Neurological deficits following permanent focal cerebral ischemia in rats--论文代写范文精选

中风是第二个最常见的死亡原因,除了心脏病导致更高的死亡率。中风最常见的结果是一个阻塞血液流动,如果不在短时间内解决,将导致梗塞的组织可能是无法治疗挽救的。下面的report代写范文讲述了这一事实。

ABSTRACT 
Recent experimental evidences indicate that pyruvate, the final metabolite of glycolysis, has a remarkable protective effect against different types of brain injury. The purpose of this study was to assess the neuroprotective effect and the neurological outcome after pyruvate administration in a model of ischemic stroke induced by permanent middle cerebral artery occlusion (pMCAO) in rats. Three doses of pyruvate (250, 500 and 1000 mg/kg; i.p.) or vehicle were administered intraperitoneally 30 min after pMCAO. In other set of experiments, pyruvate was given either before, immediately after ischemia or in a long-term administration paradigm. Functional outcome, mortality and infarct volume were determined 24 h after stroke. Even when the lowest doses of pyruvate reduced mortality and neurological deficits, no concomitant reduction in infarct volume was observed. The highest dose of pyruvate increased cortical infarction by 27 % when administered 30 min after pMCAO. In addition, when pyruvate was given before pMCAO, a significant increase in neurological deficits was noticed. Surprisingly, on the contrary of what was found in the case of transient global ischemia, present findings do not support a great neuroprotective role for pyruvate in permanent focal cerebral ischemia, suggesting two distinct mechanisms involved in the effects of this glycolytic metabolite in the ischemic brain. 
Key words: pyruvate; middle cerebral artery occlusion; neuroprotection; stroke

INTRODUCTION 
Stroke is the second most frequent cause of death and only heart disease causes higher mortality. Stroke is most commonly the result of an obstruction of blood flow in a major cerebral vessel (e.g., the middle cerebral artery), which, if not resolved within a short period of time, will lead to an infarcted tissue that may not be therapeutically salvaged [16,35,36]. Development of an effective therapeutic strategy for stroke has been a priority of neuroscientists for decades. Although some clinical benefits have been obtained with the antioxidants ebselen and edaravone [11,39], no neuroprotective agents has been shown conclusively to be clinically effective to prevent or restrict acute neuronal damage after stroke [6,12]. Ischemia-induced neuronal loss is associated with numerous biochemical events initially triggered by the extracellular accumulation of glutamate.

In turn, excitotoxicity leads to membrane depolarization, increased concentrations of intracellular calcium, overproduction of reactive oxygen species, inflammation and activation of apoptotic pathways contributing to the progression of tissue damage [2,4,5,7,25,38]. Recent evidences indicate that, in addition to calcium, endogenous zinc may play a role as an ionic mediator of neuronal death, activating various cell death cascades, such as free radical generation and caspase activation [3,21,26,41,48,53,56]. Interestingly, pyruvate, the end metabolite of the glycolytic pathway, protects striatal neurons against excitotoxicity induced by a 30-min exposure to N-methyl-D-aspartate [29,47], prevents neuronal death induced by exogenous and endogenous H2O2 in cultured neurons [8,30,31], protects almost completely against zinc neurotoxicity [23,49] and prevents H2O2-induced apoptosis [43]. In addition, results from a very recent report indicate that administration of pyruvate provides spectacular protection against hippocampal CA1 neuronal injury following transient global cerebral ischemia in rats [23]. In the light of all these evidences, the present study was conducted to assess whether pyruvate would show neuroprotective efficacy on the cerebral infarction induced by permanent middle cerebral artery occlusion (pMCAO), a clinically-relevant model of stroke.

METHODS
Animals Male Sprague-Dawley rats (CENPALAB, Havana, Cuba) weighing 280-340 g at the time of surgery were used in the present study. Our institutional animal care and use committee approved the experimental protocol (No. 03/12). The animals were quarantined for at least 7 days before the experiment. Animals were housed in groups in a room whose environment was maintained at 21-25 ºC, 45-50 % humidity and 12-h light/dark cycle. They had free access to pellet chow and water. Animal housing, care, and application of experimental procedures were in accordance with institutional guidelines under approved protocols. Permanent focal ischemia model Rats were anesthetized with chloral hydrate (300 mg/kg body weight, i.p.). Once surgical levels of anesthesia were attained (assessed by absence of hind leg withdrawal to pinch), ischemia was induced by using an occluding intraluminal suture [9,22,24]. Briefly, the right common carotid artery (CCA) was exposed by a ventral midline neck incision and ligated with a 3-0 silk suture. 
The pterygopalatine branch of the internal carotid artery was clipped to prevent incorrect insertion of the occluder filament. Arteriotomy was performed in the CCA approximately 3 mm proximal to the bifurcation and a 3-0 monofilament nylon suture, whose tip had been rounded by being heated near a flame was introduced into the internal carotid artery (ICA) until a mild resistance was felt (18-19 mm). Mild resistance to this advancement indicated that the intraluminal occluder had entered the anterior cerebral artery and occluded the origin of the anterior cerebral artery, the middle cerebral artery (MCA) and posterior communicating arteries [22]. After the advancement of the nylon suture, the ICA was firmly ligated with a 3-0 silk suture. The incision was closed and the occluding suture was left in place until sacrificing the animals. The animals were allowed to recover from anesthesia on an electrical heated blanket and to eat and drink freely.

RESULTS 
In this model of proximal pMCAO using an intraluminal nylon filament, TTC staining showed welldemarcated infarct areas in the temporoparietal cortex and in the laterocaudal part of the caudate putamen in all operated animals. High-grade neurological deficits (more than 2, see Methods section) were presented in all animals when tested at 24 h of pMCAO. Thus, no animals required exclusion on the basis of an inadequate degree of cerebral ischemia. The effect of pyruvate on neurological deficits and mortality following pMCAO is shown in Table 1. Pyruvate administration at doses of 250 and 500 mg/kg after 30 min of pMCAO slightly reduced mortality and neurological deficits, but failed to significantly reduce infarct volume (Fig. 1). However, the highest dose of pyruvate (1000 mg/kg) did not reduce the neurological deficits or mortality compared with vehicle. 

On the contrary, treatment with this dose of pyruvate significantly increased total (cortical + subcortical) infarct volume compared to the vehicle group (Fig. 1). When considered separately, mean cortical infarct volume was increased by 27% by treatment with pyruvate when administered 30 min after pMCAO compared to vehicle rats (237.3 ± 23.9 and 186.8 ± 41.9 mm3 , respectively; p=0.025). Mean subcortical infarct volume was not modified by pyruvate treatment. Then, the significant increased in cortical infarct volume accounts for the significant increased in total brain infarct observed in pyruvate-treated animals. The rostrocaudal distribution of cortical (Fig. 2A) and subcortical (Fig. 2B) infarct areas in the vehicle and pyruvate 1000 mg/kg groups is depicted in Fig. 

DISCUSSION 
The core findings of this study are: i) pyruvate administration failed to confer protection against permanent focal cerebral ischemia in rats and ii) the highest dose of pyruvate increased infarct volume in rats subjected to pMCAO when treatment is given 30 min after the onset of ischemia. This study was prompted by the encouraging results obtained by Lee et al [23], which showed that systemic administration of sodium pyruvate (500-1000 mg/kg) was remarkably neuroprotective in rats against global cerebral ischemia, a type of injury that mimics the clinical situation of cardio-respiratory arrest. We decided to explore the effects of pyruvate at the exact dose range and similar treatment schedule of those tested by Lee et al [23] in rats subjected to pMCAO, because most cases of human ischemic stroke are caused by permanent occlusion of cerebral arteries. 

Since in stroke patients a very early spontaneous recanalization of an obstructed brain vessel is only rarely found, experimental models of pMCAO may be more relevant to the clinical situation [13,15,20,40]. Apparent discrepancies between our present results and those of Lee et al [23] may be due to differences in the pathophysiological mechanisms between the two models of cerebral ischemia. It is important to emphasize that in global cerebral ischemia, delayed neuronal death occurs in selective vulnerable regions of brain, specifically in CA1 region of hippocampus through a myriad of biochemical mechanisms that predominantly lead to apoptosis of damaged neurons [17,44,57]. In pMCAO models, most of the ischemic tissue dies through a rapid necrotic mechanism, which is accompanied by a dramatic inflammatory response [19,25,58]. Probably, the mechanism of neuronal death prevailing in each model is playing a key role, since pyruvate has been proven to limit apoptotic cell death in both non-neuronal cells [43] and in hippocampal and cortical neurons following forebrain ischemia, but does not reduce necrotic neuronal death induced by a 24-h exposure to NMDA, glutamate or ionomycin (calcium-overload toxicity) [23].

In summary, the present study has evaluated by the first time the effects of pyruvate in permanent focal cerebral ischemia showing modest positive effects at low doses and detrimental effects when given at high doses. On the contrary of what was found in the case of transient global ischemia [23], present findings do not support a great neuroprotective role for pyruvate in permanent focal ischemia. We believe that it is very important to perform thorough, multifactorial and well-designed pre-clinical studies before assuming definitive conclusions on the neuroprotective effect of a given compound. In this particular case, our results and those by Lee et al [23] suggest that pyruvate could be tested in clinical trials with patients suffering from global cerebral damage but not in those with permanent stroke. Although success in animal studies does not guarantee success in clinical trials, the absence of neuroprotection or modest positive effects in animal studies indicate a lower likelihood of success in humans.

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