Neuroprotective properties of prostaglandin I2 IP receptor in focal cerebral ischemia

We and others have identified that inhibition of cyclooxygenase might not be the optimal approach to limiting brain damage after stroke. Now we are investigating the unique properties of the various prostaglandin receptors to determine whether blocking those that mediate toxicity or stimulating those that reduce toxicity will improve neurological outcomes. Here, we determined the respective contribution of the prostaglandin I₂ (PGI₂) receptor in transient middle cerebral artery (MCA) occlusion (tMCAO) and permanent MCAO (pMCAO) preclinical stroke models by using male wildtype (WT) and IP receptor knockout (IP^−/−) C57Bl/6 mice. In addition, we investigated the putative preventive and therapeutic effects of the IP receptor agonist beraprost. The infarct volumes and neurological deficit scores (NDS) were significantly greater in IP^−/− than in WT mice after both tMCAO and pMCAO. Interestingly, beraprost pretreatment (50 or 100 μg/kg p.o.) 30 min before tMCAO and post-treatment (100 μg/kg p.o.) at 2 or 4.5 h of reperfusion significantly reduced the neurological deficit score and infarct volume in WT mice. Post-treatment with beraprost (100 μg/kg p.o.) 4.5 h after pMCAO also significantly decreased neurological deficits and infarct volume in WT mice. Together, these novel findings suggest for the first time that PGI₂ IP receptor activation can attenuate anatomical and functional damage following ischemic stroke.     

Expression of the kcnk3 potassium channel gene lessens the injury from cerebral ischemia,most likely by a general influence on blood pressure

We examined the possible protective effect of TASK-1 (TWIK-related acid-sensitive potassium channel-1, kcnk3) and -3 potassium channels during stroke. TASK-1 and TASK-3, members of the two pore domain (K2P or kcnk) potassium channel family, form hetero or homodimers and help set the resting membrane potential. We used male TASK-1 and TASK-3 knockout mice in a model of focal cerebral ischemia, permanent middle cerebral artery occlusion (pMCAO). Infarct volume was measured 48 h after pMCAO. The TASK-1 knockout brains had larger infarct volumes (P=0.004), and those in TASK-3 knockouts were unchanged. As the TASK-1 gene is expressed in adrenal gland, heart and possibly blood vessels, the higher infarct volumes in the TASK-1 knockout mice could be due to TASK-1 regulating blood vessel tone and hence blood pressure or influencing blood vessel microarchitecture and blood flow rate. Indeed, we found that male TASK-1 knockout mice had reduced blood pressure, likely explaining the increased brain injury seen after pMCAO. Thus to make precise conclusions about how TASK-1 protects neurons, neural- or organ-specific deletions of the gene will be needed. Nevertheless, a consequence of having TASK-1 channels expressed (in various non-neuronal tissues and organs) is that neuronal damage is lessened when stroke occurs.     

Neuroprotective effect of cyanidin-3-O-glucoside anthocyanin in mice with focal cerebral ischemia

The present study sought to determine the neuroprotective effect of anthocyanin cyanidin-3-O-glucoside (CG), isolated and purified from tart cherries, against permanent middle cerebral artery occlusion (pMCAO) in mice and its potential mechanisms of neuroprotection. C57BL/6 mice subjected to pMCAO were treated with CG orally. Twenty-four hours after pMCAO, neurological scoring was used to evaluate functional outcome. The brains were then excised for measuring infarct volume and brain superoxide levels were determined. In a separate set of experiments, the influence of CG on cytochrome c (cyt c) and apoptosis-inducing factor (AIF) release from mitochondria under oxidative stress were assessed in isolated cortical neurons from adult mouse brains. Infarction volume was attenuated by 27% in mice pre-treated with 2 mg/kg of CG compared to vehicle-treated mice. Delayed treatment with 2 mg/kg of CG also showed 25% reduction in infarct size. Neurological functional outcome was significantly improved in mice pre- or post-treated with CG. Compared to vehicle treated mice CG treated mice had lower levels of brain superoxide. CG also blocked the release of AIF from mitochondria under oxidative stress, but did not inhibit the release of cyt c. Our data show that CG is neuroprotective against pMCAO in mice, and this beneficial effect may be mediated by attenuation of brain superoxide levels after ischemia. CG may also exert its neuroprotective effect by blocking AIF release in mitochondria.     

L-PGDS-derived PGD2 attenuates acute lung injury by enhancing endothelial barrier formation

Acute lung injury (ALI) is caused by various stimuli such as acid aspiration and infection, resulting in severe clinical outcomes with high mortality. Prostaglandin D₂ (PGD₂) is a lipid mediator produced in the lungs of patients with ALI. There are two prostaglandin D synthases (PGDS), namely, lipocalin-type PGDS (L-PGDS) and hematopoietic PGDS (H-PGDS). We previously reported the anti-inflammatory role of H-PGDS-derived PGD₂ in an endotoxin-induced murine ALI model. Therefore, in this study, we investigated the role of L-PGDS-derived PGD₂ in ALI in comparison to H-PGDS-derived PGD₂. Intratracheal administration of HCl caused lung inflammation accompanied by tissue edema and neutrophil accumulation in mouse lungs. The deficiency of both L-PGDS and H-PGDS exacerbated HCl-induced lung dysfunction to a similar extent. Furthermore, a detailed investigation revealed that L-PGDS-derived PGD₂ inhibited lung edema, while H-PGDS-derived PGD₂ inhibited neutrophil infiltration. Immunostaining showed that inflamed endothelial/epithelial cells express L-PGDS, while macrophages and neutrophils express H-PGDS. Hematopoietic reconstitution with WT bone marrow did not rescue the exacerbated lung edema in L-PGDS deficient mice, indicating the importance of nonhematopoietic endothelial/epithelial cell-expressing L-PGDS for protection against ALI. A modified Miles assay showed that L-PGDS deficiency accelerated vascular hyper-permeability in the inflamed lung, which was suppressed by the stimulation of D prostanoid (DP) receptor, a PGD₂ receptor. In vitro, DP agonism enhanced the barrier function of endothelial cells but not epithelial cells. Taken together, our results suggest that in the HCl-induced murine ALI model PGD₂ was produced locally by inflamed endothelial and epithelial L-PGDS and this enhanced the endothelial barrier through the DP receptor. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Early induction of neuronal lipocalin-type prostaglandin D synthase after hypoxic-ischemic injury in developing brains

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is up-regulated in oligodendrocytes (OLs) in mouse models for genetic neurological disorders including globoid cell leukodystrophy (twitcher) and GM₁ and GM₂ gangliosidoses and in the brain of patients with multiple sclerosis. Since L-PGDS-deficient twitcher mice undergo extensive neuronal death, we concluded that L-PGDS functions protectively against neuronal degeneration. In this study, we investigated whether L-PGDS is also up-regulated in acute and massive brain injury resulting from neonatal hypoxic-ischemic encephalopathy (HIE). Analysis of brains from human neonates who had died from HIE disclosed that the surviving neurons in the infarcted lesions expressed L-PGDS. Mouse models for neonatal HIE were made on postnatal day (PND) 7. Global infarction in the ipsilateral hemisphere was evident at 24 h after reoxygenation in this model. Intense L-PGDS immunoreactivity was already observed at 10 min after reoxygenation in apparently normal neurons in the cortex, and thereafter, in neurons adjacent to the infarcted area. Quantitative RT-PCR revealed that the L-PGDS mRNA level of the infarcted hemisphere was 33-fold higher than that of the sham-operated mouse brains at 1 h after reoxygenation and that it decreased to the normal level by 24 h thereafter. Furthermore, in both human and mouse brains, many of L-PGDS-positive cells were also immunoreactive for p53; and some of these expressed cleaved caspase-3. The expression of L-PGDS in degenerating neurons implies that L-PGDS functions as an early stress protein to protect against neuronal death in the HIE brain.     

Thalidomide protects against ischemic neuronal damage induced by focal cerebral ischemia in mice

We aimed to examine whether thalidomide might inhibit the neuronal damage resulting from focal cerebral ischemia, and if so to explore the neuroprotective mechanism. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (MCAO) in mice, and thalidomide was intraperitoneally administered a total of three times (at 10 min before, just before, and 1 h after MCAO). Thalidomide significantly reduced (a) the infarct area and volume at 24 and 72 h after MCAO and (b) the neurological score at 72 h after MCAO. Brains were also histochemically assessed for apoptosis and lipid peroxidation using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and an antibody recognizing 8-hydroxy-2′-deoxyguanosine (8-OHdG), respectively. Thalidomide reduced both the number of TUNEL-positive cells and the oxidative damage. However, post-treatment of thalidomide [20 mg/kg, three times (at just after, 1 h after, 3 h after MCAO)] did not reduce the infarct volume. In an in vitro study, we examined the effects of thalidomide on lipid peroxidation in mouse brain homogenates and on the production of various radical species. Thalidomide inhibited both the lipid peroxidation and the production of H₂O₂ and O₂ · ⁻ (but not HO⁻) radicals. We also measured the brain concentration of TNF-α by ELISA. The TNF-α level in the brain was significantly increased at 9–24 h after MCAO. However, thalidomide did not reduce the elevated TNF-α level at either 12 or 24 h after MCAO. These findings indicate that thalidomide has neuroprotective effects against ischemic neuronal damage in mice, and that an inhibitory action of thalidomide against oxidative stress may be partly responsible for these neuroprotective effects.     

Neuronal Rho GTPase Rac1 elimination confers neuroprotection in a mouse model of permanent ischemic stroke

The Rho GTPase Rac1 is a multifunctional protein involved in distinct pathways ranging from development to pathology. The aim of the present study was to unravel the contribution of neuronal Rac1 in regulating the response to brain injury induced by permanent focal cerebral ischemia (pMCAO). Our results show that pMCAO significantly increased total Rac1 levels in wild type mice, mainly through rising nuclear Rac1, while a reduction in Rac1 activation was observed. Such changes preceded cell death induced by excitotoxic stress. Pharmacological inhibition of Rac1 in primary neuronal cortical cells prevented the increase in oxidative stress induced after overactivation of glutamate receptors. However, this was not sufficient to prevent the associated neuronal cell death. In contrast, RNAi‐mediated knock down of Rac1 in primary cortical neurons prevented cell death elicited by glutamate excitotoxicity and decreased the activity of NADPH oxidase. To test whether in vivo down regulation of neuronal Rac1 was neuroprotective after pMCAO, we used tamoxifen‐inducible neuron‐specific conditional Rac1‐knockout mice. We observed a significant 50% decrease in brain infarct volume of knockout mice and a concomitant increase in HIF‐1α expression compared to littermate control mice, demonstrating that ablation of Rac1 in neurons is neuroprotective. Transmission electron microscopy performed in the ischemic brain showed that lysosomes in the infarct of Rac1‐ knockout mice were preserved at similar levels to those of non‐infarcted tissue, while littermate mice displayed a decrease in the number of lysosomes, further corroborating the notion that Rac1 ablation in neurons is neuroprotective. Our results demonstrate that Rac1 plays important roles in the ischemic pathological cascade and that modulation of its levels is of therapeutic interest.     

L-丝氨酸对永久性大脑中动脉栓塞大鼠的神经保护作用

目的: 研究L-丝氨酸对大鼠永久性脑梗死的神经保护作用、治疗剂量及有效治疗时间窗,并探讨相关作用机制。方法: 制作大鼠永久性大脑中动脉栓塞(pMCAO)模型,腹腔注射L-丝氨酸,通过神经行为学评分、脑梗死体积测定和尼氏染色法,观察L-丝氨酸的治疗剂量效应(56 mg/kg、168 mg/kg和504 mg/kg治疗组)和治疗时间窗(1 h、3 h、6 h、12 h和24 h治疗组);并测定丝氨酸消旋酶抑制剂对L-丝氨酸疗效的影响。利用激光多普勒血流监测仪观察缺血区血供及L-丝氨酸对缺血区局部脑血流量的影响。结果: 与pMCAO组相比,L-丝氨酸于pMCAO后3 h使用,168 mg/kg和504 mg/kg两个剂量都能较好地降低神经行为学评分,减少脑梗死体积,抑制海马CA1区神经细胞的丢失。在治疗时间窗的研究中,L-丝氨酸在pMCAO后6 h内治疗具有明显的神经保护作用,12 h及以后使用,神经保护作用不明显。丝氨酸消旋酶抑制剂不改变L-丝氨酸的疗效。脑缺血30 min时注射L-丝氨酸可明显增加缺血区局部脑血流量,并且这一作用不受甘氨酸受体阻断剂士的宁的影响。结论: L-丝氨酸对永久性脑梗死具有神经保护作用,其机制可能部分与增加缺血区皮质的血供有关。     

缺血预处理对局灶性脑缺血再灌注大鼠顶叶皮层bFGF表达的影响

目的研究脑缺血预处理对大鼠局灶性脑缺血再灌注损伤及脑顶叶皮层碱性成纤维细胞生长因子(bFGF)表达的影响。方法线栓法阻塞32只大鼠大脑中动脉15min作为预处理,3天后线栓法制备大鼠大脑中动脉缺血模型,缺血2h再灌注24h后,缺血再灌注组32只。观察神经功能缺损程度、脑梗死体积、脑含水量、bFGF表达的变化。结果与对照组相比,脑缺血预处理组神经功能缺损评分明显降低(P<0.01),TTC染色显示脑梗死体积明显减小(P<0.01),缺血侧的脑水肿程度明显减轻(P<0.01),免疫组化和Westernblot检测表明脑顶叶皮层缺血周围组织bFGF表达水平明显升高(P<0.01),阳性细胞以神经元和胶质细胞为主。结论预先给予短暂性脑缺血预处理可对脑缺血再灌注损伤起保护作用,bFGF过表达可能是其机制之一。     

Effect of tris-(hydroxymethyl)-aminomethane on experimental focal cerebral ischemia

Systemic and focal cerebral acidosis is considered deleterious to cell metabolism and neuronal recovery. We investigated the immediate effect of tris-(hyroxymethyl)-aminomethane (THAM), an alkalizing agent, on focal cerebral ischemia produced by occlusion of the left middle cerebral artery (MCA) in cats with systemic acidosis. Occlusion of MCA resulted in prompt decreases in local cerebral blood flow of the ipsilateral marginal and ectosylvian gyri from 47.7 ml/100 g per minute in control to 32.3 ml/100 g per minute and 8.3 ml/100 g per minute, respectively. In the control group, physiological saline was infused continuously and the treated group received 0.3 M THAM to normalize systemic and focal cerebral acidosis. There were no significant changes in the systemic arterial pressure, arterial PO₂ and PCO2 throughout the experiments in the two groups. Arterial pH decreased from 7.42 to 7.30 in the control, while it remained normal during THAM treatment. Extracellular pH of the marginal gyrus (peri-infarct zone) decreased from 7.39 to 6.87 with 6 h ischemia in the control group. In THAM infusion, extracellular pH was kept between 7.26 and 7.29, which was significantly higher than the control group. THAM significantly decreased infarct volume and lactate and water contents of the gray matter in the marginal gyrus at 6 h after occlusion. It is concluded that THAM infusion immediately after ischemia onset is considered effective in improving acidosis at the site of ischemic penumbra and consequently reduces lactate production, brain edema, and infarct volume.     

脑的不对称性影响大鼠局灶性脑缺血的结局

为评价左右侧大脑中动脉闭塞(MCAO)对右利大鼠神经行为功能和脑梗死体积的影响,本研究应用四足动物觅食实验筛选右利爪雄性SD大鼠24只,随机分为经左、右侧插线组各12只,8%水合氯醛腹腔注射(300mg/kg)麻醉,线栓法经左、右侧颈外-内动脉插入头端涂有多聚赖氨酸的4-0尼龙线,建立大鼠MCAO缺血2h模型,再灌注72h后评价动物的神经行为功能,测量脑梗死体积。结果表明,所有动物在脑缺血2h神经功能缺损评分最高,再灌注1、24、48和72h经左侧MCAO大鼠显著高于经右侧MCAO大鼠(P<0.05),后者功能明显优于前者,脑梗死体积经左侧插线的大鼠显著大于经右侧插线的大鼠(P<0.05)。研究结果提示,大鼠主侧半球大脑中动脉缺血后,神经功能缺损和脑梗死体积较对侧严重,脑的不对称性影响大鼠局灶性脑缺血的最终结局。     

Neuroprotective effect of 20(R)-ginsenoside Rg3 against transient focal cerebral ischemia in rats

Gensenosides, the active ingredients of Chinese herbal medicine Panax ginseng, have a wide spectrum of medical effects, such as anti-tumorigenic, angiosuppressive, adaptogenic, and anti-fatigue activities. In the present study, we have investigated the neuroprotective effect of 20(R)-ginsenoside Rg₃ (20(R)-Rg₃) against transient focal cerebral ischemia in male Sprague-Dawley (SD) rats. The middle cerebral artery was occluded for 2 h in rats and then reperfused for 24 h. The behavioral disturbance was evaluated according to neurological deficit scores, and the infarct volumes were evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) staining; in addition, ischemia-mediated apoptosis was examined using the method of terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick end labeling (TUNEL). The expressions of calpain I and caspase-3 mRNA in hippocampal CA1 region were further assayed using in situ hybridization, in order to clarify the neuroprotective mechanism of 20(R)-Rg₃. 20(R)-Rg₃ at the doses of 10 and 20 mg kg⁻¹ i.p., but not 5 mg kg⁻¹, showed significant neuroprotective effect in rats against focal cerebral ischemic injury by markedly reducing cerebral infarct volumes and degrading infarct rate of TTC-stained coronal brain sections, and improving behavior of the animals. Our results also suggested that 20(R)-Rg₃ (10 and 20 mg kg⁻¹) could significantly suppress the expressions of calpain I and caspase-3 mRNA. These results indicated that 20(R)-Rg₃ attenuates the neuronal apoptosis caused by cerebral ischemia–reperfusion injury and its neuprotective effect may be involved in the downregulation of calpain I and caspase-3.     

Effects of transient focal cerebral ischemia in mice deficient in puma

Bcl-2 homology domain 3 (BH3)-only pro-apoptotic proteins may play an important role in upstream cell death signaling pathways underlying ischemic brain injury. Puma is a potent BH3-only protein that can be induced via p53, FoxO3a and endoplasmic reticulum stress pathways and is upregulated by global cerebral ischemia. To more completely define the contribution of Puma to ischemic brain injury we measured the expressional response of Puma to transient focal cerebral ischemia in mice and also compared infarct volumes in puma-deficient versus puma-expressing mice. Real-time quantitative PCR determined puma mRNA levels were significantly increased 8 h after 90 min middle cerebral artery (MCA) occlusion in the ipsilateral cortex, while expression remained unchanged contralaterally. Puma protein levels were also increased in the ischemic cortex over the same period. However, cortical and striatal infarct volumes were not significantly different between puma-deficient and puma-expressing mice at 24 h, and no differences between genotypes were found for post-ischemic neurological deficit scores. These data demonstrate that focal cerebral ischemia is associated with puma induction but suggest that Puma does not contribute significantly to lesion development in the present model.     

Toll-like receptor 4 (TLR4), but not TLR3 or TLR9, knock-out mice have neuroprotective effects against focal cerebral ischemia

Toll-like receptors (TLRs) are signaling receptors in the innate immune system that is a specific immunologic response to systemic bacterial infection. We investigated whether cerebral ischemia induced by the middle cerebral artery occlusion (MCAO) for 2 h differed in mice that lack a functional TLR3, TLR4, or TLR9 signaling pathway. TLR4, but not TLR3 or TLR9, knock-out (KO) mice had significantly smaller infarct area and volume at 24 h after ischemia-reperfusion (I/R) compared with wild-type mice. In addition, TLR4 KO mice improved in neurological deficits after I/R compared with wild-type mice. Moreover, we investigated the expression of TLR4 in the ischemic brain with immunohistochemistry. The number of TLR4-positive cells gradually increased from 1 h after MCAO to 22 h after I/R. We also examined the localization of TLR4 in the ischemic area. TLR4 was localized with CD11b-positive microglial cells in the ischemic striatum and the number of CD11b-positive microglial cells was smaller in TLR4 KO mice than in wild-type mice. In addition, we investigated the translocation of NF-κB among TLR3, 4, and 9 KO mice after I/R injury using western blotting. NF-κB's p65 subunit was decreased in TLR4 KO mice compared to wild-type mice, but not TLR3 or 9 KO mice. These data suggest that TLR4 knockout, but not TLR3 or TLR9 knockout, may play a neuroprotective role in ischemic brain injury induced by MCAO in mice.     

Comparison between coated vs. uncoated suture middle cerebral artery occlusion in the rat as assessed by perfusion/diffusion weighted imaging

Differences among models in the temporal evolution of ischemia after middle cerebral artery occlusion (MCAO) in rats may considerably influence the results of experimental treatment studies. Using diffusion and perfusion imaging, we compared the spatiotemporal evolution of ischemia in Sprague-Dawley rats after permanent MCAO (pMCAO) with different types of sutures. Male Sprague-Dawley rats were randomly assigned to pMCAO produced with either 4-0 silicone coated (n=8), or 3-0 uncoated monofilaments (n=8). Serial determination of quantitative cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) maps were performed up to 3 h after pMCAO. Lesion volumes were calculated by using previously validated thresholds and correlated with infarct volume corrected for edema defined by 2,3,5-triphenyltetrazolium chloride (TTC) staining at 24 h after MCAO. The ADC/CBF-defined mismatch volume in the 4-0 coated suture model was present significantly longer (up to 120 min) compared to the uncoated 3-0 suture model (30 min). The TTC-derived infarct volume was significantly larger in the coated model (290.3+/-32.8 mm(3)) relative to the uncoated model (252.3+/-34.6 mm(3)). This study demonstrates that the type of suture may significantly influence the spatiotemporal evolution of the ADC/CBF-mismatch as well as the final infarct volume. These inter-model variations must be taken into account when assessing new therapeutic approaches on ischemic lesion evolution in the rat MCAO model.     

RAGE expression is up-regulated in human cerebral ischemia and pMCAO rats

To determine whether the receptor for advanced glycation endproducts (RAGE) contributes to cerebral ischemia, we evaluated RAGE expression in human cerebral ischemia and a model of permanent middle cerebral artery occlusion (pMCAO) in rats. Biopsy specimens were obtained from 12 patients with unilateral cerebral infarction. For the pMCAO model, the middle cerebral artery (MCA) of Sprague-Dawley (SD) rats was permanently occluded. Immunohistochemistry and Western blotting were used to measure RAGE expression in the ischemic hemisphere relative to the normal hemisphere. PC12 cells subjected to oxygen and glucose deprivation (OGD) were used to evaluate the role of RAGE in cell injury. As expected, cerebral ischemia patients expressed elevated levels of RAGE in the ischemic hemisphere. In 1 and 2 days pMCAO rats, levels of RAGE were higher in the ischemic hemisphere relative to the non-ischemic hemisphere, and expression was primarily located in the penumbra of the ischemic hemisphere. In PC12 cells, levels of RAGE increased after 7h of OGD culture. Notably, blockade of RAGE with a selective RAGE antibody in vitro reduced the cytotoxicity caused by OGD. The present data suggest that RAGE is up-regulated in human cerebral ischemia and pMCAO rats, suggesting a role for RAGE in brain ischemia.     

Granzyme-b is involved in mediating post-ischemic neuronal death during focal cerebral ischemia in rat model

Although peripheral immune cells infiltrate ischemic infarct tissue and elicit immune injury, the role of Cytotoxic T Lymphocytes (CTLs) and the toxins they release in mediating neuronal death is not well understood. Granzyme-b (Gra-b), a serine protease found in the cytoplasmic granules of CTLs and natural killer cells, plays an important role in inducing target cell death by activating several caspases and by initiating caspase-independent pathways that contribute to target cell death. To determine if CTLs and Gra-b are involved in post-ischemic cerebral cell death; we investigated the role of CD8⁺ CTLs and Gra-b in ischemic rat brain infarct after transient middle cerebral artery occlusion (tMCAO) and in sham-operated animals. We observed that CTLs infiltrate the ischemic infarct within 1 h of reperfusion. There was a significant increase in Gra-b levels in the ischemic region starting from 1 h until 3 day which correlated with increased levels of chemokines (IP-10/CXCL10, IL-2) and TNF-α. Co-immunoprecipitation experiments show that Gra-b interacts with Bid, PARP, and caspase-3 in ischemic samples. Immunofluorescence analysis of Gra-b and TUNEL showed that Gra-b is present both in apoptotic and necrotic cells. Triple immunostaining further confirmed that the Gra-b positive degenerating cells were neurons. CTLs in close spatial proximity to degenerating neurons, increased levels of Gra-b, localization in neurons positive for TUNEL, and interaction with other pro-apoptotic proteins indicate that Gra-b and CTLs play a significant role in neuronal death following cerebral ischemia in the rat brain after tMCAO. Based on the above findings we support our hypothesis that Gra-b secreted from activated CTLs might be involved in aggravating post-ischemic damage by mediating neuronal death.     

Neuroprotective effects of the novel brain-penetrating antioxidant U-101033E and the spin-trapping agent α-phenyl-N-tert-butyl nitrone (PBN)

Literature on the therapeutic efficacy of free radical scavengers suggests that drugs that are able to cross the blood-brain barrier are more effective in protecting the brain from ischemic damage. However, the exact mechanisms by which brain-penetrating antioxidants act have yet not been delineated. We compared the neuroprotective potential of the newly discovered pyrrolopyrimidine U-101033E with that of α-phenyl-N-tert-butyl nitrone (PBN) and investigated their influence on cerebral blood flow. Thirty male Sprague-Dawley rats were subjected to 90 min of middle cerebral artery (MCA) occlusion by an intraluminal filament. Local cerebral blood flow (LCBF) was bilaterally recorded by laser Doppler flowmetry. Neurological deficits were quantified daily. Infarct volume was assessed after 7 days. MCA occlusion reduced ipsilateral LCBF to 20–30% of baseline. After reperfusion, postischemic hyperemia was followed by a decrease in LCBF to about 70% of baseline. There was no difference in LCBF among groups. U-101033E improved neurological function and reduced infarct volume by 52% (P<0.05). Improvement of neurological function and reduction of infarct volume (–25%) in animals treated with PBN was not significant. We conclude that U-101033E has superior neuroprotective properties compared with PBN. Neither drug improves blood flow during ischemia and 1 h of reperfusion. The mechanisms by which these brain-penetrating antioxidants act remain to be clarified. Received: 17 March 1999 / Accepted: 12 July 1999     

Hyperbaric oxygen preconditioning reduces ischemia–reperfusion injury by inhibition of apoptosis via mitochondrial pathway in rat brain

This study examined the hypothesis that apoptotic inhibition via mitochondrial pathway was involved in hyperbaric oxygen preconditioning (HBO-PC)–induced neuroprotection on ischemia–reperfusion injury in rat brain. Male Sprague–Dawley rats (250∼280 g, n=144) were divided into control, middle cerebral artery occlusion (MCAO) for 90 min, and HBO-PC plus MCAO groups. HBO-PC was conducted four times by giving 100% oxygen at 2.5 atm absolute (ATA), for 1 h at 12 h intervals for 2 days. At 24 h after the last HBO-PC, MCAO was performed and at 24 h after MCAO, neurological function, brain water content, infarct volume, and cell death were evaluated. Enzymatic activity of capase-3 and −9, and expression of cytochrome c, Bcl-2 and Bax proteins were performed in the samples from hippocampus, ischemic penumbra and core of the brain cortex, respectively. HBO-PC reduced brain edema, decreased infarction volume, and improved neurological recovery. HBO-PC reduced cytoplasm cytochrome c levels, decreased caspase enzyme activity, upregulated the ratio of Bcl-2 and Bax expression, and abated the apoptosis of ischemic tissue. HBO-PC protects brain tissues from ischemia–reperfusion injury by suppressing mitochondrial apoptotic pathways.