Evidence of apoptosis in chronic alcoholic skeletal myopathy

Apoptosis is a common mechanism of programmed cell death that has been implicated in the pathogenesis of alcohol-induced organ damage. Experimental studies have suggested alcohol-mediated apoptosis in cardiac muscle. The relationship between skeletal and cardiac muscle damage in alcoholism led us to consider the possible role of apoptosis in the pathogenesis of skeletal myopathy. We prospectively evaluated apoptosis in skeletal muscle biopsies of 30 consecutively selected male high-dose well-nourished chronic alcohol consumers and 12 nonalcoholic controls. Alcohol consumption, evaluation of muscle strength by myometry, and deltoid muscle biopsy with immunohistochemical and morphometric analysis were performed. Apoptosis was assessed by TUNEL, BAX, and BCL-2 immunohistochemical assays. Chronic alcoholics compared with controls showed a significantly higher apoptotic index in TUNEL (2.35% +/- 0.25% versus 0.18% +/- 0.03%, P < 0.001), BAX (9.16% +/- 2.00% versus 0.66% +/- 0.22%, P < 0.001), and BCL-2 muscle assays (8.08% +/- 0.20% versus 0.83% +/- 0.20%, P = 0.001), respectively. In addition, these apoptotic indexes were higher in alcoholics with skeletal myopathy compared with in those without skeletal myopathy (3.04% +/- 0.36% versus 1.65% +/- 0.26%, P = 0.004 for TUNEL; 17.00% +/- 2.78% versus 1.33% +/- 0.22%, P < 0.001 for BAX; and 15.13% +/- 3.2% versus 1.03% +/- 0.33%, P < 0.001 for BCL-2 assays, respectively). We conclude that apoptosis is present in the skeletal muscle of high-dose alcohol consumers, mainly in those affected by myopathy. However, the specific pathogenic mechanism of apoptosis in chronic skeletal myopathy in alcoholics remains to be elucidated.     

Evidence for apoptosis in human atherogenesis and in a rat vascular injury model.

Apoptosis is a physiological cell death process important for normal development and involved in many pathological conditions. In atherosclerosis, pathological accumulation of cells in the intima has been attributed to the migration and proliferation of smooth muscle cells, macrophages, and lymphocytes. In this report, we explored the possibility that apoptosis may also contribute to the pathogenesis of this disease. We examined 35 human atherosclerotic lesion samples and identified a substantial number of cells undergoing apoptosis in 25 of the samples. Furthermore, in a rat vascular injury model, apoptotic cells were specifically identified in the neointima. The presence of apoptotic cells was demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, nuclear staining with propidium iodide, and electron microscopy. Immunostaining with cell-type-specific markers and subsequent terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling analysis on the same sample revealed that the majority of the apoptotic cells were modulated smooth muscle cells as well as macrophages. These results indicate that apoptosis occurs in cells of the injured blood vessel as well as the advanced atherosclerotic lesion and that physiological cell death may have an important role in determining the course of atherogenesis.     

结直肠腺瘤及癌变组织CHOP蛋白表达与细胞凋亡的相关性研究

目的: 检测和探讨结直肠腺瘤及癌变组织C/EBP同源蛋白(CHOP)表达和细胞凋亡及其与临床病理的关系,并分析CHOP与细胞凋亡指数(AI)的相关性。方法: 采用免疫组织化学SABC法和原位末端标记法分别检测59 例正常肠黏膜、67 例结直肠腺瘤和56 例结直肠腺癌组织CHOP表达及细胞凋亡。结果: CHOP在伴高级别上皮内瘤变和恶变的腺瘤和腺癌组织阳性表达率均显著高于正常肠黏膜和早期腺瘤(P<0.05),且与腺瘤大小、病理类型和肠癌大小、浸润深度相关(P<0.05);伴高级别上皮内瘤变和恶变的腺瘤和腺癌组织AI显著高于正常肠黏膜和早期腺瘤(P<0.05),AI与腺瘤大小、病理类型和肠癌大小有关(P<0.05);伴高级别上皮内瘤变和恶变的腺瘤和腺癌组织CHOP阳性表达者AI显著高于阴性表达者(P<0.05),在具有某相同临床病理特征的腺瘤和肠癌组织中CHOP阳性表达者AI显著高于CHOP阴性表达者(P<0.05)。结论: CHOP和细胞凋亡异常共同参与结直肠腺瘤癌变,CHOP表达与细胞凋亡增加有关,CHOP可能通过促细胞凋亡介导腺瘤癌变。     

纳秒级陡脉冲可用于治疗裸鼠人恶性黑色素瘤

 摘要 目的:观察纳秒级陡脉冲(Nanosecond Pulsed Electric Fields, nsPEF)治疗裸鼠皮下人恶性黑色素瘤的效果及其机制。方法：建立裸鼠皮下人黑色素瘤模型,随机分为长期治疗组及对照组各10只,观察肿瘤生长、裸鼠存活率时间,和短期治疗组(=10;分2h、4d组及对照组(分别为4、6和6只),以HE染色检查细胞形态,DNA琼脂糖凝胶电泳、TUNEL法分析细胞凋亡,Western blot法、免疫组织化学方法分析组织肿瘤Bax、Bcl-2的表达。两组均用电场峰值40kV/cm、脉冲宽度200ns、频率1Hz、脉冲次数1000个的nsPEF进行治疗。结果：长期治疗后即刻看到肿瘤区均变为灰白色,表面皮肤均没有出血现象,肿瘤质地变硬;随生存时间延长,肿瘤体积较对照组明显缩小(P<0.01);平均生存时间（天）较对照组（天）明显延长(P<0.01);短期治疗组,4d时坏死区域增多;4d时琼脂糖凝胶电泳显示较对照组有明显“ladder”状分布,凋亡率明显高于对照组(P<0.01),较对照组相比Bax表达明显增高(P<0.01)、而Bcl-2表达明显降低(P<0.01)。结论：nsPEF对裸鼠皮下人黑色素瘤有明显的治疗效果,可能是通过调控Bax、Bcl-2 基因表达诱导细胞凋亡。     

葛根素减轻乙醇导致大鼠生精细胞的凋亡

目的 观察乙醇导致大鼠生精细胞的凋亡及葛根素的干预。方法 将大鼠30 只,随机均分为对照组、乙醇组及葛根素干预组。于实验第40天免疫组织化学法（SABC）检测左侧睾丸各组Bcl-2、Bax 蛋白在生精细胞的表达; RT-PCR检测右侧睾丸各组Bcl-2及Bax mRNA的表达;TUNEL 法检测生精细胞的凋亡。结果 醇组平均每个生精小管断面中Bcl-2 蛋白阳性细胞数和A值低于对照组(P<0.01),而平均每个生精小管断面中Bax 蛋白的阳性细胞数和A值高于对照组(P<0.01);乙醇组Bax mRNA表达较葛根素干预组及对照组强（P<0.05）,而Bcl-2 mRNA表达较葛根素干预组及对照组弱（P<0.05）;乙醇组每个生精小管横切面中的凋亡细胞数目高于对照组(P< 0.01),葛根素干预组显著缓解上述变化。结论 根素对乙醇导致的大鼠生精细胞凋亡有干预作用。     

Caspase-3 activation in the guinea pig cochlea exposed to salicylate

In the current study, we explored whether chronic salicylate exposure could induce apoptosis in outer hair cells (OHCs) and spiral ganglion neurons (SGNs) of the cochlea. Guinea pig received sodium salicylate (400 mg/kg/d) or saline vehicle for 10 consecutive days. Programmed cell death (PCD) executioner was evaluated with immunohistochemistry detection of activated caspase-3. Apoptosis was examined with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Repeated salicylate administration activated caspase-3 and caused apoptosis in OHCs and SGNs (p < 0.01 vs. saline control for both measures and in both cell types). Cell counting showed a significant loss in OHCs (p < 0.01 vs. saline control), but not in inner hair cells (IHCs). Transmission electron microscopy (TEM) revealed chromatin condensation and nucleus margination in salicylate-treated cochlea. Scanning electron microscopy (SEM) demonstrated stereociliary bundles breakdown and fusion at the apical of OHCs, villous matter was discovered to attach on the surface of SGNs. These findings suggest that long-term administration of high-dose salicylate can activate caspase-3 pathway to induce OHC and SGN apoptosis.     

The potential role of BAX and BCL-2 expression in diffuse alveolar damage.

Apoptosis of type II pneumocytes has been identified in diffuse alveolar damage (DAD), is associated with p53 and WAF1 expression, and may be of pathogenetic importance. BAX, a homologue of BCL-2, is induced by p53 and is a promoter of apoptosis. The proapoptotic effect of BAX is negatively regulated by its binding with BCL-2. In this study, we sought to investigate that role of BAX and BCL-2 in DAD. We hypothesized that alterations in BAX and BCL-2 expression may be important in determining the susceptibility of type II pneumocytes and interstitial cells to apoptosis. Twenty-eight cases of DAD and 16 control cases (i.e., lung tissues adjacent to resected tumors) were retrieved from the files of the University of Utah and the Armed Forces Institute of Pathology. Immunohistochemical stains were performed with antigen retrieval by microwave using antibodies recognizing BAX and BCL-2. The percentage of positively staining pneumocytes and interstitial cells was estimated in each case to the nearest 10%. BAX expression was markedly increased in pneumocytes and interstitial cells in DAD compared with control lung tissues. In DAD, BAX was identified on an average of 80% of alveolar pneumocytes (range 30 to 100%) and 70% of interstitial cells (range 20 to 90%). In control lungs, BAX was identified on an average of 10% of pneumocytes (range 0 to 20%) but not in interstitial cells. Focal BCL-2 staining was identified in interstitial myofibroblasts in 7 of 25 cases of DAD but was only identified in bronchiolar epithelium of control lungs. These results suggest that the induction of BAX in DAD may enhance the susceptibility of alveolar epithelial cells to apoptosis, whereas BCL-2 expression may contribute to the absence of apoptosis in interstitial myofibroblasts. Expression of BCL-2 in interstitial myofibroblasts may contribute to the development of pulmonary fibrosis in some patients.     

Overexpression of BCL-2, BCL-X, and BAX in primary central nervous system lymphomas that occur in immunosuppressed patients.

In contrast to primary central nervous system lymphomas (PCNSLs) that occur in immunocompetent patients, most of those that occur in immunosuppressed patients are associated with Epstein-Barr virus (EBV). BCL-2-related proteins either block or promote cell death, forming homo- or heterodimers with each other. LMP-1, EBV latent protein, has been shown to upregulate BCL-2 and BCL-XL. This observation suggests that these proteins may be involved in the transformation process of EBV-infected cells. Twenty-three cases of PCNSLs were studied: 12 of the patients were immunosuppressed, and 11 were immunocompetent. For all cases, we collected clinical information, histologic data, and immunophenotype and tested for the presence of EBV (EBER-1, LMP-1). Apoptosis was assessed by the TdT-mediated dUTP-biotin nick-end labeling method and quantified by image analysis. In three cases, electron microscopy was performed. The BCL-2 family proteins (BCL-2, BCL-X, MCL1, and BAX) and p53 expression were studied by immunohistochemistry on paraffin slides. All cases were classified as diffuse large B-cell lymphomas. PCNSLs in immunosuppressed patients were characterized by EBV association, necrosis, important gliosis, and numerous macrophages. There was no significant difference between the two groups regarding the TdT-mediated dUTP-biotin nick-end labeling staining (P = .08). In contrast, PCNSLs in immunosuppressed patients were shown to express high levels of BCL-2, BCL-X, and BAX in more than 80% of tumor cells in 7, 10, and 11 cases, respectively. In immunocompetent patients, only one case showed a high level of BCL-2 expression in more than 80% of the cells, whereas BCL-X and BAX were overexpressed in two cases. These differences are significant (P < .05). In contrast, there was no significant difference between the two groups in MCL-1 expression. Besides EBV association and necrosis, PCNSLs related to immunosuppression are characterized by an overexpression of BCL-2-related proteins, without dramatically modifying their susceptibility for apoptosis.     

大鼠肢体缺血再灌注后肺组织BAX基因表达上调

目的探讨在体大鼠肢体缺血再灌注(LIR)后肺组织BAX和BCL-2基因表达的变化。方法在大鼠肢体缺血再灌注(LIR)损伤动物模型上,用TUNEL法、电泳法及免疫组织化学等技术观察LIR后肺损伤发生过程中,肺组织细胞凋亡变化以及BAX和BCL-2蛋白质表达的改变。结果大鼠LIR后,肺血管内皮细胞及附壁的炎细胞凋亡明显增加;肺组织BCL-2表达的变化不大,但BAX蛋白质表达明显上调,DNA断链率升高,活性氧(ROS)含量增加,且与肺组织细胞凋亡的增加相一致。结论肺组织细胞凋亡以及BAX和BCL-2表达的变化可能参与LIR后肺损伤的发生。     

Apoptosis in the developing mouse heart.

Apoptosis occurs at high frequency in the myocardium of the developing avian cardiac outflow tract (OFT). Up- or down-regulating apoptosis results in defects resembling human conotruncal heart anomalies. This finding suggested that regulated levels of apoptosis are critical for normal morphogenesis of the four-chambered heart. Recent evidence supports an important role for hypoxia of the OFT myocardium in regulating cell death and vasculogenesis. The purpose of this study was to determine whether apoptosis in the outflow tract myocardium occurs in the mouse heart during developmental stages comparable to the avian heart and to determine whether differential hypoxia is also present at this site in the murine heart. Apoptosis was detected using a fluorescent vital dye, Lysotracker Red (LTR), in the OFT myocardium of the mouse starting at embryonic day (E) 12.5, peaking at E13.5-14.5, and declining thereafter to low or background levels by E18.5. In addition, high levels of apoptosis were detected in other cardiac regions, including the apices of the ventricles and along the interventricular sulcus. Apoptosis in the myocardium was detected by double-labeling with LTR and cardiomyocyte markers. Terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) and immunostaining for cleaved Caspase-3 were used to confirm the LTR results. At the peak of OFT apoptosis in the mouse, the OFT myocardium was relatively hypoxic, as indicated by specific and intense EF5 staining and HIF1alpha nuclear localization, and was surrounded by the developing vasculature as in the chicken embryo. These findings suggest that cardiomyocyte apoptosis is an evolutionarily conserved mechanism for normal morphogenesis of the outflow tract myocardium in avian and mammalian species.     

大鼠应激性胃黏膜损伤时细胞发生凋亡

目的探讨急性胃黏膜损伤时细胞凋亡形式及作用。方法制备大鼠水浸-束缚应激(WRS)模型,计算溃疡指数(UI);TUNEL法检测胃黏膜细胞凋亡;透射电镜观察细胞形态;免疫组化法检测BCl-2/BAX蛋白的表达。结果与正常大鼠比较,WRS后2h,黏膜损伤严重(P<0.01),凋亡细胞明显增多(P<0.01),BCl-2表达明显减弱(P<0.05),BAX表达明显增加(P<0.01);WRS后24h,黏膜修复,凋亡细胞仍高于正常水平(P<0.05),BCl-2表达基本恢复正常,BAX表达仍高于正常水平(P<0.05)。透射电镜下可见WRS组发生凋亡的细胞形态各异。结论细胞凋亡是急性胃黏膜损伤过程中细胞死亡的重要形式。     

Transurethral balloon laser thermotherapy induces long-lasting apoptosis of prostatic smooth muscle cells in human prostatic hyperplasia

Transurethral balloon laser thermotherapy (TUBAL-T) has been developed recently as a noninvasive treatment for benign prostatic hyperplasia (BPH), but the cellular events occurrmg after TUBAL-T were not yet fully understood. We examined prostate tissues by electron microscopy and the DNA nick end-labeling method at 2 and 4 weeks after a single TUBAL-T treatment in an attempt to elucidate the mechanism by which TUBAL-T progressively improves obstructive complications of patients with BPH. Condensation of chromatin at the nuclear margin was observed in prostatic smooth muscle cells (PSMCs) located in the area adjacent to periurethral coagulative necrosis. PSMCs with condensed chromatin were often accompanied by cytoplasmic vacuoles in perinuclear areas. Application of the nick end-labeling method at the light microscopic level revealed that most PSMC nuclei were positive for terminal TdT-mediated dUTP-dioxygenic nick end-labeling (TUNEL) staining. These results strongly suggested that long-standing apoptosis is induced in PSMCs by TUBAL-T. as well as massive coagulative necrosis of prostatic tissues. The role of apoptosis in PSMCs in the clinical improvement of outlet obstruction after TUBAL-T is discussed.     

Pathology of the thyroid in severe acute respiratory syndrome

The severe acute respiratory syndrome (SARS) epidemic started in November 2002 and spread worldwide. The pathological changes in several human organs of patients with SARS have been extensively described. However, to date, little has been reported about the effects of this infection on the thyroid gland. Femoral head necrosis and low serum triiodothyronine and thyroxine levels, commonly found in patients with SARS, raise the possibility of thyroid dysfunction. We have undertaken this study to evaluate for any potential injury to the thyroid gland caused by SARS on tissue samples obtained from 5 SARS autopsies. The terminal deoxynucleotidyl transferase-mediated dUPT nick end-labeling assay was performed to identify apoptotic cells. The follicular epithelium was found to be damaged with large numbers of cells exfoliated into the follicle. The terminal deoxynucleotidyl transferase-mediated dUPT nick end-labeling assay demonstrated many cells undergoing apoptosis. Follicular architecture was altered and showed distortion, dilatation, and collapse. No distinct calcitonin-positive cells were detectable in the SARS thyroids. In conclusion, both parafollicular and follicular cells were injured. This may provide an explanation both for low serum triiodothyronine and thyroxine levels and the osteonecrosis of the femoral head associated with patients with SARS. Apoptosis may play a role in the pathogenesis of SARS associated coronavirus infection in the thyroid gland.     

氯沙坦降低心力衰竭大鼠心肌细胞凋亡

目的 观察氯沙坦对心力衰竭大鼠心肌细胞凋亡的影响,方法 选8周龄雄性SD大鼠,分为对照组（C组）、心衰组（HF组）和治疗组（LS组）。采用腹腔注射阿霉素造模,LS组同时氯沙坦灌胃。透射电镜观察心肌超微结构, 检测血清中CPK、CK-MB及LDH的含量。用原位脱氧核糖核酸酶未端标记法（TUNEL法）检测大鼠心肌细胞凋亡,免疫组化法检测大鼠心肌Bax、Bcl-2蛋白的表达。结果 HF组与C比,心肌细胞损伤明显,并可见凋亡小体,血清中CPK、CK-MB和LDH升高,LS组与HF组比,心肌细胞凋亡指数下调(P<0.01),心肌细胞Bc1-2表达增加、Bax表达减少。结论 氯沙坦可有效抑制心力衰竭过程中发生的心肌细胞凋亡,逆转心肌损伤,改善心衰进程。     

The hippocampus in spontaneously hypertensive rats: a quantitative microanatomical study

The influence of hypertension on the morphology of hippocampus was assessed in spontaneously hypertensive rats of two, four and six months and in age-matched normotensive Wistar-Kyoto rats. Values of systolic pressure were slightly increased in two-month-old spontaneously hypertensive rats in comparison with age-matched Wistar-Kyoto rats and augmented progressively with age in spontaneously hypertensive rats. No microanatomical changes were observed in the hippocampus of spontaneously hypertensive rats of two months in comparison with age-matched Wistar-Kyoto rats, whereas a decrease of white matter volume was observed in the CA(1) subfield and in the dentate gyrus of four-month-old spontaneously hypertensive rats. In the hippocampus of six-month-old spontaneously hypertensive rats a reduction of grey matter volume both in the CA(1) subfield and in the dentate gyrus, a loss of neurons affecting to a greater extent the CA(1) subfield and an increase of glial fibrillary acid protein-immunoreactive astrocytes was found. The occurrence of apoptosis and/or necrosis identified using the terminal deoxyribonucleotidyl transferase-mediated biotin-16-dUTP nick end labelling technique was also observed in the CA(1) subfield and to a lesser extent in the dentate gyrus. The only change noticeable in the CA(3) subfield of six-month-old spontaneously hypertensive rats was a slight increase in the number of glial fibrillary acid protein-immunoreactive astrocytes.These findings indicate the occurrence of neuronal loss and of astrocyte changes in the hippocampus of spontaneously hypertensive rats of six months, being the CA(1) subfield the area most affected. The relevance of these neurodegenerative changes in hypertension and the possible occurrence of apoptosis and/or necrosis as expression of hypertensive brain damage is discussed.     

RNA干扰沉默Bax基因对线粒体途径软骨细胞凋亡的影响

 【摘要】 目的 利用体外培养的鼠软骨细胞,研究RNA干扰沉默Bax基因表达对经线粒体途径细胞凋亡的影响。方法 体外分离培养SD大鼠软骨细胞;Bax siRNA干扰沉默Bax基因表达。RT-PCR和Western blot检测mRNA及蛋白表达水平;MTT法检测细胞活力;Annexin V-FITC/PI双标记法检测细胞凋亡率;Western blot检测Bcl-2、Cytochrome C蛋白的表达。结果 Bax siRNA干扰24h后,Bax的mRNA和蛋白的表达水平均明显降低。细胞凋亡受到明显抑制,细胞存活率增高。并且,在Bax基因沉默的细胞中,Cytochrome C蛋白表达水平降低,同时Bcl-2蛋白表达水平升高。结论 RNA干扰沉默Bax基因可抑制鼠软骨细胞凋亡并且促进其存活,其机制可能与线粒体途径相关。     

Peroxynitrite generated in the rat spinal cord induces apoptotic cell death and activates caspase-3

We previously demonstrated that the peroxynitrite concentration increases after impact spinal cord injury. This study tests whether spinal cord injury-elevated peroxynitrite induces apoptotic cell death. Peroxynitrite was generated at the concentration and duration produced by spinal cord injury by administering S-morpholinosydnonimine through a microdialysis fiber into the gray matter of the rat spinal cord. Fragmented DNA was visualized by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling. Transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling-positive neurons were quantitated by counting the transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling and neuron-specific enolase double-stained neurons along the fiber track in the sections removed at 6, 12, 24 and 48 h post-peroxynitrite exposure. Peroxynitrite significantly increased transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling-positive neurons at all time points examined (P< or =0.001) compared with artificial cerebrospinal fluid controls (Two-way analysis of variance followed by Tukey test), peaking at 24 h post-exposure. Electron microscopic observation of characteristic features of apoptosis confirmed peroxynitrite-induced neuronal apoptosis. Total transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling-positive cells were counted in areas near and 0.2 mm away from the fiber track. The counts both peaked at 24 h with no significant difference between the two areas. However, at 6 and 12 h post-exposure the counts were significantly higher near than away from the fiber track (P=0.03 and P=0.007 respectively, paired t test). Immunohistochemical staining indicates caspase-3 was activated by peroxynitrite; this activation peaked at 6 h post-exposure, suggesting that activation of caspase-3 might be an early event in the apoptotic cell death cascade. We conclude that 1) peroxynitrite generated in the cord at the level produced by spinal cord injury induces neuronal apoptosis, indicating a role for peroxynitrite in secondary spinal cord injury; 2) caspase activation might be involved in peroxynitrite-induced neuronal apoptosis; 3) therefore removal of peroxynitrite should reduce secondary cell death after spinal cord injury.     

Molecular evidence for apoptosis in microfilariae of <Emphasis Type="Italic">Wuchereria bancrofti</Emphasis> induced by diethylcarbamazine

Apoptosis, a programmed cell death, is characterized by chromatin condensation, numerous vacuoles, reduction in cell volume, and endonuclease cleavage DNA degradation detected in gel electrophoresis as nucleosomal ladder. Here we report that diethylcarbamazine induces DNA fragmentation in microfilariae of Wuchereria bancrofti revealed by ligation-mediated polymerase chain reaction and by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling at the light and electron transmission level.     

FAS和FASL基因在大鼠胸腺细胞凋亡时的表达及其意义

目的检测FAS和FASL基因在Wistar大鼠胸腺细胞凋亡的表达,探讨细胞凋亡调控基因在大鼠胸腺细胞凋亡时的作用。方法采用原位标记(TUNEL)法检测DNA单链或双链的裂解;应用免疫组织化学染色法,观察不同剂量糖皮质激素诱导的大鼠胸腺细胞凋亡时FAS和FASL基因的表达。结果镜观察地塞米松组TUNEL阳性细胞较多并且分散在皮质各处。免疫组织化学结果表明,正常对照组胸腺内FAS呈低表达,随着地塞米松剂量的增加FAS的表达成递增趋势,地塞米松各组与对照组比较差异有统计学意义(P0.05);正常对照组胸腺内FASL呈高表达,随着地塞米松剂量的增加FASL的表达成递减趋势,地塞米松各组与对照组比较差异有统计学意义(P0.05)。结论促凋亡蛋白FAS和抗凋亡蛋白FASL在糖皮质激素诱导引起的胸腺细胞凋亡调控中起重要作用。     

Early signs of neuronal apoptosis in the substantia nigra pars compacta of the progressive neurodegenerative mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model of Parkinson's disease

Parkinson's disease is associated with a progressive loss of substantia nigra pars compacta dopaminergic neurons. The cellular and molecular mechanisms underlying Parkinson's disease neurodegeneration have not been fully determined. Clinical investigations and subacute in vivo studies using the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine have generated some observations suggesting that apoptosis is involved in neurodegeneration; however, this view remains equivocal. In this study, the substantia nigra pars compacta neurodegenerative process was examined in the chronic mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model of Parkinson's disease treated with 10 doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (25 mg/kg) and probenecid (250 mg/kg) over five weeks. One day after chronic treatment, numerous terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells were detected specifically in the substantia nigra pars compacta displaying shrunken volume, chromatin condensation, and DNA fragmentation. The number of apoptotic cells declined over time. No terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells were found in untreated or probenecid-treated control animals. Cytomorphometric analysis of substantia nigra pars compacta nuclear loci revealed eccentric nucleoli dislocation and vesicular degranulation in all of the apoptotic neurons for the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model for Parkinson's disease. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells phenotypically showed neuronal origin (NeuN-positive) with a loss of tyrosine hydroxylase immunoreactivity. While the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells were not co-localized with astroglial (GFAP-positive) cells, some apoptotic cells were clearly associated with the activated microglial (macrophage antigen complex-1 and isolectin B(4)-positive) cells suggesting an active process of dead cell removal. In the one-day and seven-day post-treated mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model for Parkinson's disease, marked depression of tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta and striatum was observed, which was correlated with significant reductions of striatal dopamine content and uptake. These results suggest that initial neuronal apoptosis and morphological changes are involved, at least in part, in the chronic neurodegeneration of mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model for Parkinson's disease.