肌苷促进脑损伤后神经功能恢复的实验研究

目的：中枢损伤是目前致残率最高的疾病之一,肌苷对于神经损伤后功能恢复的促进作用已经成为研究热点,本研究拟建立一侧前肢瘫痪的大鼠脑外伤模型,证实肌苷治疗促进中枢损伤后上肢功能恢复的有效性,同时初步探索其机制。方法：建立一侧运动皮层冲击损毁的大鼠模型,通过肢体不对称实验、抓取实验等行为学观察证实其惠侧上肢功能受损,后在实验组进行肌苷药物14天,观察28天内上肢功能的恢复情况,与对照组作对比。证实其行为学上的有效性,同时对损伤侧大脑进行顺行BDA染色,探索其内在机制。结果：通过28天的观察发现经过肌苷治疗的的实验组大鼠肢体不对称实验、抓取实验等行为学评分明显好于隐性对照组,顺行BDA染色证实其有促进损伤周围健存皮层突触再生和代偿的作用。结论：肌苷可以促进中枢损伤后大鼠残存神经元得突触再生,使其大脑能在最大程度上代偿其丧失的功能,该药物可能会成为一种新的中枢损伤治疗的前体药物。     

肌苷促进脑损伤后神经功能恢复的实验研究

目的:中枢损伤是目前致残率最高的疾病之一,肌苷对于神经损伤后功能恢复的促进作用已经成为研究热点,本研究拟建立一侧前肢瘫痪的大鼠脑外伤模型,证实肌苷治疗促进中枢损伤后上肢功能恢复的有效性,同时初步探索其机制。方法:建立一侧运动皮层冲击损毁的大鼠模型,通过肢体不对称实验、抓取实验等行为学观察证实其患侧上肢功能受损,后在实验组进行肌苷药物14天,观察28天内上肢功能的恢复情况,与对照组作对比,证实其行为学上的有效性,同时对损伤侧大脑进行顺行BDA染色,探索其内在机制。结果:通过28天的观察发现经过肌苷治疗的的实验组大鼠肢体不对称实验、抓取实验等行为学评分明显好于隐性对照组,顺行BDA染色证实其有促进损伤周围健存皮层突触再生和代偿的作用。结论:肌苷可以促进中枢损伤后大鼠残存神经元得突触再生,使其大脑能在最大程度上代偿其丧失的功能,该药物可能会成为一种新的中枢损伤治疗的前体药物。     

Immediate recovery from spinal cord injury through molecular repair of nerve membranes with polyethylene glycol.

A brief application of the hydrophilic polymer polyethylene glycol (PEG) swiftly repairs nerve membrane damage associated with severe spinal cord injury in adult guinea pigs. A 2 min application of PEG to a standardized compression injury to the cord immediately reversed the loss of nerve impulse conduction through the injury in all treated animals while nerve impulse conduction remained absent in all sham-treated guinea pigs. Physiological recovery was associated with a significant recovery of a quantifiable spinal cord dependent behavior in only PEG-treated animals. The application of PEG could be delayed for approximately 8 h without adversely affecting physiological and behavioral recovery which continued to improve for up to 1 month after PEG treatment.     

Rationalization of membrane protein crystallization with polyethylene glycol using a simple depletion model

Based on the importance of crystallizing membrane proteins in a rational way, cytochrome bc(1) complex (BC1) was crystallized using polyethylene glycol (PEG) as a sole crystallization agent. Interaction between protein-detergent complexes of BC1 was estimated by dynamic light scattering, and was compared with the numerical calculation using the Derjaguin-Landau-Verwey-Overbeek potential plus a depletion potential, without considering specific surface properties of the protein-detergent complexes. The experiments and calculation were found to be consistent and we obtained a relation between PEG molecular weight M and the range of depletion zone delta as delta approximately M(0.48+/-0.02). The stability of liquid phase of BC1 solutions was controlled by a ratio of (the range of depletion zone)/(the radius of a BC1 particle), which was consistent with recent theoretical predictions. The crystallization was most successful under a condition where the stability of the liquid phase changed from stable to unstable. The PEG molecular weight that fulfilled this condition coincided with the one used empirically to crystallize BC1 in the past by a number of groups. These results are compared to the fact that membrane proteins were often successfully crystallized close to the detergent cloud point.     

A simple method of rat renal brush border membrane preparation using polyethylene glycol precipitation

A simple method for preparation of brush border membranes (BBM) from rat kidney using polyethylene glycol (PEG) precipitation has been described. This method avoids the use of cations for the preparation, which might alter membrane lipid composition. These preparations were assessed for enrichment of marker enzymes, contamination by subcellular structures, lipid composition and transport function. An enrichment of 11.8910-fold of alkaline phosphatase, 13.9500-fold of amino peptidase and 13.6500-fold of gamma-glutamyl transpeptidase and an approximate yield of 60% were seen in the final membrane preparation as compared to the homogenate. There was very little contamination of basolateral membranes, peroxisomes, microsomes or lysosomes in the final membrane preparation. Analysis of sugars indicated high content of fucose and sailic acid as compared to hexoses. Isolated membranes appeared as vesicles as seen by electron microscopy. Lipid analysis indicated the presence of various neutral and phospholipids with a high content of sphingomyelin along with a cholesterol/phospholipid ratio of 0.4850. The isolated membrane vesicles were able to transport glucose. This study has shown a simple method of renal brush border membrane preparation, which is comparatively pure and functionally active.     

Isolation and purification of bacterial ribosomes from endotoxin using polyethylene glycol

The difficulties arising in the study of the immunogenicity of bacterial ribosomes and in their possible use as vaccines are due to the fact that preparative ultracentrifugation, constituting a necessary stage in most of the methods used for the isolation of ribosomes, has a low productive capacity. To develop a more effective method for obtaining Shigella ribosomal vaccines, an attempt to use the method of precipitation with 10% polyethylene glycol (PEG), proposed by Expert-Bezan?on et al., has been made. The serological determination of O antigen has shown that nearly contained in the supernatant fluid S-30 can be detected in precipitated ribosomes. Taking into account the wide spectrum of the biological activity of bacterial endotoxin, it must be removed from the vaccine. The study has revealed that precipitation by means of ethanol (15-35%), low pH (4,2-4,7) and PEG (4-8%) can be used for this purpose. In accordance with the chosen method, the clarified material obtained by precipitation with 10% PEG is fractionated by means of 5% PEG which causes the complete precipitation of ribosomes, thus leaving endotoxin in the solution. Centrifugation in the density gradient of saccharose and electron microscopy have demonstrated that ribosomes isolated by this method possess typical sedimentation properties and structure. The yield of ribosomes is 3 times greater than that obtained by ultracentrifugation. Fractionation with PEG may be used as the method of the mass production of ribosomal vaccines.     

Mitochondrial dysfunction early after traumatic brain injury in immature rats

Mitochondria play central roles in acute brain injury; however, little is known about mitochondrial function following traumatic brain injury (TBI) to the immature brain. We hypothesized that TBI would cause mitochondrial dysfunction early (<4 h) after injury. Immature rats underwent controlled cortical impact (CCI) or sham injury to the left cortex, and mitochondria were isolated from both hemispheres at 1 and 4 h after TBI. Rates of phosphorylating (State 3) and resting (State 4) respiration were measured with and without bovine serum albumin. The respiratory control ratio was calculated (State 3/State 4). Rates of mitochondrial H(2)O(2) production, pyruvate dehydrogenase complex enzyme activity, and cytochrome c content were measured. Mitochondrial State 4 rates (ipsilateral/contralateral ratios) were higher after TBI at 1 h, which was reversed with bovine serum albumin. Four hours after TBI, pyruvate dehydrogenase complex activity and cytochrome c content (ipsilateral/contralateral ratios) were lower in TBI mitochondria. These data demonstrate abnormal mitochondrial function early (相似文献   

Change in plasma visfatin level after severe traumatic brain injury

Higher plasma visfatin concentration has been associated with ischemic stroke. Thus, we sought to investigate change in plasma visfatin level after traumatic brain injury and to evaluate its relation with disease outcome. Seventy-six healthy controls and 98 patients with acute severe traumatic brain injury were recruited. Twenty-seven patients (27.6%) died and 48 patients (49.0%) suffered from unfavorable outcome (Glasgow outcome scale score of 1–3) in 6 months. On admission, plasma visfatin level was increased in patients than in healthy controls and was highly correlated with Glasgow Coma Scale score. A multivariate analysis identified plasma visfatin level as an independent predictor for 6-month mortality and unfavorable outcome. According to receiver operating characteristic curve analysis, the predictive value of the plasma visfatin concentration was similar to Glasgow Coma Scale score's. In a combined logistic-regression model, visfatin did not improve the predictive value of Glasgow Coma Scale score. Thus, increased plasma visfatin level is associated with 6-month clinical outcomes after severe traumatic brain injury.     

BDNF polymorphism predicts general intelligence after penetrating traumatic brain injury

Neuronal plasticity is a fundamental factor in cognitive outcome following traumatic brain injury. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays an important role in this process. While there are many ways to measure cognitive outcome, general cognitive intelligence is a strong predictor of everyday decision-making, occupational attainment, social mobility and job performance. Thus it is an excellent measure of cognitive outcome following traumatic brain injury (TBI). Although the importance of the single-nucleotide polymorphisms polymorphism on cognitive function has been previously addressed, its role in recovery of general intelligence following TBI is unknown. We genotyped male Caucasian Vietnam combat veterans with focal penetrating TBI (pTBI) (n = 109) and non-head injured controls (n = 38) for 7 BDNF single-nucleotide polymorphisms. Subjects were administrated the Armed Forces Qualification Test (AFQT) at three different time periods: pre-injury on induction into the military, Phase II (10–15 years post-injury, and Phase III (30–35 years post-injury). Two single-nucleotide polymorphisms, rs7124442 and rs1519480, were significantly associated with post-injury recovery of general cognitive intelligence with the most pronounced effect at the Phase II time point, indicating lesion-induced plasticity. The genotypes accounted for 5% of the variance of the AFQT scores, independently of other significant predictors such as pre-injury intelligence and percentage of brain volume loss. These data indicate that genetic variations in BDNF play a significant role in lesion-induced recovery following pTBI. Identifying the underlying mechanism of this brain-derived neurotrophic factor effect could provide insight into an important aspect of post-traumatic cognitive recovery.     

Plasma copeptin concentration and outcome after pediatric traumatic brain injury

Higher plasma copeptin level has been associated with poor outcomes of critical illness. The present study was undertaken to investigate the plasma copeptin concentrations in children with traumatic brain injury (TBI) and to analyze the correlation of copeptin with disease outcome. Plasma copeptin concentrations of 126 healthy children and 126 children with acute severe TBI were measured by enzyme-linked immunosorbent assay. Twenty-one patients (16.7%) died and 38 patients (30.2%) had an unfavorable outcome (Glasgow Outcome Scale score of 1–3) at 6 months. Plasma copeptin level was obviously higher in patients than in healthy children (46.2 ± 20.8 pmol/L vs. 9.6 ± 3.0 pmol/L, P < 0.001). Plasma copeptin level was identified as an independent predictor for 6-month mortality [odds ratio (OR) 1.261, 95% confidence interval (CI) 1.112–1.538, P = 0.005] and unfavorable outcome (OR 1.313, 95% CI 1.146–1.659, P = 0.003). The predictive value of copeptin was similar to that of Glasgow Coma Scale (GCS) score for 6-month mortality [area under curve (AUC) 0.832, 95% CI 0.755–0.892 vs. AUC 0.873, 95% CI 0.802–0.926, P = 0.412] and unfavorable outcome (AUC 0.863, 95% CI 0.790–0.918 vs. AUC 0.885, 95% CI 0.816–0.935, P = 0.596). Copeptin improved the AUC of GCS score for 6-month unfavorable outcome (AUC 0.929, 95% CI 0.869–0.967, P = 0.013), but not for 6-month mortality (AUC 0.887, 95% CI 0.818–0.936, P = 0.600). Thus, plasma copeptin level represents a novel biomarker for predicting 6-month clinical outcome in children with TBI.     

Increased expression of BAG-1 in rat brain cortex after traumatic brain injury

BAG-1 protein was initially identified as a Bcl-2-binding protein. It was reported to enhance Bcl-2 protection from cell death, suggesting that BAG-1 represents a new type of anti-cell death gene. Moreover, recent study has shown that BAG-1 can enhance the proliferation of neuronal precursor cells, attenuate the growth inhibition induced by siah1. However, its function and expression in the central nervous system lesion are not been understood very well. In this study, we performed a traumatic brain injury (TBI) model in adult rats and investigated the dynamic changes of BAG-1 expression in the brain cortex. Double immunofluorescence staining revealed that BAG-1 was co-expressed with NEURON and glial fibrillary acidic protein (GFAP). In addition, we detected that proliferating cell nuclear antigen had the co-localization with GFAP, and BAG-1. All our findings suggested that BAG-1 might involve in the pathophysiology of brain after TBI.     

The expression changes of Numblike in rat brain cortex after traumatic brain injury

Numblike (Numbl) plays an important role in ependymal wall integrity and subventricular zone neuroblast survival. And Numbl is specifically expressed in the brain. However, its expression and function in the central nervous system lesion are still unclear. In this study, we performed a traumatic brain injury (TBI) model in adult rats and investigated the dynamic changes of Numbl expression in the brain cortex. Western blot and immunohistochemistry analysis revealed that Numbl was present in normal brain. It gradually decreased, reached the lowest point at day 3 after TBI, and then increased during the following days. Double immunofluorescence staining showed that Numbl immunoreactivity was found in neurons, but not astrocytes and microglia. Moreover, the 3rd day post injury was the apoptotic peak implied by the alteration of caspase-3. All these results suggested that Numbl may be involved in the pathophysiology of TBI and further research is needed to have a good understanding of its function and mechanism.     

Regulated expression of pancreatic triglyceride lipase after rat traumatic brain injury

Pancreatic triglyceride lipase (PTL), an enzyme of digestive system, plays very important roles in the digestion and absorption of lipids. However, its distribution and function in the central nervous system (CNS) remains unclear. In the present study, we mainly investigated the expression and cellular localization of PTL during traumatic brain injury (TBI). Western blot and RT–PCR analysis revealed that PTL was present in normal rat brain cortex. It gradually increased, reached a peak at the 3rd day after TBI, and then decreased. Double immunofluorescence staining showed that PTL was co-expressed with neuron, but had a few colocalizations in astrocytes. When TBI occurred in the rat cortex, the expression of PTL gradually increased, reached the peak at the 3rd day after TBI, and then decreased. Importantly, more PTL was colocalized with astrocytes, which is positive for proliferating cell nuclear antigen (PCNA). In addition, Western blot detection showed that the 3rd day post injury was not only the proliferation peak indicated by the elevated expression of PCNA, glial fibrillary acidic protein (GFAP) and cyclin D1, but also the apoptotic peak implied by the alteration of caspase-3 and bcl-2. These data suggested that PTL may be involved in the pathophysiology of TBI and PTL may be complicated after injury, more PTL was colocalized with astrocytes. Importantly, injury-induced expression of PTL was colabelled by proliferating cell nuclear antigen (proliferating cells marker), and the western blot for GFAP, PCNA and cyclin D1, showed that 3 days post injury was the proliferation peak, in coincidence to it, the protein level change of caspase-3 and bcl-2 revealed that the stage was peak of apoptotic too. These data suggested that PTL may be involved in the pathophysiology of TBI and that PTL may be implicated in the proliferation of astrocytes and the recovery of neurological outcomes. But the inherent mechanisms remained unknown. Further studies are needed to confirm the exact role of PTL after brain injury.     

Caspase-3 mediated neuronal death after traumatic brain injury in rats

During programmed cell death, activation of caspase-3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of caspase-activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis. The participation of caspase-3 activation in the evolution of neuronal death after traumatic brain injury in rats was examined. Cleavage of pro-caspase-3 in cytosolic cellular fractions and an increase in caspase-3-like enzyme activity were seen in injured brain versus control. Cleavage of the caspase-3 substrates DNA-dependent protein kinase and inhibitor of caspase-activated deoxyribonuclease and co-localization of cytosolic caspase-3 in neurons with evidence of DNA fragmentation were also identified. Intracerebral administration of the caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (480 ng) after trauma reduced caspase-3-like activity and DNA fragmentation in injured brain versus vehicle at 24 h. Treatment with N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone for 72 h (480 ng/day) reduced contusion size and ipsilateral dorsal hippocampal tissue loss at 3 weeks but had no effect on functional outcome versus vehicle. These data demonstrate that caspase-3 activation contributes to brain tissue loss and downstream biochemical events that execute programmed cell death after traumatic brain injury. Caspase inhibition may prove efficacious in the treatment of certain types of brain injury where programmed cell death occurs.