus exposure to endogenous or exogenous ovarian hormones appears to modulate cerebrovascular inflammation. Anti-inflammatory effects of estrogen would attenuate ischemic brain injury; however, this vasoprotective benefit may be diminished in the presence of progestagens.

Effects of reperfusion on ADC and CBF pixel-by-pixel dynamics in stroke: characterizing tissue fates using quantitative diffusion and perfusion imaging.

The effects of reperfusion on the spatiotemporal dynamics of transient (60 minutes) focal ischemic brain injury in rats were evaluated on a pixel-by-pixel basis using quantitative cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) measurements every 30 minutes for 3 hours and compared to post-mortem histology at 24 hours. Four biologically relevant clusters were classified based on ADC (0.53 +/- 0.02 x 10mm/s, SD) and CBF (0.30 +/- 0.09 ml/g/min) viability thresholds, namely: (1) the "normal" cluster with ADC and CBF > thresholds; (2) the "mismatch" cluster with ADC > threshold but CBF < threshold; (3) the "core" cluster with ADC and CBF < thresholds; and (4) "non-nourishing reperfusion zone" where ADC < threshold but CBF > threshold. The spatio-temporal progression of tissue volumes, ADC and CBF of each cluster were evaluated. Pixels of each cluster on the CBF-ADC space were mapped onto the image space. Following reperfusion, 28% of the "core" pixels and 90% of the "mismatch" (defined at 60 minutes) pixels were salvaged at 180 minutes, which correlated with histology. The ADC and CBF of subsequently salvaged tissues were significantly higher than those became infarcted. Salvaging "core" pixels indicated that reduced ADC was not synonymous with irreversible injury; duration of exposure and severity of reduced ADC and CBF were likely critical. Projection profiles showed a bimodal ADC, but uni-modal CBF, distributions. The ADC bimodal minima, obtained without histological correlation, were similar to the histology-derived ADC and CBF viability thresholds, and could have potential clinical applications. This study demonstrated a simple but powerful approach to evaluate, on a pixel-by-pixel basis, the spatio-temporal evolution of ischemic brain injury, and a potential for statistical prediction of tissue fate.

Induced hyperthermia exacerbates neurologic neuronal histologic damage after asphyxial cardiac arrest in rats.

Temperature is an important modulator of the evolution of ischemic brain injury--with hypothermia lessening and hyperthermia exacerbating damage. We recently reported that children resuscitated from predominantly asphyxial arrest often develop an initial spontaneous hypothermia followed by delayed hyperthermia. The initial hypothermia observed in these children was frequently treated with warming lights which, despite careful monitoring, often resulted in overshoot hyperthermia. We have previously reported in a rat model of asphyxial cardiac arrest that active warming, to prevent spontaneous hypothermia, worsens brain injury. We sought to determine whether delayed induction of hyperthermia would worsen brain injury after asphyxial arrest in rats. Male Sprague-Dawley rats were asphyxiated for 8 mins and resuscitated. An implantable temperature probe was placed into the peritoneum before asphyxia. The probe is a component of a computer-based, radiofrequency, telemetry system (Minimitter, Sunriver, OR) that allowed continuous acquisition and manipulation (via heating and cooling devices) of core (intraperitoneal) body temperature. Body temperature was monitored but not manipulated for the first 24 hrs of recovery. Rats were assigned to: no temperature manipulation (n = 21), induced hyperthermia (40 +/- 0.5 degrees C) for 3 hrs beginning at 24 hrs (n = 21), or induced hyperthermia at 48 hrs (n = 10). Control groups included sham rats (all surgical procedures except asphyxia) treated with induced hyperthermia at 24 hrs (n = 4) or 48 hrs (n = 4) and naïve rats (n = 4). Rats were killed at 7 days and injured neurons in hematoxylin and eosin stained coronal brain sections through dorsal hippocampus were scored in a semiquantitative manner on a scale of 0 to 10 (0 = normal; 1 = up to 10% neurons with ischemic neuronal changes; 10 = 90-100% neurons with ischemic neuronal changes). Normal-appearing neurons were also counted in CA1. The number of normal-appearing neurons in a 20x field in CA1 were also counted. All naïve and sham hyperthermia control rats survived the protocol. There was a trend toward a larger mortality rate in asphyxiated rats treated with induced hyperthermia at 24 hrs (9 of 21 died) vs. asphyxiated rats without induced hyperthermia (3 of 21) or with hyperthermia induced at 48 hrs (3 of 10) (Kaplan-Meier p=.0595). Asphyxiated rats with hyperthermia induced at 24 hrs had larger (worse) histopathology damage scores than rats subjected to asphyxia without induced hyperthermia (9.3 +/- 1.5 vs. 6.2 +/- 2.6; p=.001). Histopathology damage scores in asphyxiated rats with hyperthermia induced at 48 hrs did not differ from those in rats asphyxiated without induced hyperthermia (6.4 +/- 3.0 vs. 6.2 +/- 2.6; p=.907). There were fewer normal-appearing CA1 neurons in asphyxiated rats with hyperthermia induced at 24 hrs vs. rats subjected to asphyxia without induced hyperthermia (33 +/- 13 vs. 67 +/- 36; p=.002). The number of normal-appearing CA1 neurons in asphyxiated rats with hyperthermia induced at 48 hrs did not differ from that in rats asphyxiated without induced hyperthermia (59 +/- 21 vs. 67 +/- 36; p=.885). Induced hyperthermia when administered at 24 hrs, but not 48 hrs, worsens ischemic brain injury in rats resuscitated from asphyxial cardiac arrest. This may have implications for postresuscitative management of children and adults resuscitated from cardiac arrest. The common clinical practice of actively warming patients with spontaneous hypothermia might result in iatrogenic injury if warming results in hyperthermic overshoot. Avoidance of hyperthermia induced by active warming at critical time periods after cardiac arrest may be important.

Temporal evolution of apparent diffusion coefficient and T2 value following transient focal cerebral ischemia in gerbils.

We examined the time course of apparent diffusion coefficient (ADC) and T2 values in gerbils subjected to transient focal cerebral ischemia and compared them with histopathologic changes. Ten gerbils were subjected to two times 10-min occlusions of the left common carotid artery and examined with diffusion-weighted and T2-weighted MR imaging, at the interocclusion period and 1 hour, 2 hours. 1 day, 2 days, 4 days, and 7 days after the second occlusion. Immediately after the last MR imaging, their brains were examined histopathologically. ADC values decreased 1 hour after the second occlusion and continued to decrease up to 1 or 2 days later. ADC values remained lowered up to 4 days and slightly recovered at 7 days. T2 values were normal at 1 and 2 hours and began to increase at 1 day. T2 values began to recover at 4 days. Histopathologically, infarction was confined in the fronto-parietal cortex, dorsolateral caudate nucleus and dorsolateral thalamus, and neuronal necrosis was found in the pyramidal cell layer of the hippocampus. This study indicates that sequential ADC and T2 studies of gerbils subjected to transient focal cerebral ischemia provide a useful tool for evaluating temporal evolution of ischemic brain injury and edema, including cytotoxic and vasogenic edema.

Unique profile of spreading depression in a primate model.

Spreading depression (SD) is considered to play a role in pathologic conditions of humans such as in the evolution of ischemic brain injury and migraine aura. Because many studies have demonstrated spreading hypoperfusion in patients with migraine and persistent hypoperfusion in nonprimate animal models of SD, these changes in cerebral blood flow (CBF) were regarded as an epiphenomenon of SD. However, there is no direct evidence of the occurrence of SD in primates. The authors attempted to elicit SD by applying 3.3 mol/L potassium chloride to the cerebral cortex of nine male cynomolgus monkeys. The CBF was monitored by positron emission tomography in five animals. Propagated direct-current shifts were found by the two neighboring microelectrodes only in one animal. The direct-current wave propagated at a speed of 4 mm/min and its amplitude was 20 mV, being consistent with the SD findings. Except in one animal with 6 SD episodes, SD waves were recorded infrequently at the rostral site (none in three animals, once in three, and twice in two). Focal hyperemia accompanied SD. Neither spreading hypoperfusion nor persistent hypoperfusion was found. These unique features of SD in primates raise a doubt as to whether the role of SD in nonprimate animals is the same as that in stroke and migraine in humans.

Analysis of high intracellular [Na+]-induced release of [3H]noradrenaline in rat hippocampal slices.

Our aim was to investigate the mechanisms involved in the high intracellular sodium-induced transmitter release in the CNS through the characterisation of the veratridine-evoked (40 microM) noradrenaline release from rat hippocampal slices. The response to veratridine was completely inhibited by tetrodotoxin (1 microM), indicating that the effect is due to the activation of sodium channels. Omission of Ca2+ from the superfusion fluid inhibited the veratridine-evoked release by 72%, showing that the majority of release results from external Ca2+-dependent exocytosis. The residual Ca2+-independent release was not blocked by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (100 microM) suggesting that intracellular Ca2+ stores are not involved in this component of veratridine effect. The noradrenaline uptake blockers, desipramine (10 microM) and nisoxetine (10 microM), inhibited the external Ca2+-independent release by 50 and 46%, respectively, indicating that the release partly originates from the reversal of transporters (carrier-mediated release). In contrast to uptake blockers, lowering the temperature, another possibility to inhibit transporter function, completely inhibited the effect of veratridine in the absence of Ca2+. Further experiments revealed that low temperature (20 and 12 degrees C) reduces the veratridine-induced increase of intracellular sodium concentration ([Na+]i) in rat cortical synaptosomes (68 and 78% inhibition, respectively). The clinical relevance of our data is that during ischemia a massive release of transmitters occurs mainly due to the elevation of [Na+]i, which contributes to the development of ischemic brain injury. Our results show that low temperature may be a better therapeutic approach to the treatment of ischemia because it has a dual action on this process. Firstly, it inhibits the function of uptake transporters and hence reduces the carrier-mediated outflow of transmitters. Secondly, it inhibits the sodium influx and therefore prevents the unwanted elevation of [Na+]i. Our data also suggest that veratridine stimulation can be a suitable model for ischemic conditions.

Effect of reduced flow on blood–brain barrier transport systems

Pathological states (i.e. stroke, cardiac arrest) can lead to reduced blood flow to the brain potentially altering blood–brain barrier (BBB) permeability and regulatory transport functions. BBB disruption leads to increased cerebrovascular permeability, an important factor in the development of ischemic brain injury and edema formation. In this study, reduced flow was investigated to determine the effects on cerebral blood flow (CBF), pressure, basal BBB permeability, and transport of insulin and K + across the BBB. Anesthetized adult female Sprague–Dawley rats were measured at normal flow (3.1 ml min −1), half flow (1.5 ml min −1), and quarter flow (0.75 ml min −1), using bilateral in situ brain perfusion for 20 min followed by capillary depletion analysis. Reduction in perfusion flow rates demonstrated a modest reduction in CBF (1.27–1.56 ml min −1 g −1), a decrease in pressure, and no significant effect on basal BBB permeability indicating that autoregulation remained functional. In contrast, there was a concomittant decrease in BBB transport of both insulin and K + with reduced flow. At half and quarter flow, insulin transport was significantly reduced ( R Br%=17.2 and R Br%=16.2, respectively) from control ( R Br%=30.4). Additionally, a significant reduction in [ 86Rb +] was observed at quarter flow ( R Br%=2.5) as compared to control ( R Br%=4.8) suggesting an alteration in ion homeostasis as a result of low flow. This investigation suggests that although autoregulation maintains CBF, BBB transport mechanisms were significantly compromised in states of reduced flow. These flow alterations may have a significant impact on brain homeostasis in pathological states.

ω-Agatoxin IVA-sensitive Ca 2+ channel blocker, α-eudesmol, protects against brain injury after focal ischemia in rats

ω-Agatoxin IVA-sensitive Ca 2+ channels have been thought to be involved in physiological excitatory amino acid glutamate release and these channels may also contribute to the development of ischemic brain injury. Recently, we demonstrated that α-eudesmol from Juniperus virginiana Linn. (Cupressaceae) inhibits potently the presynaptic ω-agatoxin IVA-sensitive Ca 2+ channels. In the present study, we investigated the effects of α-eudesmol on brain edema formation and infarct size determined after 24 h of reperfusion following 1 h of middle cerebral artery occlusion in rats. We first found that α-eudesmol concentration-dependently inhibited glutamate release from rat brain synaptosomes and that its inhibitory effect was Ca 2+-dependent. In the middle cerebral artery occlusion study, intracerebroventricular (i.c.v.) treatment with α-eudesmol significantly attenuated the post-ischemic increase in brain water content. α-Eudesmol also significantly reduced the size of the infarct area determined by triphenyltetrazolium chloride staining after 24 h of reperfusion. Using a microdialysis technique, we further demonstrated that α-eudesmol inhibits the elevation of the extracellular concentration of glutamate during ischemia. From these results, we suggest that α-eudesmol displays an ability to inhibit exocytotic glutamate release and to attenuate post-ischemic brain injury.

C-Jun N-terminal kinase (JNK) and JNK interacting protein response in rat brain after transient middle cerebral artery occlusion

c-Jun response is involved in the development of ischemic brain injury, which is activated by c-Jun N-terminal kinase-1 (JNK-1). The activity of JNK-1 is strictly regulated, and only the phosphorylated form of JNK (phospho-JNK) which is translocated to the nucleus has an ability to activate c-Jun response. There is a protein which inhibits JNK-1 activation, and known as JNK interacting protein-1 (JIP-1). In this study, we investigated change in JNK-1, phospho-JNK, and JIP-1 immunoreactivity in rat brain after transient middle cerebral artery (MCA) occlusion. Immunoreactive JNK-1 was scant in the sham-control brain, but it was induced at 1 h after reperfusion, which was slightly increased at 3 h of reperfusion. By contrast, phospho-JNK remained negative till 3 h. At 8 h, JNK-1 and phospho-JNK became distinctly positive, and nuclei as well as cytoplasm were stained. Thereafter, immunoreactivity for JNK-1 and phospho-JNK became furthermore dense, and most neurons revealed positively stained nuclei. Immunoreactivity for JIP-1 remained negative till 8 h of reperfusion, but at 24 and 72 h, cytoplasm of cortical neurons at the MCA boundary area was positively stained. This JIP-1 induction got behind the JNK-1 activation, and therefore, may be a vain effort for neurons to survive. Inhibition of JNK-1 activation might become an innovative means of therapy for stroke treatment in the future.

Induction of tolerance against ischemia/reperfusion injury in the rat brain by preconditioning with the endotoxin analog diphosphoryl lipid A.

Inflammatory responses and oxygen free radicals have increasingly been implicated in the development of ischemic brain injury. In some cases, an attenuation of inflammation or free-radical injury can provide tissue protection. Diphosphoryl lipid A (DPL) is a detoxified derivative of a lipopolysaccharide (endotoxin) of Salmonella minnesota strain R595, which is capable of stimulating the immune system without eliciting direct toxic effects. In this study the authors examined the influence of preconditioning with DPL on ischemia/reperfusion injury in rats. Sprague-Dawley rats were injected intravenously with either DPL or vehicle. Twenty-four hours later, some animals were tested for superoxide dismutase (SOD) activity. Others were subjected to a 3-hour period of focal cerebral ischemia and, after a reperfusion period of 24 hours, were killed. Infarction volume, SOD activity, and myeloperoxidase (MPO) activity were assayed in the postischemic animals. Pretreatment with DPL produced significant reductions in cerebral infarction and MPO activity in the ischemic penumbra. A significant enhancement of basal SOD activity was observed 24 hours after DPL treatment (that is, before ischemia), and a further enhancement of SOD activity was seen in the ischemic penumbra 24 hours after reperfusion. These data provide the first evidence of a neuroprotective effect of preconditioning with DPL in an in vivo model of cerebral ischemia. Although the precise mechanisms through which DPL exerts its neuroprotective influence remain to be established, an inhibition of the complex inflammatory response to ischemia and an enhancement of endogenous antioxidant activity are leading candidates.

Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia.

Focal cerebral ischemia is associated with expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), enzymes whose reaction products contribute to the evolution of ischemic brain injury. We tested the hypothesis that, after cerebral ischemia, nitric oxide (NO) produced by iNOS enhances COX-2 activity, thereby increasing the toxic potential of this enzyme. Cerebral ischemia was produced by middle cerebral artery occlusion in rats or mice. Twenty-four hours after ischemia in rats, iNOS-immunoreactive neutrophils were observed in close proximity (<20 micrometer) to COX-2-positive cells at the periphery of the infarct. In the olfactory bulb, only COX-2 positive cells were observed. Cerebral ischemia increased the concentration of the COX-2 reaction product prostaglandin E2 (PGE2) in the ischemic area and in the ipsilateral olfactory bulb. The iNOS inhibitor aminoguanidine reduced PGE2 concentration in the infarct, where both iNOS and COX-2 were expressed, but not in the olfactory bulb, where only COX-2 was expressed. Postischemic PGE2 accumulation was reduced significantly in iNOS null mice compared with wild-type controls (C57BL/6 or SV129). The data provide evidence that NO produced by iNOS influences COX-2 activity after focal cerebral ischemia. Pro-inflammatory prostanoids and reactive oxygen species produced by COX-2 may be a previously unrecognized factor by which NO contributes to ischemic brain injury. The pathogenic effect of the interaction between NO, or a derived specie, and COX-2 is likely to play a role also in other brain diseases associated with inflammation.

Effects of NO-generating compounds on the uptake and release of taurine in the mouse brain.

Nitric oxide (NO) has been ascertained to play an important role in signal transduction in the central nervous system, functioning as a short-lived modulator and/or second messenger1,32. There is a functional link between NO and glutamatergic neurotransmission. NO is involved in synaptic plasticity, for example, in long-term potentiation, memory and learning, in the hippocampus4, which phenomena represent physiological effects of glutamate as a transmitter. Glutamate also participates in the development of ischemic brain injury, and therefore, NO is likely to be associated with mechanisms of neuronal damage4.