大鼠耳蜗雪旺细胞的体外培养和纯化

目的:探讨利用免疫磁珠从新生SD大鼠耳蜗螺旋神经节分离培养获得大量、高纯度雪旺细胞的方法。方法:选用1-3d SD大鼠,无菌条件下暴露双侧听泡,在高倍镜下仔细剥离蜗壳,开放耳蜗,完整取出耳蜗组织,分离并且除去膜蜗管外侧壁的血管纹和基底膜组织,然后剪碎。用0.25%的胰蛋白酶消化,用胎牛血清中止消化,离心以后加入DMEM/F12培养液培养。3-5天后对细胞应用免疫磁珠阳性分选方法进行纯化,培养2天后进行传代接种,培养过程中对提纯后的大鼠耳蜗雪旺细胞进行形态学观察、并绘制其生长曲线,采用细胞免疫荧光染色对细胞进行S-100免疫荧光鉴定并且计算细胞纯度。结果:分离培养后所得的细胞即为雪旺细胞;利用免疫磁珠阳性分选法对培养所得的细胞进行纯化,纯化后的大鼠耳蜗雪旺细胞纯度为97%±1.2%。结论:免疫磁珠法是一种有效的分离纯化新生大鼠仔鼠耳蜗螺旋神经节雪旺细胞的方法。所得耳蜗雪旺细胞活力强、纯度高,可以用于耳蜗雪旺细胞与螺旋神经节轴突的生长和再生等相关研究。     

The cannibalistic behaviour of two Gammarus species (Crustacea: Amphipoda)

This study examines the cannibalistic behaviour of the freshwater amphipods Gammarus duebeni celticus Stock & Pinkster, 1970 and G. pulex (L., 1758). In the first experiment, interactions were staged among all combinations of single adult males, single adult females, adults in the precopulatory mate-guarding phase and juveniles. Cannibalism by inter-moult individuals on newly moulted conspecifics occurred in all interaction categories in both species. Gammarus d. celticus , however, were significantly more cannibalistic than G. pulex. Cannibalism between and within sex and size categories (males > females > juveniles) was facilitated by the vulnerability of individuals at moult. Individuals of smaller size categories, however, did not cannibalize newly moulted conspecifics of larger size categories. Males were less cannibalistic on newly moulted females than on newly moulted males and juveniles and, when in the precopulatory condition, appeared to defend females from cannibalistic attacks. In a second experiment, stream conditions were simulated in the laboratory and replicated populations monitored for nine weeks. High levels of cannibalism, and the species and sex differences in cannibalism identified in the first experiment, were confirmed under these heterogeneous conditions. Cannibalism by males on their newly moulted female mating partners, termed 'reversed' sexual cannibalism, was further investigated. When males were deprived of foraging opportunities, cannibalism of precopulatory partners was significantly more frequent. The occurrence of 'reversed' sexual cannibalism is thus interpreted as a conflict between motivation to feed and motivation to mate.     

Effects of acute hypoxia and hyperoxia on respiratory rate in albino rats chronically exposed to hypoxia

Changes in respiratory frequencies with hypoxic or hyperoxic exposure were studied in: 12 normoxic control rats (N) born and raised in normoxic environment at sea level; 12 rats (A) born and raised in normoxic environment at sea level exposed to normobaric hypoxia (10% O2 in N2) as adults; 12 rats of first generation (G1) raised in the above mentioned hypoxic environment since a few hours after birth; 12 rats of third generation (G3) conceived and born in the hypoxic environment of hypoxic parents of second generation and maintained continuously under hypoxic conditions until their utilization. The response of A rats to 10% O2 and 7% O2 breathing was elevated (57% and 86% over air breathing). The mean respiratory frequency of A rats exposed to 7% O2 rose to a greater extent than did that of N rats. The G1 and G3 rats were less responsive to 7% O2 (64% and 37% over air breathing, respectively) than N and A rats; however, in G1 rats the exposure to 7% O2 produced a greater rise of frequency than in G3 rats. Furthermore A rats, G1 rats and G3 rats were less responsive to 97% O2 breathing (19%, 19% and 11% below air breathing, respectively). Comparing these data with previous findings we suggest that, with chronic exposure to hypoxia, changes in ventilatory response to hypoxia and hyperoxia occur in the following manner: I) loss of response to hypoxia if chronic exposure is begun in the immediate postnatal period; 2) degree of response to hypoxia or hyperoxia influenced by duration of chronic exposure.     

Demyelination-Induced Inflammation Attracts Newly Born Neurons to the White Matter

There is compelling evidence that microglial activation negatively impacts neurogenesis. However, microglia have also been shown to promote recruitment of newly born neurons to injured areas of the gray matter. In the present study, we explored whether demyelination-triggered inflammation alters the process of neurogenesis in the white matter. A 2-μl solution of 0.04 % ethidium bromide was stereotaxically injected into the corpus callosum of adult male rats. Brain inflammation was dampened by daily injections of progesterone (5 mg/kg, s.c.) for 14 days. Control rats received oil (s.c.). Newly born neurons (DCX and Tbr2), microglia (Iba-1), astrocytes (vimentin or GFAP), oligodendrocyte progenitor cells (OPCs; NG2), and mature oligodendrocytes (CC-1) were monitored in the vicinity of demyelination site using immunofluorescent staining. Western blot was used to explore microglial polarization using M1 (iNOS) and M2 (arginase-1) markers. Focal demyelination elicited strong microglial and astroglial activation and reduced the number of OPCs at the site of demyelination. This inflammatory response was associated with enhanced number of newly born neurons in the white matter and the subventricular zone (SVZ). A proportion of newly born neurons within the white matter showed features of OPCs. Interestingly, blunting brain inflammation led to reduced neurogenesis around the demyelination area and in the SVZ. These data suggest that the white matter inflammation creates a conducive environment for the recruitment of newly born neurons. The fact that a sizable fraction of these newly born neurons adopt OPC features suggests that they could contribute to the remyelination process.     

Live and carcass weights of giant rat Cricetomys gambianus Waterhouse and domestic rabbit Oryctolagus cuniculus L

This paper presents a record of the live and carcass weights of the African giant rat Cricetomys gambianus Waterhouse and the domestic rabbit Oryctolagus cuniculus L. Male and female giant rats have an average killing out percentage of 51.4% (n= 10, S.D. ± 3.8) and 516% (n = 7, S.D. ± 3.47) respectively. The difference between the killing-out percentages of both sexes was not statistically significant at 5% probability level. There was also no significant difference at 5% probability level between the killing-out percentages of giant rats and domestic rabbit. Very low fat content was observed in the carcasses of wild giant rats.     

Role of the renal kallikrein-kinin system in the development of salt-sensitive hypertension

The role of the renal kallikrein-kinin system in the development of salt-sensitive hypertension was studied using mutant kininogen-deficient Brown-Norway Katholiek (BN-Ka) rats, which generate no kinin in their urine, and other hypertensive rat models. It was found that ingestion of a low sodium diet or infusion of NaCl in doses slightly above 0.15 M caused hypertension and sodium accumulation in erythrocytes and the cerebrospinal fluid of kininogen-deficient BN-Ka rats. Development of hypertension in the deoxycorticosterone-acetate-salt model was completely prevented by administration of a newly discovered inhibitor, ebelactone B, of carboxypeptidase Y-like exopeptidase (an urinary kininase). The urinary kallikrein excretion of spontaneously hypertensive rats was lower than that of Wistar Kyoto rats at 4 weeks of age and did not increase by administration of furosemide, a diuretic agent, although approximately 50% of the diuretic action of this agent was dependent upon the renal kallikrein-kinin system in normal rats. In conclusion, the renal kallikrein-kinin system works as a safety valve for excess sodium intake.     

Efficient transgenic rat production by a lentiviral vector

A lentiviral construct for an enhanced green fluorescent protein (eGFP) driven by a chicken beta-actin promoter, cytomegalovirus enhancer, and intronic sequences from rabbit beta-globin (CAG) was used to produce transgenic lines of rats for evaluation of the usefulness of this approach in gene function studies. Fertilized eggs were collected from inbred Dahl S and outbred Sprague-Dawley rats, and approximately 100 pl of concentrated virus were microinjected into the perivitrelline space of one-cell embryos. Of 121 embryos injected, 60 pups (49.6%) were born. Transgenic rates averaged 22% in Dahl S and 14% in Sprague-Dawley rats. Copy number ranged from one to four in the founders, and the inheritance of the transgene in a subsequent F(1) population was 48.2%. The small number of insertion sites enabled us to derive inbred transgenic lines with a single copy of the transgene within one generation. Sequencing of each transgene insertion site revealed that they inserted as single copies with a preference for the introns of genes. The CAG promoter drove high levels of eGFP expression in brain, kidney, heart, and vasculature, making it very suitable for exploring the cardiovascular function of newly discovered genes. The pattern of eGFP expression was similar across five different F(1) transgenic lines, indicating that the expression of the transgene was independent of its chromosomal position. Thus lentiviral transgenesis provides a powerful tool for the production of transgenic inbred rats and will enhance the usefulness of this species in gene discovery and target validation studies.     

Experimental congenital toxoplasmosis in Wistar and Holtzman rats

Congenital toxoplasmosis was evaluated in Wistar and Holtzman rats using two strains of Toxoplasma gondii isolated in Brazil. Pregnant rats were inoculated by subcutaneous or intraperitoneal routes with 10(6) or 8 x 10(6) tachyzoites of N strain (virulent for mice) and by subcutaneous or oral routes with 10(2) or 1.2 x 10(3) cysts of P strain (avirulent for mice). The tissues of rat pups born from these rats were bioassayed for T. gondii infection. T. gondii was not observed in the pups born from rats inoculated with N strain. In the animals inoculated with P strain, congenital toxoplasmosis occurred in 22.8% (Wistar rats inoculated with 10(2) cysts by the subcutaneous route), 11.4% (Wistar rats inoculated with 10(2) cysts by the oral route), 21.2% (Wistar rats inoculated with 1.2 x 10(3) cysts by the oral route) and 2.9% of fetal infection (Holtzman rats inoculated with 10(2) cysts by the oral route). None of the pups born from chronically infected mother were infected with T. gondii.     

Neurogenesis response of middle-aged hippocampus to acute seizure activity

Acute Seizure (AS) activity in young adult age conspicuously modifies hippocampal neurogenesis. This is epitomized by both increased addition of new neurons to the granule cell layer (GCL) by neural stem/progenitor cells (NSCs) in the dentate subgranular zone (SGZ), and greatly enhanced numbers of newly born neurons located abnormally in the dentate hilus (DH). Interestingly, AS activity in old age does not induce such changes in hippocampal neurogenesis. However, the effect of AS activity on neurogenesis in the middle-aged hippocampus is yet to be elucidated. We examined hippocampal neurogenesis in middle-aged F344 rats after a continuous AS activity for >4 hrs, induced through graded intraperitoneal injections of the kainic acid. We labeled newly born cells via daily intraperitoneal injections of the 5'-bromodeoxyuridine (BrdU) for 12 days, commencing from the day of induction of AS activity. AS activity enhanced the addition of newly born BrdU+ cells by 5.6 fold and newly born neurons (expressing both BrdU and doublecortin [DCX]) by 2.2 fold to the SGZ-GCL. Measurement of the total number of DCX+ newly born neurons also revealed a similar trend. Furthermore, AS activity increased DCX+ newly born neurons located ectopically in the DH (2.7 fold increase and 17% of total newly born neurons). This rate of ectopic migration is however considerably less than what was observed earlier for the young adult hippocampus after similar AS activity. Thus, the plasticity of hippocampal neurogenesis to AS activity in middle age is closer to its response observed in the young adult age. However, the extent of abnormal migration of newly born neurons into the DH is less than that of the young adult hippocampus after similar AS activity. These results also point out a highly divergent response of neurogenesis to AS activity between middle age and old age.     

Aldose reductase deficiency protects sugar-induced lens opacification in rats

Aldose reductase (AKR1B1), which catalyzes the reduction of glucose to sorbitol and lipid aldehydes to lipid alcohols, has been shown to be involved in secondary diabetic complications including cataractogenesis. Rats have high levels of AKR1B1 in lenses and readily develop diabetic cataracts, whereas mice have very low levels of AKR1B1 in their lenses and are not susceptible to hyperglycemic cataracts. Studies with transgenic mice that over-express AKR1B1 indicate that it is the key protein for the development of diabetic complications including diabetic cataract. However, no such studies were performed in genetically altered AKR1B1 rats. Hence, we developed siRNA-based AKR1B1 knockdown rats (ARKO) using the AKR1B1-siRNA-pSuper vector construct. Genotyping analysis suggested that more than 90% of AKR1B1 was knocked down in the littermates. Interestingly, all the male animals were born dead and only 3 female rats survived. Furthermore, all 3 female animals were not able to give birth to F1 generation. Hence, we could not establish an AKR1B1 rat knockdown colony. However, we examined the effect of AKR1B1 knockdown on sugar-induced lens opacification in ex vivo. Our results indicate that rat lenses obtained from AKR1B1 knockdown rats were resistant to high glucose-induced lens opacification as compared to wild-type (WT) rat lenses. Biochemical analysis of lens homogenates showed that the AKR1B1 activity and sorbitol levels were significantly lower in sugar-treated AKR1B1 knockdown rat lenses as compared to WT rat lenses treated with 50mM glucose. Our results thus confirmed the significance of AKR1B1 in the mediation of sugar-induced lens opacification and indicate the use of AKR1B1 inhibitors in the prevention of cataractogenesis.     

Gender differences in myosin heavy chain-beta and phosphorylated phospholamban in diabetic rat hearts

The objective of this study was to determine whether a gender difference exists in myosin heavy chain (MHC) isoform or sarcoplasmic reticulum protein levels in diabetic rat hearts. As is the case with normal rodent hearts, all four chambers of the control rat hearts expressed almost 100% MHC-alpha. In 6-wk diabetic rats, MHC-beta expression in ventricles of males was significantly greater (78 +/- 7%) than in females (50 +/- 5%). The cardiac sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) protein level was decreased and the phospholamban (PLB) protein level was increased in the left ventricle of diabetic rats, but there was no difference between male and female diabetic rats. The phosphorylated PLB level was decreased more in male than in female diabetic rats. Insulin treatment completely normalized blood glucose level, cardiac SERCA2a and PLB protein levels, and the decrease in MHC-beta levels in both male and female diabetic rats. Insulin treatment completely normalized serum insulin and almost completely normalized phosphorylation of PLB at serine 16 in male diabetic rats. Although insulin treatment completely normalized serum insulin levels in male diabetic rats, in females it only partially normalized serum insulin levels. Also, insulin treatment almost completely normalized phosphorylation of PLB at threonine 17 in female diabetic rats; however, the increase was significantly greater than that identified for insulin-treated male diabetic rats. We conclude that higher levels of MHC-beta and dephosphorylated PLB may contribute to more contractile dysfunction in male than in female diabetic rat hearts, and that phosphorylation of PLB at threonine 17 is more responsive to insulin in female diabetic rat hearts.     

Brown and white adipose tissue lipid composition in three successive progenies of rats: Effects of ethanol exposure

The effect of ethanol exposure on the fatty acid composition of brown and white adipose tissue in three successive rat progenies at the end of an experimental period (24 weeks) was studied. Ethanol‐treated rats received a standard rat chow diet and 5, 10 and 15% ethanol in the ad libitum drinking fluid over 3 successive weeks. Then a concentration of 20% ethanol was maintained for 5 additional weeks up to the end of the experimental period. The males and females in the ethanol treated group were mated to obtain the 1st generation of offspring. Then female and male rats from the 1st generation were mated to obtain the 2nd generation. Finally, males and females from the 2nd generation were mated to obtain the 3rd generation of ethanol treated rats. Another group served as control and received only water and a standard rat chow diet. The control group was handled in the same way as the other experimental groups. In the 1st and 2nd generations the percentage of stearic acid (18:0) decreased and palmitoleic (16:ln7) and oleic acid (18:ln9) increased in both adipose tissues of ethanol‐treated rats with respect to control. Additionally, n‐3 and n‐6 series were reduced both in brown and white adipose tissues. In the 3rd generation the fatty acid composition of the white adipose tissue was similar to that of control rats. Thus, no significant difference in essential fatty acids and oleic acid (18:ln9) were found. However, the fatty acid composition of the brown adipose tissue, in the 3rd generation, was similar to that observed in the 1st and 2nd generation. Thus, a decrease in essential fatty acids and an increase in oleic acid (18:ln9) was found. This suggests adaptation to ethanol consumption during successive progenies in white adipose tissue. However, in brown adipose tissue the values indicate a triglyceride storing during the thermogenesis, which is more important to newborns.     

Nature of the regions involved in the insertion of newly synthesized protein into the outer membrane of Escherichia coli.

Outer membrane proteins are synthesized by cytoplasmic membrane-bound polysomes, and inserted at insertion sites which cover about 10% of the total outer membrane when cells grow with a generation time of 1 h. A membrane fraction enriched in outer membrane insertion regions was isolated and partly characterized. The rat at which newly inserted proteins are transferred from such insertion regions into the rest of the outer membrane was found to be very fast; the new protein content of insertion regions and that of the remaining outer membrane equilibrate completely within about 20 s at 25 degrees C. Given the rather rigid structure of the outer membrane and the multiple interactions between outer membrane components and the murein layer, lateral diffusion of newly inserted proteins from insertion sites to the remaining outer membrane is not likely to explain this rapid equilibration. Instead, the data support a model in which insertion regions move along the cell surface, leaving behind stationary, newly inserted outer membrane proteins.     

Atypical miniature end plate currents in neuromuscular synapses of the rat under normal conditions, after acetylcholinesterase inhibition and cooling

In the end-plates of rat diaphragm among atypical miniature end-plate currents (MEPCs) 2.9% were giant and 5.1% were slowly rising. The frequency of the giant MEPCs was decreased when temperature was lowered and increased when acetylcholinesterase (AChE) was inhibited; the latter effect was reversed if d-tubocurarine was added. Frequency of the slowly rising MEPCs changed insignificantly by all conditions. It is suggested that a highly temperature-dependent presynaptic mechanism of giant MEPC generation does exist which is activated by acetylcholine (ACh). Data about changes in the time course of the slowly rising MEPCs by AChE inhibition and lowering of temperature make it possible to suggest that the slowly rising MEPCs may be accounted for either slow release of ACh quanta or release of quanta on large distances from synaptic cleft and postsynaptic cholinoreceptors. The latter is possible if ACh quanta are released from synaptic Schwann cell to periaxonial space.     

Rotenone model of Parkinson disease: multiple brain mitochondria dysfunctions after short term systemic rotenone intoxication

Chronic infusion of rotenone (Rot) to Lewis rats reproduces many features of Parkinson disease. Rot (3 mg/kg/day) was infused subcutaneously to male Lewis rats for 6 days using Alzet minipumps. Control rats received the vehicle only. Presence of 0.1% bovine serum albumin during the isolation procedure completely removed rotenone bound to the mitochondria. Therefore all functional changes observed were aftereffects of rotenone toxicity in vivo. In Rot rat brain mitochondria (Rot-RBM) there was a 30-40% inhibition of respiration in State 3 and State 3U with Complex I (Co-I) substrates and succinate. Rot did not affect the State 4Deltapsi of RBM and rat liver mitochondria (RLM). However, Rot-RBM required two times less Ca2+ to initiate permeability transition (mPT). There was a 2-fold increase in O*2- or H2O2 generation in Rot-RBM oxidizing glutamate. Rot infusion affected RLM little. Our results show that in RBM, the major site of reactive oxygen species generation with glutamate or succinate is Co-I. We also found that Co-II generates substantial amounts of reactive oxygen species that increased 2-fold in the Rot-RBM. Our data suggest that the primary mechanism of the Rot toxic effect on RBM consists in a significant increase of O*2- generation that causes damage to Co-I and Co-II, presumably at the level of 4Fe-4S clusters. Decreased respiratory activity diminishes resistance of RBM to Ca2+ and thus increases probability of mPT and apoptotic cell death. We suggest that the damage to Co-I and Co-II shifts O*2- generation from the CoQ10 sites to more proximal sites, such as flavines, and makes it independent of the RBM functional state.     

Evidence of Cannibalism in Macrolophus pygmaeus, a Natural Enemy of Whiteflies

Cannibalism and the effects of host plant, sex, time and food resources on its expression were studied for the zoophagous mirid Macrolophus pygmaeus Wagner (Hemiptera: Miridae). Cannibalistic behaviour was studied by offering 5 conspecific larvae (first instar) to newly emerging adults. Four treatments were studied: without water, with water only, with a host plant (tobacco) and with both a host plant and prey (eggs of Ephestia kuehniella). Cannibalism was observed in all treatments. In the “host plant + eggs of E. kuehniella” treatment, very few individuals displayed cannibalistic behaviour. The proportion of cannibalism was only reduced when eggs of E. kuehniella were offered. Water (free or via a host plant) was very important for both survival and feeding. The cannibalistic behaviour of M. pygmaeus should be taken into account when planning a release strategy in the context of biological control.     

Two cases of spontaneous malignant fibrous histiocytoma in Wistar rats

Spontaneous malignant fibrous histiocytoma (MFH) of subcutaneous tissues was found in 2 Wistar rats of a 18-month-old male and a 21-month-old female. These tumors consisted of spindle, oval, myxoid and giant cells. In the female rat, a similar histological pattern was found in the metastatic tumor nodules in the lung and liver. In the electron microscopy, four differentiated cells of fibroblast-like, histiocyte-like, giant and undifferentiated cells were identified in these tumors. Spontaneous MFH of Wistar rats in the present study was quite similar to those reported in rats of other strains.     

P50 values in hypoxic albino rats of first and second generation.

We determined the "in vivo" (arterial pH and PCO2) and standard (pH = 7.4, PCO2 = 40 mm Hg) PO2 at 50% O2 saturation of hemoglobin (P50, vv and P50, st) in Wistar albino rats when living in a normobaric hypoxic environment. Two generations of hypoxic rats were observed for changes in their P50, vv, P50, st, (n50) 2,3-diphosphoglycerate (2,3-DPG), hemoglobin (Hb) and DPG-Hb ratio: the first generation (H1) and the second generation (H2). A few hours after birth, the H1 rats were placed and raised in a normobaric hypoxic environment (10% O2 in N2). The H2 rats were born from hypoxic parents of first generation and were raised in the same hypoxic environment. The control group had a normoxic environment. The P50, st was significantly higher in H1 rats than both H2 and controls. P50, st was similar in H2 and control rats. The P50, vv was significantly higher in H1 rats than both H2 and controls but it was significantly lower in H2 when compared with both controls and H1. Hb and 2,3-DPG had values significantly greater for both H1 and H2 when compared with their controls. However, the values of H2 were significantly lower than H1. The effectiveness of an increase in Hb-O2 affinity as an adaptive mechanism in H2 rats is discussed.