Preservation of visual responsiveness in the superior colliculus of RCS rats after retinal pigment epithelium cell transplantation

The dystrophic RCS rat undergoes progressive photoreceptor degeneration due to a primary defect in retinal pigment epithelial (RPE) cells. This has a major impact on central visual responsiveness. Here we have examined how functional deterioration is contained by subretinal transplantation of immortalized human RPE cells. Transplantation was done at three to four weeks of age prior to significant photoreceptor loss and recipients were kept on cyclosporin. At six months of age, sensitivity maps and multi-unit response properties were obtained across the visual field by recording at 76 equidistant sites encompassing the whole superior colliculus.A significant degree of functional protection, both in terms of area of responsive retina and response characteristics was observed following RPE transplantation. At best, the sensitivity, latency of onset, and response rise time were all maintained within normal ranges and this was achieved with no more than half of the normal complement of photoreceptors. Although partial, the degree of anatomical preservation (both in terms of outer nuclear layer thickness and area of rescue) correlated well with the level of preserved visual sensitivities. Sham injections also resulted in rescue, though the area of preservation was strictly confined to the needle injury site and the response properties were significantly worse than with RPE injections.This study shows that central physiological responsiveness and correlated retinal morphology can be preserved in an animal model of retinal disease by implantation of an immortalized cell line. The use of retinal sensitivity measurements provides a background for assessing higher visual functions in these animals and a direct comparison for human perimetry measures.     

大鼠冻存卵巢组织自体异位移植后长期功能评估

目的 评估成年大鼠冻存卵巢组织自体异位移植后的长期生殖、内分泌功能. 方法 78只Wistar成年大鼠,随机分为假手术组(1组,15只)、新鲜组织移植组(2组,24只)、冻存组织移植组(3组,24只)、去势组(4组,15只).将去势后成年大鼠的新鲜卵巢组织及冻存卵巢组织自体异位移植于肾被膜下,分别于移植后5、8、10个月处死各处理组1/3数量动物,取卵巢及子宫组织作组织学检查.通过阴道脱落细胞学检查、动情期雌、孕激素水平监测评估移植后卵巢组织的生殖内分泌功能. 结果 所有动物模型中,新鲜及冻存卵巢组织移植后都可观察到存活组织块.2、3组动情期雌、孕激素水平与假手术组无显著性差异(P>0.05),且均明显高于去势组(P<0.01).冷冻复苏后组织与新鲜组织各级卵泡构成比无显著性差异(P>0.05),各组不同时间取得的卵巢组织各级卵泡构成比相比较均无显著性差异(P>0.05).移植后5个月,2、3组原始卵泡明显减少,分别为假手术组的59.1%和54.5%. 结论 小块卵巢组织可耐受冻融过程;冻存卵巢组织自体肾被膜下移植后,卵巢组织形态无明显改变,原始卵泡总数虽然明显减少,但有成熟卵泡发育并排卵,并能长期维持生殖内分泌功能.     

基因转染的虹膜色素上皮细胞移植后RCS鼠视网膜BDNF表达观察

目的探讨脑源性神经营养因子（brain-derived neurotrophic factor,BDNF）基因转染的虹膜色素上皮细胞（AAV-BDNF-IPE）移植入皇家外科学院（royal college of surgeons,RCS）大鼠视网膜下腔后,不同时期视网膜组织BDNF表达变化。方法通过外路途径将BDNF基因转染的虹膜色素上皮细胞移植到RCS大鼠视网膜下腔,术后3、5、7、9、11周分别取RCS大鼠手术眼及对照组动物眼视网膜组织,用酶联免疫吸附法（Elisa）检测视网膜组织中BDNF的表达水平,比较分析这些数据。结果对照组RCS大鼠出生后3周龄时视网膜组织中BDNF仍保持较高水平,其后迅速降低,其中3周龄组与其它周龄组比较,P〈0.01;手术组RCS大鼠术时、术后3、5、7、9、11周各组间两两比较,BDNF表达无显著差异（P〉0.05）;出生后6周龄直到14周龄的不同时期,AAV-BDNF-IPE移植手术组RCS大鼠视网膜BDNF表达水平均明显高于对照组（其中6周龄组P〈0.05,其它各周龄组P〈0.01）。结论 BDNF基因转染的虹膜色素上皮细胞在RCS大鼠视网膜下腔移植后,视网膜组织中BDNF可以持续稳定高水平表达,这为临床开发新的神经营养因子给药方式提供了实验依据。     

Caspase-9抑制剂对RCS大鼠感光细胞凋亡抑制作用的研究

目的：采用Z-LEHD-FMK进行体内实验，观察caspase-9抑制剂对RCS大鼠感光细胞凋亡的抑制作用。方法：32只 18 d RCS大鼠随机分4组，检查ERG后随机选择一眼为实验眼给予玻璃体内注射Z-LEHD-FMK 4 μg，对侧眼给予4 μg 2%DMSO作对照。各组分别在术后2 d、7 d、12 d、17 d行ERG检查，然后摘取双眼球，石蜡切片行HE染色及感光细胞凋亡的TUNEL检测，透射电镜观察视网膜超微结构。结果：实验眼注药后 7 d ERG b波振幅达到最大(137.35±7.41)mV，17 d时b波振幅为(57.91±9.27)mV，对照眼7 d及以后各组ERG接近熄灭型；实验眼注药后12 d视网膜外颗粒层才开始出现凋亡阳性细胞，17 d更明显，对照眼强荧光的凋亡阳性细胞在术后7 d已经很明显；光镜下注药后17 d实验眼感光细胞外颗粒层细胞数尚保持有7-8层，对照眼仅余下2-4层细胞，视网膜厚度变薄；透射电镜下实验眼注药后17 d可见部分感光细胞胞核、核仁固缩，对照眼从术后 7 d 开始见感光细胞呈现凋亡改变。结论：Z-LEHD-FMK能够延缓RCS大鼠感光细胞凋亡，合适的caspases抑制剂在适宜的时机应用对视网膜感光细胞凋亡具有一定的抑制作用。     

骨髓间充质干细胞调节心脏移植大鼠的免疫功能

BACKGROUND:Heart transplantation is an effective method for treatment of end-stage heart failure, but immune rejection that seriously impact therapeutic effacicy is easy to occur after transplantation. OBJECTIVE:To investigate the regulatory effect of bone marrow mesenchymal stem cells on the immune function of rats undergoiong heart transplantation. METHODS:Twenty Lewis rats were enrolled as donors, and 20 Wistar rats as recipients. Heart transplantation models were established in the Wistar rats. These 20 model rats were randomized into cell transplantation and control group with 10 rats in each group. Forty-eight hours after heart transplantation, rats in the cell transplantation group were given bone marrow mesenchymal stem cell suspension (1 mL, 2×10⁸ cells/L) via the tail vein, while rats in the control group were given normal saline in the same dose. Then, the expression levels of serum interleukin-2, interleukin-10 and percentage of CD4⁺, CD8⁺, CD4⁺/CD8⁺, CD4⁺CD25^high, CD4⁺CD25^high Foxp3⁺ T cells in the venous blood were detected in the two groups at 7 days after cell transplantation. Additionally, rat myocardial tissues were taken and observed pathologically. RESULTS AND CONCLUSION:The survival time of the cell transplantation group was significantly longer than that of the control group (P < 0.05). The expression level of interleukin-2 showed no significant difference between the two groups (P > 0.05), but the level of interleukin-10 in the cell transplantation group was significantly higher than that in the control group (P < 0.05). Compared with the control group, the percentage of CD4⁺/CD8⁺, CD4⁺CD25^high, CD4⁺CD25^high Foxp3⁺ and CD4⁺ T cells was significantly higher, and the percentage of CD8⁺ T cells was significantly lower in the cell transplantation group (P < 0.05). Histopathological findings showed that there were a small amount of infiltrated lymphocytes in the cell transplantation group with the presence of slight bleeding and edema, and these inflammatory reactions were milder than those in the control group. These findings indicate that bone marrow mesenchymal stem cell transplantation can effectively reduce the rejection in rats undergoing heart transplantation.     

Long-term preservation of antigenicity on tissue microarrays

Tissue microarrays have facilitated the evaluation of large cohort studies; however, there is little data on the best method for preserving sections once they are cut. We assessed three methods of storing precut breast cancer microarray slides: paraffin coating and storage in a nitrogen desiccator, either alone or in combination. We tested the durability of three antigens, cytokeratin, estrogen receptor, and Ki-67 on microarrays stored under these conditions for 3 months at room temperature. Staining was assessed with both manual scoring using traditional brown stain (0-3+) as well as automated scoring using fluorescently stained sections. Staining intensity was compared to that obtained from freshly cut slides. Slides stored under ambient conditions (room temperature and air) for 3 months exhibited marked degradation of all target antigens, in some cases resulting in slides that were virtually unreadable. We found that combined paraffin coating and nitrogen storage resulted in the best preservation of antigenicity, with retention of 72-99% of the antigenicity of a freshly cut slide, depending upon the marker and detection system used. The use of either paraffin coating or nitrogen storage alone protected slides to a lesser degree.     

Aberrant T-cell function in vitro and impaired T-cell dependent antibody response in vivo in vitamin A-deficient rats.

We have previously reported that vitamin A deficiency resulted in a reduced IgA antibody response to cholera toxin (CT) after per-oral immunization. In the present investigation we have studied the in vivo and in vitro immune response in vitamin A-deficient rats to two parenterally applied antigens, beta-lactoglobulin (beta-LG) and picrylsulphonic acid (TNP)-Ficoll. The serum IgG and IgM antibody responses to the T-cell dependent antigen beta-LG were significantly lower in the vitamin A-deficient rats than in the pair-fed control rats. No such differences were seen with the IgG and IgM responses to the T-cell independent antigen TNP-Ficoll. However, the biliary IgA and the serum IgE antibodies against both antigens were decreased in the vitamin A-deficient rats. In vitro lymphocyte stimulation with concanavalin A (Con A) or beta-LG gave higher T-cell proliferation rates in the vitamin A-deficient than in the control rats. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) levels in supernatants from Con A-stimulated mesenteric lymph node cells were also higher in the vitamin A-deficient rats, while IL-6 levels were decreased, which is consistent with an up-regulated Th1 activity. Proliferation studies on purified accessory cells and T cells from the deficient and the control rats, mixed in different combinations, showed that the T cells, but not the accessory cells, were disturbed in the vitamin A-deficient rats. Despite the increased T-cell activity in vitro the vitamin A-deficient rats had a lower delayed-type hypersensitivity (DTH) reaction than the pair-fed control rats. In conclusion, the increased IL-2 and IFN-gamma levels may reflect an up-regulation of Th1 cell function, while the decreased IgA, IgE and IL-6 levels indicate a suppression of Th2 cells. The disturbed T-lymphocyte function is manifested in vivo as a decreased DTH reaction and suppressed antibody production, the latter possibly due to a lack of B-cell switching and proliferation factors in vitamin A-deficient rats.     

Differential modulation of host macrophage function by AK-5 tumor is dependent on the site of tumor transplantation

We have studied the mechanisms involved in the activation or suppression of the immune response by the tumor cells, which is also dependent on the site of tumor transplantation. AK-5 tumor cells when transplanted s.c. activated peritoneal macrophages through the induction of different cytokines, whereas tumor cells when transplanted i.p. suppressed the activity of macrophages as well as the production of cytokines. Suppression of macrophage function was also observed when they were cocultured with tumor cells in vitro. Our studies suggest the capability of AK-5 tumor in driving the immune response either towards activated status or towards immune suppression, depending on the site of transplantation. Activated macrophages produced free radicals, which were cytotoxic to the tumor cells.     

Restoration of visual orienting into a cortically blind hemifield by reversible deactivation of posterior parietal cortex or the superior colliculus

A contralateral hemineglect of the visual field can be induced by unilateral cooling deactivation of posterior middle suprasylvian (pMS) sulcal cortex of the posterior parietal region, and this neglect can be reversed by additional cooling deactivation of pMS cortex in the opposite hemisphere. The purpose of the present study was to test whether an enduring hemianopia induced by removal of all contiguous visual cortical areas of one hemisphere could be reversed by local cooling of pMS cortex in the opposite hemisphere. Two cats sustained large unilateral ablations of the contiguous visual areas, and cooling loops were placed in the pMS sulcus, and in contact with adjacent area 7 or posterior ectosylvian (PE) cortex of the opposite hemisphere. In both instances cooling of pMS cortex, but neither area 7 nor PE, restored a virtually normal level of orienting performance to stimuli presented anywhere in the previously hemianopic field. The reversal was highly sensitive to the extent of cooling deactivation. In a third cat, cooling deactivation of the superficial layers of the contralateral superior colliculus also restored orienting performance to a cortical ablation-induced hemianopia. This reversal was graded from center-to-periphery in a temperature-dependent manner. Neither the cortical ablation nor any of the cooling deactivations had any impact on an auditory detection and orienting task. The deactivations were localized and confirmed by reduced uptake of radiolabeled 2-deoxyglucose to be limited to the immediate vicinity of each cooling loop. The results are discussed in terms of excitation and disinhibition of visual circuits.     

State dependent activity in monkey visual cortex

Summary This study examined the extent to which the responses of single cells in the striate cortex (V1) and the extrastriate cortex (V4) of the alert rhesus monkey are modulated by visual stimuli whose relevance in a behavioral task is varied. The animal had to detect the repetition of a visual pattern (i.e. detect similarity), preceded by a randomized number of alternations between two different patterns. The responses produced by the last, reward contingent stimulus were compared with responses obtained to that same stimulus earlier in the sequence. Modulatory effects in V1 were moderate: 31% of the cells (63 of 200) showed response increments of 20% or more to the last, reward contingent stimulus. In V4 the effects were much more pronounced: 72% of the cells (110 of 154) showed modulatory effects of more than 20%. In V4 but not in V1 orientation tuning curves showed a significant narrowing as well as a peak response increment to the behaviorally salient stimulus, suggesting a feature specific mechanism associated with the detection of similarity. Although a response decrement was observed in many cells during the repeated alternations, this effect was significantly smaller than the modulation produced by the detection of similarity. Controls included the presentation of novel stimuli during the presentation sequence which did not produce an enhanced response. It is hypothesized that the feature specific effects reported here are produced by higher order feedback systems.     

State dependent activity in monkey visual cortex

Responses were recorded from isolated neurons in the visual cortex of rhesus monkeys while they performed an orientation match to sample task. In each trial the animal was first cued with randomly selected orientation, and then presented with a sequence of gratings whose orientations were randomly selected. The animal was required to release a switch when it saw a grating that matched the cued orientation. For some recordings the animal was given a tactile cue by having it feel the orientation of a grooved plate that it could not see. In other experiments the cue orientation was presented visually on the screen in front of the animal and then removed before the sequence of gratings was presented. Using this task it was possible to determine if a neuron's response to a particular orientation was affected by whether or not it was the orientation for which the animal was looking. Over half the neurons examined in V4 (110/192) responded differently to the visual stimuli when the animal was cued to look for different orientations. For some neurons responses to all stimuli were strong when the animal was cued to look for a particular orientation, but weak when the same stimuli were presented in trials where the animal had been cued to look for another orientation. This type of sensitivity was found in neurons recorded while the animal was given a tactile cue, and also in other neurons tested when a visual cue was used, suggesting that the activity was not of direct sensory origin. In support of this, neurons in V4 were not strongly affected when the animal felt the grooved plate while not performing the orientation matching task. The prevalence of behavioral effects that was found using the orientation matching task suggests that extraretinal signals represent a prominent component of the activity in V4 of the behaving monkey.     

Long-term group isolation is a factor of dysregulation of reproductive function in rats

In the condition of prolonged group sex starvation of rats variations were seen in behavioral reactions, fertility, extent of weight gain, litter, blood alpha-tocopherol level, eosinophil count, lipid peroxidation, evoked brain potentials. All these processes are the sign of stress development in rats. Thus, stress in mature rats influences litter and fertility, newborn rats had underdeveloped nervous system. Changes in the activity of endogenic antioxidant system in this experiment are discussed.     

Reversal-rate dependent differences in the EEG gamma-band during multistable visual perception.

It is an often reported observation in the literature on multistable perception that the reversal rate within a given observation time is subject to a high interindividual variability. Recently, we reported frontal gamma-band enhancement during multistable visual perception. The aim of the present study was to investigate whether changes in the gamma-band correspond to the variability of the reversal rate. Therefore, a total of 25 observers were divided into two subgroups according to their reversal rate during a 400-s observation period of a reversible pattern based on apparent motion. Subjects with more than 40 reversals within the 400-s were defined as high-rate switchers (HRS). Subjects with a reversal rate below 40 switches were defined as low-rate switchers (LRS). EEG was recorded from frontal, central, parietal, and occipital locations of both hemispheres. The results showed significantly higher gamma activity for the HRS in both phase-locked and non-phase-locked oscillations. Both subgroups showed the highest gamma amplitudes at frontal locations. The results support the involvement of attentional top-down processing in figure reversal. It is concluded that the higher gamma activity for the HRS reflects states of higher arousal, alertness and/or attention according to their fast reversal rate.     

Casein-induced experimental amyloidosis. IX. Alterations in marrow dependent function.

CBA/J mice receiving multiple injections of sodium caseinate (CAS) or bovine serum albumin (BSA) were assayed for marrow dependent functions by measuring their ability (i) to reject bone marrow allografts and (ii) to resist Friend virus (FV)-induced suppression of lymphocyte mitogenesis. Mice that developed amyloidosis following 25-30 injections completely lost the ability to reject allogeneic marrow cells, whereas nonamyloid BSA-treated mice had enhanced rejection of marrow allografts. There was increased resistance to the suppressive effects of FV in spleen cells from 'preamyloid' mice receiving CAS injections and nonamyloid mice receiving 10-40 BSA injections. Amyloid mice appeared to be as susceptible to the effects of FV-induced suppression as control (untreated) animals. These data indicate that alterations in marrow dependent function may be related to the pathogenesis of amyloid disease.     

Functional development of the visual system in normal and protein-deprived rats. IV. Latencies in the specific visual pathway of adult rats

It was recently shown that an increase in latency of the cortical visual evoked response (VER) seen in young malnourished animals persists in adult rats given a low protein diet. In the present paper the VER latencies of the specific visual pathway were measured in order to establish the level at which the latency increase occurs in protein-deprived rats. The VER in the dorsal lateral geniculate nucleus (dLGN) showed no significant differences to onset or peak latencies between control (C) and the protein-deprived (PD) rats. The dLGN activity was higher in C rats than in PD rats. Late components differed in median values between the two groups, but the individual variations were large. Generally, the VER of the dLGN in PD rats was more stereotype compared with the C rats. Intracortical VER at a depth of 0.4-0.5 mm showed a small negative component of short onset latency in both C and PD rats. This component preceded the onset of the initial positivity recorded from the cortical surface by 1-2 ms in C rats and by 3-6 ms in PD rats. Following electrical stimulation of the dLGN, no differences in onset latency of the first cortical activity (monosynaptic response) were recorded between C and PD rats, whereas later activity was significantly delayed in PD compared with C rats. The laminar potential pattern was less distinct in PD compared with C rats, and the late components of the evoked response from deep cortical layers were markedly attenuated or lacking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term preservation of human saphenous vein by green tea polyphenol under physiological conditions

Polyphenolic compounds are well known as a functional food with various bioactivities. However, less attention has been paid to the effect of phenolic antioxidants on the preservation of blood vessels. In this study, the possible effects of green tea polyphenolic compounds (GTPCs) on the longterm preservation of the human saphenous vein (HSV) were investigated under physiological conditions. HSV segments were pretreated with GTPCs (0.5 or 1.0 mg/mL) for 1 day and then incubated for 1, 2, 4, or 8 weeks. After incubation, cellular viability, endothelial nitric oxide synthase (eNOS) expression level, biomechanical properties, and vein histology were evaluated. When HSV segments were incubated without GTPC treatment, endothelial cell viability was significantly (p < 0.05) reduced with incubation time, and none of the endothelial cells expressed eNOS after 2 weeks. Furthermore, nontreated veins demonstrated appreciable inferiority in such mechanical properties as failure strength, elastic modulus, and compliance, compared with fresh veins. These results were confirmed by histological observations, which showed severe structural changes in nontreated veins. On the other hand, these phenomena were markedly prevented by preincubating veins with GTPCs (1.0 mg/mL) at 37 degrees C in a CO(2) incubator for 1 day. GTPC-pretreated veins could be preserved for at least 2 weeks under physiological conditions, retaining cellular viability and eNOS expression level and maintaining both biomechanical properties and vascular structures without any morphological alterations. These results demonstrate that GTPC treatment may be a useful method for preserving the HSV and could be exploited to craft strategies for the long-term preservation of other tissues under physiological conditions.