脑活素治疗新生大鼠缺血缺氧性脑病机理的研究

7日龄Wister大鼠结扎左侧颈总动脉后吸入氧氮混合气体（8％O2和92％N2）2h,制成缺血缺氧性脑病动物模型,测定大鼠脑组织中丙二醛（MDA）的含量和超氧化物歧化酶（SOD）的活性。结果显示,缺血缺氧后6h,缺血缺氧组MDA含量显著升高,24h达到高峰,以后逐渐下降,96h与对照组比较无统计学差异;缺血缺氧脑活素治疗组MDA含量于缺血缺氧后6h已经显著下降,72h与对照组比较无统计学差异。缺血缺氧组SOD活力值在缺血缺氧后6h已经下降,24h降到最低点,以后逐渐回升,96h与对照组比较无统计学差异;缺血缺氧脑活素治疗组SOD活力值在缺血缺氧后6h明显提高,于缺血缺氧后96h与对照组比较无统计学差异。结果提示,缺血缺氧引起新生大鼠脑组织的氧化-抗氧化系统失衡,氧自由基大量产生,参与了新生大鼠缺血缺氧性脑病脑损伤过程;脑活素能够抑制氧自由基的生成,提高SOD的活力值,对缺血缺氧性脑病具有神经保护作用。     

Effects of nitric oxide on hepatic encephalopathy in cirrhotic rats induced by LPS

AIM: To investigate the effects of nitric oxide (NO) on hepatic encephalopathy in cirrhotic rats induced by LPS. METHODS: The cirrhotic model of rats was established by complex pathogeny. Since the end of the 8 th week, the rats were intragastrically-infused with 0.9% salt, L-arginine(L-arg) and LNNA respectively for 2 weeks.The hepatic encephalopathy in cirrhotic rats were induced by 3 mg/kg LPS (ip) 4 hours before the rats were sacrificed. RESULTS: The normal behaviors and electroencephalograph were appeared in L-arg group. LNNA group showed hepatic encephalopathy. The content of NO₂^-/NO₃^- of brain tissue was markedly higher in L-arg group than LNNA group(P<0.05), but the content of histamine in brain tissue was lower in L-arg group than LNNA group(P<0.05). There was a negative correlation between the content of histamine in brain tissue and the content of NO₂^-/NO₃^- of brain tissue. CONCLUSION: NO can prevent hepatic encephalopathy in cirrhotic rats induced by LPS.     

Experimental transmission of encephalopathy and retinopathy induced by nodavirus to sea bream, Sparus aurata L., using different infection models

The transmission of viral encephalopathy and retinopathy (VER) was investigated in juvenile sea bream, Sparus aurata L. Two different infection routes [intraperitoneal (i.p.) and intramuscular injection (i.m.)] were tested at two different temperatures (20 and 26 °C) using sea bream of mean weight 0.7, 2 and 4 g, as well as an immersion challenge performed at 26 °C with sea bream of 0.7 g. Successful transmission of the disease was only achieved by i.m. injection. Mortalities of 100% occurred in sea bream of 0.7 g at day 15 post-infection and 47% in sea bream of both 2 and 4 g at day 30 post-infection in all the experimental infections at 26 °C. No mortalities were ever observed with infections at 20 °C. When mortalities were observed, the virus was detected by immunoperoxidase staining in the SSN-1 cell line inoculated with tissues from infected fish. Histological examination of both normal and infected fish showed a vacuolization in the bipolar and granular layers of the retina of the infected sea bream. This is the first experimentally induced transmission of VER in sea bream. Differences were observed at the time of disease onset depending on water temperature, the route of infection and the age of the juvenile fish.     

Protective immunity of sevenband grouper, Epinephelus septemfasciatus Thunberg, against experimental viral nervous necrosis

This paper describes the protective immune responses of sevenband grouper, Epinephelus septemfasciatus Thunberg, immunized with live piscine nodavirus, the causative agent of viral nervous necrosis (VNN), or the Escherichia coli – expressed recombinant coat protein. Nodavirus-neutralizing antibodies were detected at titres ranging from 1:158 to 1:1257 in serum of sevenband grouper which survived intramuscular injection with the virus, by a cell culture assay system. The virus-neutralizing ability of immune serum was also confirmed by injecting virus previously treated with serum into fish. This indicates establishment of acquired immunity in survivors and thus explains why survivors from natural infection are resistant to recurrence of the disease. Young sevenband grouper were immunized twice by intramuscular injections with the recombinant coat protein. Immunized fish produced neutralizing antibodies at high titres for at least 110 days and showed significantly lower mortalities in virus challenge tests. These results suggest the potential for vaccination against VNN in sevenband grouper, which is susceptible to piscine nodavirus at all life-stages.     

Comparative pathogenicity study of ten different betanodavirus strains in experimentally infected European sea bass,Dicentrarchus labrax (L.)

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a severe pathological condition caused by RNA viruses belonging to the Nodaviridae family, genus Betanodavirus. The disease, described in more than 50 fish species worldwide, is considered as the most serious viral threat affecting marine farmed species in the Mediterranean region, thus representing one of the bottlenecks for further development of the aquaculture industry. To date, four different genotypes have been identified, namely red‐spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus and barfin flounder nervous necrosis virus, with the RGNNV genotype appearing as the most widespread in the Mediterranean region, although SJNNV‐type strains and reassortant viruses have also been reported. The existence of these genetically different strains could be the reason for the differences in mortality observed in the field. However, very little experimental data are available on the pathogenicity of these viruses in farmed fish. Therefore, in this study, the pathogenicity of 10 isolates has been assessed with an in vivo trial. The investigation was conducted using the European sea bass, the first target fish species for the disease in the Mediterranean basin. Naive fish were challenged by immersion and clinical signs and mortality were recorded for 68 days; furthermore, samples collected at selected time points were analysed to evaluate the development of the infection. Finally, survivors were weighed to estimate the growth reduction. The statistically supported results obtained in this study demonstrated different pathogenicity patterns, underlined the potential risk represented by different strains in the transmission of the infection to highly susceptible species and highlighted the indirect damage caused by a clinical outbreak of VER/VNN.     

Ellagic acid attenuates hypoxic-ischemic brain injury by alleviating autophagy

AIM:To investigate whether ellagic acid (EA) attenuates hypoxic-ischemic encephalopathy (HIE) by down-regulating autophagy. METHODS:In vivo, Sprague-Dawley rats (n=17) were randomly divided into 3 groups:5 rats for sham group, 6 rats for HIE group and 6 rats for HIE+EA pretreatment group. The rats in HIE+EA pretreatment group were treated with EA (10 mg/kg, 10 mL/kg, suspended in corn oil, ig). After 24 h of operation, the rats from each group were sacrificed and their brains were collected. TTC staining and HE staining were used to define the infarct areas and brain structure. The autophagy-related proteins beclin-1, P62, LC3-Ⅱ/-I and Atg5 in the cortex in each group were compared by Western blot. In vitro, PC12 cells were divided into 3 groups:control group, CoCl₂ group and CoCl₂+EA pretreatment group. CoCl₂ at 800 μmol/L was added to the PC12 cells to induce an anoxic environment. The PC12 cells were pretreated with EA at 8 μmol/L and the cell viability was measured by CCK-8 assay. The production of reactive oxidative species (ROS) in the cells was detected by flow cytometry with DCFH-DA staining. MDC staining and TMRE staining were applied to reflect the extent of autophagy and the state of apoptosis, respectively. The autophagy-related proteins in PC12 cells were also investigated. RESULTS:In HIE group, 7-day-old rats were given the operations and the their large infarct areas in the hemisphere were observed by TTC staining. HE staining displayed the injured hemispheres which contained few neurons, and exhibited edema status and serious structural damage. EA pretreatment decreased the infarct area and alleviated the damage to hemisphere with more visible neurons, compared with HIE group. Compared with sham group, the levels of autophagy-related proteins Atg5, beclin-1 and LC3-Ⅱ/-I in the cortex were increased (P<0.01), and P62 protein expression was decreased (P<0.01) in HIE group. Compared with HIE group, the protein expression of Atg5, beclin-1 and LC3-Ⅱ/-I was decreased (P<0.01) and P62 protein expression was increased in HIE+EA pretreatment group (P<0.01). In vitro, compared with CoCl₂ group, the PC12 cells in CoCl₂+EA pretreatment group showed a lower ROS level. Moreover, the cells in CoCl₂+EA pretreatment group exhibited higher mitochondrial membrane potential than that in CoCl₂ group. MDC staining in CoCl₂ group showed high value of fluorescence and increased number of autophagosomes. EA pretreatment reduced the number of autophagosomes and the extent of autophagy to protect PC12 cells. Furthermore, the protein levels of Atg5, beclin-1 and LC3-Ⅱ/-I in CoCl₂ group were higher (P<0.01), and the protein expression of P62 was lower (P<0.01) than those in control group. In CoCl₂+EA pretreatment group, the protein levels of Atg5, beclin-1 and LC3-Ⅱ/-I were decreased (P<0.01) and the protein expression of P62 was increased as compared with CoCl₂ group (P<0.01). CONCLUSION:EA pretreatment attenuates autophagy to protect the neurons against HIE injury.     

Protective effect of deferoxamine on hypoxic-ischemic encephalopathy in neonatal rat

AIM:To investigate the possible mechanism of deferoxamine on angiogenesis in rat hypoxic-ischemic encephalopathy (HIE). METHODS:SD rats (7 days of age) were used to make HIE model. Model group and treatment group were injected with deferoxamine or normal saline alone 24 hours before hypoxic-ischemic insult. Rats were sacrificed at 1,3,7 or 14 days after hypoxic-ischemic insult. Brain capillary density index (BCDI),the number of proliferating capillary,brain water content and extent of brain atrophy were determined. The expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α(HIF-1α) mRNA was measured. RESULTS:Early water content and late atrophic ratio of the left brain were significantly improved in the treatment group compared to model group (P<0.01). The number of proliferating capillary in the treatment group was significantly higher than that in the model group ［(2.01±0.31)/HPF vs　(0.90±0.25)/HPF,P<0.01］. Deferoxamine markedly up-regulated the expression of VEGF and HIF-1α mRNA in the brain ［VEGF at 12 h: (1.41±0.07) vs (1.10±.15),P<0.05; HIF-1α at 12 h: (1.49±0.12) vs (1.11±0.16),P<0.05］.CONCLUSION:Deferoxamine may promote angiogenesis and attenuate hypoxic-ischemic induced brain injury via up-regulation of HIF-1α and VEGF expression.     

A Syndrome Resembling Idiopathic Noncirrhotic Portal Hypertension in 4 Young Doberman Pinschers

We describe 4 young male Doberman Pinschers (3 littermates and 1 unrelated dog) with a syndrome resembling idiopathic or noncirrhotic portal hypertension of humans. Each dog was evaluated for a hepatopathy resulting in portal hypertension, development of portosystemic collateral vessels, and hepatic encephalopathy. These dogs differ from previous reports of young dogs with hepatic insufficiency associated with portal hypertension and acquired portal systemic shunting by their lack of intrahepatic arteriovenous fistulae, portal vein atresia, or intrahepatic fibrosis. Clinicopathologic features included erythrocyte microcytosis, normal to mildly increased liver enzyme activities, increased concentrations of serum bile acids, reduced plasma indocyanine green clearance, and normal total bilirubin concentration. Abdominal ultrasonography disclosed a small liver and portosystemic collateral vessels. Radiographic imaging studies confirmed hepatofugal portal circulation and discounted hepatic arteriovenous fistulae. Histopathologic features in liver tissue from each dog were similar and consistent in all sections examined. Common findings included increased cross-sectional views of hepatic arterioles; hepatic lobular atrophy; scanty increase in connective tissue around some large portal triads; and absence of inflammation, disturbed lobular architecture, bile duct proliferation, or intrahepatic cholestasis.