Nicotinamide prevents the down-regulation of MEK/ERK/p90RSK signaling cascade in brain ischemic injury

Nicotinamide attenuates neuronal cell death related to focal cerebral ischemic injury. This study investigated whether nicotinamide exerts a neuroprotective effect through the activation of Raf- mitogen-activated protein kinase kinase (MEK)-ERK and its downstream targets, including p90 ribosomal S6 kinase (p90RSK) and Bad. Adult male Sprague-Dawley rats were treated with nicotinamide (500 mg/kg) or vehicle 2 hr after the onset of middle cerebral artery occlusion (MCAO). Brains were collected 24 hr after MCAO. In the present study, nicotinamide significantly reduces the volume of infarct regions and decreases the number of positive cells by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in the cerebral cortex. Nicotinamide prevents injury-induced decrease in Raf-1, MEK1/2, and ERK1/2 phosphorylation. As part of the downstream cascade, nicotinamide inhibits the injury-induced decrease in p90RSK and Bad phosphorylation. Moreover, nicotinamide prevents the injury-induced increase in cleaved caspase-3 levels. These findings suggest that nicotinamide protects neuronal cells against cerebral ischemic injury and that MEK-ERK-p90RSK cascade activation by nicotinamide contributes to these neuroprotective effects.     

Melatonin regulates nitric oxide synthase expression in ischemic brain injury

Nitric oxide (NO) is produced by three NO synthases (NOS), iNOS, eNOS, and nNOS. Production of NO by iNOS plays key roles in neurodegeneration, while eNOS is a protective enzyme. This study investigated the neuroprotective effect of melatonin and the levels of NOS isoforms induced by melatonin in ischemic brain injury. Adult male rats were treated with melatonin (5 mg/kg) or vehicle prior to middle cerebral artery occlusion (MCAO). Brain samples were collected at 24 hr after the onset of occlusion. Results confirmed that melatonin significantly reduces infarct area. Western blot analysis was used to evaluate the expression levels of iNOS, eNOS, and nNOS. The level of iNOS and nNOS increased in vehicle-treated animals, while melatonin prevented injury-induced increase of iNOS. In contrast to iNOS levels, eNOS levels decreased in vehicle-treated animals, while melatonin prevented the injury-induced decrease of eNOS. This study provides further evidence that melatonin exerts neuroprotective effects, and the regulation of NOS isoforms by melatonin may contribute to the neuroprotective effects.     

Estrogen modulates Bcl-2 family proteins in ischemic brain injury

Estradiol acts as a neuroprotective factor against brain injury. This study investigated whether estradiol modulates the Bcl-2 family proteins in ischemic brain injury. Adult female rats were ovariectomized and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24 hr after MCAO, and infarct volumes were analyzed. Estradiol significantly reduces the infarct volume and decreases the positive cells of TUNEL staining in cerebral cortex. In ischemic cerebral cortex, the level of Bcl-2 was decreased, and the level of Bax was significantly increased. Estradiol prevents the injury-induced decrease of Bcl-2 and increase of Bax. In conclusion, our findings suggest that estradiol plays a potent protective role in brain injury through the regulation of Bcl-2 family proteins.     

17beta-estradiol pretreatment prevents the global ischemic injury-induced decrease of Akt activation and bad phosphorylation in gerbils

Estradiol acts as a neuroprotective factor against ischemic brain injury. This study investigated whether estradiol modulates neuroprotective mechanism through the activation of Akt and its downstream target such as Bad in global ischemic injury. Adult female gerbils were ovariectomized and treated with estradiol prior to ischemic injury. Transient cerebral ischemia was accomplished by bilateral clipping of the common carotid artery for 5 min. Brains were collected on 1, 3, 5 day after injury. In hippocampal CA1 region of non-treated gerbils, most of neuronal cells exhibited pyknotic nuclei and showed the positive reaction of TUNEL staining on 5 day after injury. However, estradiol significantly reduced the neuronal cell death. Potential activation was measured by phosphorylation of Akt at Ser473 and Bad at Ser136 using western blot analysis. The levels of pAkt and pBad were significantly decreased in non-treated gerbils on 1-5 day after injury. However, estradiol prevents the global ischemic injury-induced decrease of pAkt and pBad. Our findings suggest that estradiol prevents cell death due to global ischemic injury and that Akt activation and Bad phosphorylation by estradiol mediated these protective effects.     

Identification of regulated proteins by resveratrol in glutamate-induced cortical injury of newborn rats

Glutamate induces neuronal damage by generating oxidative stress and neurotoxicities. The neurological damage caused by glutamate is more severe during brain development in newborns than in adults. Resveratrol is naturally present in a variety of fruits and medicinal plants and exerts a neuroprotective effect against brain damage. The goal of this study was to evaluate the neuroprotective effects of resveratrol and to identify changed proteins in response to resveratrol treatment during glutamate-induced neonatal cortical damage. Sprague-Dawley rat pups (7 days old) were randomly divided into vehicle, resveratrol, glutamate, and glutamate and resveratrol groups. The animals were intraperitoneally injected with glutamate (10 mg/kg) and/or resveratrol (20 mg/kg) and their brain tissue was collected 4 hr after drug administration. Glutamate exposure caused severe histopathological changes, while resveratrol attenuated this damage. We identified regulated proteins by resveratrol in glutamate-induced cortical damaged tissue using two-dimensional gel electrophoresis and mass spectrometry. Among identified proteins, we focused on eukaryotic initiation factor 4A2, γ-enolase, protein phosphatase 2A subunit B, and isocitrate dehydrogenase. These proteins decreased in the glutamate-treated group, whereas the combination treatment of glutamate and resveratrol attenuated these protein reductions. These proteins are anti-oxidant proteins and anti-apoptotic proteins. These results suggest that glutamate induces brain cortical damage in newborns; resveratrol exerts a neuroprotective effect by controlling expression of various proteins with anti-oxidant and anti-apoptotic functions.     

牛Myf5基因克隆及蛋白质生物学特性分析

本试验旨在扩增牛Myf5基因,并对其蛋白质生物学特性进行分析。根据GenBank中已公布的牛Myf5基因序列设计1对引物,利用RT-PCR方法从牛背腰最长肌组织扩增牛Myf5基因,连接PUCm-T载体,进行酶切和测序鉴定,同时利用在线工具对牛Myf5蛋白的基本性质、二级结构和磷酸化位点进行预测。获得的牛Myf5基因编码区序列长度为768 bp,编码255个氨基酸。蛋白质生物学特性分析结果表明,Myf5具有该家族基因典型的碱性螺旋—环—螺旋结构域,氨基酸组成上丝氨酸(Ser)含量最高,其磷酸化位点分别位于丝氨酸(Ser)、苏氨酸(Thr)和酪氨酸(Tyr)残基上。     

Effects of resveratrol on the insulin signaling pathway of obese mice

The present study was conducted to investigate the effects of resveratrol on the insulin signaling pathway in the liver of obese mice. To accomplish this, we administered resveratrol to high fat diet-induced obese mice and examined the levels of protein phosphorylation in the liver using an antibody array. The phosphorylation levels of 10 proteins were decreased in the high fat diet and resveratrol (HFR) fed group relative to the levels in the high fat diet (HF) fed group. In contrast, the phosphorylation levels of more than 20 proteins were increased in the HFR group when compared with the levels of proteins in the HF group. Specifically, the phosphorylation levels of Akt (The308, Tyr326, Ser473) were restored to normal by resveratrol when compared with the levels in the HF group. In addition, the phosphorylation levels of IRS-1 (Ser636/Ser639), PI-3K p85-subunit α/γ(Tyr467/Tyr199), PDK1 (Ser241), GSK-3α (S21) and GSK-3 (Ser9), which are involved in the insulin signaling pathway, were decreased in the HF group, whereas the levels were restored to normal in the HFR group. Overall, the results show that resveratrol restores the phosphorylation levels of proteins involved in the insulin signaling pathway, which were decreased by a high fat diet.     

The effect of thrombin on astrocyte stellation with regional specificity

In this study, we have examined the possible existence of astrocyte regional heterogeneity in thrombin effect on astrocyte stellation. Neonatal astrocytes were cultured for 2 weeks from six different regions of the neonatal rat brain, including the cerebral cortex, hippocampus, brainstem, midbrain, hypothalamus and cerebellum. Culture medium was changed to DMEM containing 8-CPT-cyclic AMP (cAMP) or isoproterenol plus various concentrations of thrombin for 2 hr. Thrombin effectively blocked both cAMP- and isoproterenol-induced cell stellation in a dose-dependent manner in all regional astrocytes except cerebellar astrocytes. RT-PCR analysis showed that thrombin receptor mRNA was expressed in all regional astrocytes, suggesting that cerebellar astrocytes may maintain a unique signaling pathway downstream of the thrombin receptor.     

Ethanol exposure suppresses survival kinases activation in adult rat testes

The present study was designed to evaluate whether ethanol suppresses survival-signaling pathways in rat testes. Ethanol (1.5 g/kg or 3 g/kg i.p., 15% v/v in saline) was administrated to adult male rats for 10 days. Ethanol treatment significantly increased the number of TUNEL-positive cells in rat testes. Potential activation was measured by phosphorylation of Akt and Erk1/2 using Western blot analysis. Ethanol decreased the levels of activated survival kinases, pAkt and pErk1/2. The phosphorylation of Bad at Ser112 and Ser136 was decreased in ethanol-treated animals in comparison to saline-treated animals. Moreover, the interaction of pBad with 14-3-3 was decreased by ethanol exposure. In conclusion, our findings suggest that ethanol induces apoptotic cell death by suppressing the activation of survival kinases and the phosphorylation of their downstream targets in rat testes.     

大鼠局灶性脑缺血后适应动物模型的建立与评估

试验旨在从方法学的角度比较3种有脑保护效应的大鼠局灶性脑缺血后适应(ischemic postconditioning,IPOC)模型的成功率、稳定性及其保护作用,以期为IPOC机制的研究建立良好的动物模型。将SD大鼠随机分为模型1组(开颅电凝大脑中动脉+双侧颈总动脉夹闭30 min)、模型2组(线栓法大脑中动脉阻塞60 min)、模型3组(线栓法大脑中动脉阻塞100 min),3个组再按假手术组、缺血再灌组及缺血后处理组各分为3个亚组,计9组,每亚组10只,分别在灌注2和24 h后进行神经功能缺损评分;在灌注24 h后取大脑进行2,3,5-3氯4氮唑(TTC)染色确定脑梗死体积百分比,并进行统计学分析;比较3种模型后处理后大鼠脑梗死体积的变异系数。结果显示,3种大鼠模型均呈现了不同程度的神经功能缺损体征,且IPOC均不同程度的降低脑梗死体积,改善了神经功能缺损评分,其中电凝法缺血30 min时建模的成功率最高。电凝法缺血30 min后适应模型脑梗死体积下降42.9%;线栓法缺血60 min后适应模型脑梗死体积下降15.9%;线栓法缺血100 min后适应模型脑梗死体积下降33.4%。电凝法缺血30 min后适应模型脑梗死体积的变异系数最低。开颅电凝法缺血30 min再灌30 s/缺血10 s,反复3次的IPOC模型不仅保护作用强,而且模型成功率高、稳定性好,可作为研究IPOC机制较好的动物模型。     

Anesthetic management of the head trauma patient

Objective: To describe the optimal anesthetic management of patients with brain injury, with emphasis on the support of oxygen delivery to the brain, and the effects of anesthetic agents on cerebral perfusion. Data sources: Clinical and experimental studies from both the human and veterinary neuroanesthesia literature. Summary: The management of patients following primary traumatic brain injury (TBI) significantly impacts outcome. Outcome can be improved by strategies that improve oxygen delivery to the brain and prevent cerebral ischemia. Anesthetic agents have widely variable effects on the blood supply to the brain and, therefore, choice of anesthetic agent can influence neurological outcome. Although in the past, anesthetic agents have been selected for their neuroprotective properties, it is increasingly being recognized that the support of cerebral perfusion during anesthesia contributes more significantly to a positive outcome for these patients. Support of cardiorespiratory function is, therefore, highly important when anesthetizing patients with TBI. Conclusion: Choice of anesthetic agent is determined by the extent of brain injury and intracranial pressure (ICP) elevation. Factors that should be considered when anesthetizing head trauma patients include the effects of anesthetic agents on the cardiac and respiratory systems, their effects on cerebral blood flow (CBF), ICP, and possible neuroprotective benefits offered by certain agents.     

Aquaporin-4 in acute cerebral edema produced by Clostridium perfringens type D epsilon toxin

Sheep, particularly lambs, with high circulating levels of Clostridium perfringens type D epsilon toxin develop severe neurologic signs and often die suddenly. On microscopic examination, in the brain, there is microvascular endothelial injury and diffuse vasogenic edema. The aquaporin (AQP) family of membrane water-channel proteins, especially AQP-4, is important in the regulation of water balance in the brain and facilitates reabsorption of excess fluid. In rats given epsilon toxin, generalized cerebral edema was demonstrated by marked albumin extravasation and was correlated with widespread upregulation of AQP-4 in astrocytes. These results suggest that AQP-4 has a role in the clearance of edema fluid from brains damaged by this clostridial toxin.     

PKA implicated in the phosphorylation of Cx43 induced by stimulation with FSH in rat granulosa cells

Connexin 43 (Cx43)-mediated gap junctional communication in granulosa cells is crucial for germ line development and postnatal folliculogenesis. We previously showed that follicle-stimulating hormone (FSH) promoted phosphorylation of Cx43 in rat primary granulosa cells. We further identified Ser365, Ser368, Ser369, and Ser373 in the carboxy-terminal tail as the major sites of phosphorylation by FSH, and found that the phosphorylation of these residues was essential for channel activity. In this study, we investigated the protein kinase(s) responsible for FSH-induced phosphorylation. H89, a cyclic AMP-dependent protein kinase (PKA) inhibitor, inhibited FSH-induced phosphorylation both in vivo and in vitro, whereas PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, had little effect on the phosphorylation level. Ca2+-dependent protein kinase (PKC) appeared to negatively regulate phosphorylation. Phosphopeptide mapping with or without H89 treatment indicated that PKA could be responsible for phosphorylation of the four serine residues. In addition, the purified catalytic subunit of PKA could phosphorylate the recombinant C-terminal region of Cx43, but not the variant in which all four serine residues were substituted with alanine. These results suggest that FSH positively regulates Cx43-mediated channel formation and activity through phosphorylation of specific sites by PKA.     

The neuroprotective effects of lidocaine and methylprednisolone in a rat model of retinal ischemia-reperfusion injury

Retinal ischemia is a common cause of visual impairment for humans and animals. The neuroprotective effects of lidocaine (LDC) and methylprednisolone (MP) upon retinal ischemic injury were investigated in a rat model. Sprague-Dawley rats were divided into 3 groups, the IR control, LDC and MP. A very high intraocular pressure (HIOP) and retinal ischemia were induced. In LDC group, LDC bolus (1.5 mg/kg) was i.v. injected 30 min before ischemia and then a constant rate infusion (CRI) with 2 mg/kg/hr was given until 60 min after reperfusion. In MP group, MP bolus (30 mg/kg) was i.v. administered twice at 2 min before and immediately after ischemia, respectively. The HIOP damage to retina was evaluated by electroretinogram (ERG) and morphometrical histology. The functional analysis of the retina by ERG revealed a 35.2% reduction of a-wave in the IR group, 49.7% reduction in the LDC group but no significant change in the MP group compared to normal controls. An 81.0% reduction of b-wave was observed in the IR group, 80.7% reduction in the LDC group and 17.6% reduction in the MP group. In the morphometrical histology, the retinal inner plexiform layer/outer nuclear layer (IPL/ONL) ratio was reduced to 48.8% in the IR group, 80.1% in the LDC group and 96.2% in MP group. In conclusion, the MP showed significantly good neuroprotective effects on retinal IR injury, and the LDC showed moderate neuroprotective effects demonstrated in retinal structure but not in retinal function.     

Mechanistic target of rapamycin is activated in bovine granulosa cells after LH surge but is not essential for ovulation

The LH surge induces functional and morphological changes in granulosa cells. Mechanistic target of rapamycin (mTOR) is an integrator of signalling pathways in multiple cell types. We hypothesized that mTOR kinase activity integrates and modulates molecular pathways induced by LH in granulosa cells during the preovulatory period. Cows were ovariectomized and granulosa cells collected at 0, 3, 6, 12 and 24 hr after GnRH injection. While RHEB mRNA levels increased at 3 and 6 hr, returning to basal levels by 12 hr after GnRH treatment, RHOA mRNA levels increased at 6 hr and remained high thereafter. Western blot analyses revealed increased S6K phosphorylation at 3 and 6 hr after GnRH injection. Similarly, mRNA levels of ERK1/2, STAR and EGR‐1 were higher 3 hr after GnRH treatment. Rapamycin treatment inhibited mTOR activity and increased AKT activity, but did not alter ERK1/2 phosphorylation and EGR1 protein levels in cultured bovine granulosa cells. Rapamycin also inhibited LH‐induced increase in EREG mRNA abundance in granulosa cells in vitro. However, intrafollicular injection of rapamycin did not suppress ovulation. These findings suggest that mTOR is involved in the control of EREG expression in cattle, which may be triggered by LH surge stimulating RHEB and S6K activity.     

家蚕丝氨酸蛋白酶抑制剂基因serpin-6在不同发育时期的表达分析

丝氨酸蛋白酶抑制剂(serpin)是一类具有重要生理调控功能的蛋白酶超家族。为研究家蚕serpin-6基因(Bmserpin-6)在蚕体内的功能,利用半定量RT-PCR方法,分析该基因mRNA在家蚕不同发育时期的转录水平。Bmserpin-6基因在家蚕各个发育阶段均有表达,在4龄眠期、5龄6d、吐丝48h(化蛹前)和化蛹6d的转录水平较高,在5龄4d、吐丝初期和蛹末期的转录水平较低。Bmserpin-6基因在5龄期家蚕精巢和卵巢中的表达均呈现先下降后上升的趋势。研究结果表明,Bmserpin-6基因mRNA的转录具有明显的时空特异性,推测其在家蚕不同的变态发育期执行不同的功能。     

Dietary curcumin supplementation attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in C57BL mice

Studies in vivo and in vitro suggest that curcumin is a neuroprotective agent. Experiments were conducted to determine whether dietary supplementation with curcumin has neuroprotective effects in a mouse model of Parkinson’s disease (PD). Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) significantly induced the loss of dopaminergic cells in the substantia nigra and deletion of dopamine in the striatum, which was attenuated by long-term (7 weeks) dietary supplementation with curcumin at a concentration of 0.5% or 2.0% (w/w). Although curcumin did not prevent the MPTP-induced apoptosis of neuroblasts in the subventricular zone (SVZ), it promoted the regeneration of neuroblasts in the anterior part of the SVZ (SVZa) at 3 days after MPTP treatment. Furthermore, curcumin enhanced the MPTP-induced activation of microglia and astrocytes in the striatum and increased the expression of glial cell line-derived neurotrophic factor (GDNF) and transforming growth factor-β1 (TGFβ1) in the striatum and SVZ. GDNF and TGFβ1 are thought to play an important role in protecting neurons from injury in the central and peripheral nervous systems. These results suggest that long-term administration of curcumin blocks the neurotoxicity of MPTP in the nigrostriatal dopaminergic system of the mouse and that the neuroprotective effect might be correlated with the increased expression of GDNF and TGFβ1. Curcumin may be effective in preventing or slowing the progression of PD.     

Pharmacokinetics of piperacillan after intramuscular injection in red-tailed hawks (Buteo jamaicensis) and great horned owls (Bubo virginianus).

This study characterized and compared the pharmacokinetics of piperacillin after single 100 mg/kg i.m. injections in nine red-tailed hawks (Buteo jamaicensis) and five great horned owls (Bubo virginianus) over 48 hr by a modified agar well diffusion microbial inhibition assay. The mean maximum plasma piperacillin concentrations were 204 microg/ml and 221 microg/ml for the hawks and owls, respectively, and times of maximum concentrations were 15 min and 30 min, respectively. The calculated mean terminal elimination half-lives were 77 min in the hawks and 118 min in the owls. Area-under-the-curve values were 218 +/- 52 microg x hr/ml in the hawks and 444 +/- 104 microg x hr/ml in the owls. On the basis of the most common minimal inhibitory concentration (90%) for various bacterial isolates from clinical samples of 8 microg/ml, analysis of the data suggests that the maximum dosing interval for piperacillin at 100 mg/kg in medium sized raptors should be 4-6 hr.