Bax and Bcl-2 expression and TUNEL labeling in lumbar enlargement of neonatal rats after sciatic axotomy and melatonin treatment

Peripheral axotomy in neonatal rats induces neuronal death. We studied the anti-apoptotic protein Bcl-2 and cell death promoter Bax in spinal cord of neonatal rats after sciatic transection and treatment with melatonin, a neuroprotective substance. Pups were unilaterally axotomized at P2 and received melatonin (1 mg/kg; sc) or vehicle 1 h prior to lesion, immediately after, at 1 h, 2 h and then once daily. Rats were sacrificed at 3 h, 6 h, 24 h, 72 h and 5 days postaxotomy. Intact animals were used as controls. Lumbar enlargement was processed for Nissl staining, immunohistochemistry and RT-PCR for Bax or Bcl-2 and TUNEL reaction. Motoneurons (MN) of lesioned (L) and normal (N) sides were counted, and MN survival ratio (MSR=L/N) was calculated. Bax and Bcl-2 showed cytoplasmic immunoreactivity (IR). Bax IR was noticeable in small cells but less evident in MN. In unlesioned pups, some Bax-positive small cells (B+) and TUNEL-positive nuclei (T+) were mainly seen in the dorsal horn. In lesioned animals given vehicle, Bax mRNA levels and numbers of B+ and T+ were increased in comparison with intact controls at 24 h postaxotomy. The basal IR for Bax in MN was not changed by axotomy. Bcl-2 IR was noted in all cells and, like Bcl-2 mRNA, was unaltered after lesion. Melatonin reduced MN loss at 24 h, 72 h and 5 days and T+ at 24 h after lesion but did not interfere with Bax or Bcl-2 expression. These results suggest that (1) sciatic transection at P2 increases Bax mRNA and the amount of B+ and T+ in the lumbar enlargement, (2) Bax IR in immature MN is not altered by axotomy and (3) melatonin protects MN and dorsal horn cells through a mechanism independent of Bax and Bcl-2.     

Localization of mRNAs for six ARFs (ADP-ribosylation factors) in the brain of developing and adult rats and changes in the expression in the hypoglossal nucleus after its axotomy

ADP-ribosylation factors (ARFs) play crucial roles in the intracellular vesicular transport and in regulation of phospholipid-modifying enzyme activities and cytoskeletons. Using in situ hybridization histochemistry, the gene expression for six isoforms of ARF was examined during normal development of rats and in the hypoglossal nucleus following its axotomy. In the embryonic brain, the expression for ARF-1, -4, -5, -6 mRNAs was distinct in the ventricular germinal zone while that for ARF-3, -4, -5 in the mantle zone. In early postnatal brain, the expression for six ARFs was seen widely in various loci of the gray matter with different intensity, and the expression of ARF-4, -5, -6 mRNAs was evident in the cerebellar external granule cell layer. In the adult brain, the gene expression for all ARF isoforms decreased more or less in most gray matters and the distinct expression was maintained mainly in the hippocampal and dentate neuronal layers and cerebellar cortex. The expression levels of ARF-2 and -4 mRNAs in affected hypoglossal nucleus increased after 24 h up to 7 days following axotomy of the hypoglossal nerve, while no such changes were seen in the expression levels for the other ARFs. The present findings suggest that ARFs are differentially involved in some processes essential to nerve regeneration as well as neuronal differentiation and maturation.     

Increased expression of phosphotyrosine after axotomy in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus

To investigate the role of tyrosine kinase underlying glial cell proliferation after axotomy, the localization of phosphotyrosine was studied immunohistochemically in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus after nerve transection in adult rats. An anti-phosphotyrosine antibody weakly stained the cytoplasm of the neurons and some glial cells on the control side of both nuclei, while preferentially staining the plasma membrane of perineuronal microglial cells and neurons weakly on the severed side 2 days after axotomy and intensely between 3 and 7 days. Some of the microglial cells reacted positively with both anti-bromodeoxyuridine and antiphosphotyrosine antibodies, suggesting that tyrosine kinase is involved in microglial cell proliferation. Proliferation of numerous microglial cells was observed in the severed nuclei between 2 and 4 days after axotomy, while only a few were detected on days 5 and 7. These findings suggest that tyrosine kinase is involved in not only the proliferation of perineuronal microglial cells but also in some retrograde neuronal reactions such as differentiation and regeneration.     

Glutamate mediates cell death and increases the Bax to Bcl-2 ratio in a differentiated neuronal cell line

Excessive stimulation of the NMDA receptor by glutamate induces cell death and has been implicated in the development of several neurodegenerative diseases. While apoptosis plays a role in glutamate-mediated toxicity, the mechanisms underlying this process have yet to be completely determined. Recent evidence has shown that exposure to excitatory amino acids regulates the expression of the antiapoptotic protein, Bcl-2, and the proapoptotic protein, Bax, in neurons. Since it has been suggested that the ratio of Bax to Bcl-2 is an important determinant of neuronal survival, the reciprocal regulation of these Bcl-2 family proteins may play a role in the neurotoxicity mediated by glutamate. Here, we have used a differentiable neuronal cell line, N1E-115, to investigate the molecular properties of glutamate-induced cell death. Annexin V staining was used to determine apoptotic cell death between 0 and 5 days differentiation with DMSO/low serum. Immunoblot analysis was used to determine whether the expression of Bcl-2 or Bax was modulated during the differentiation process. Bcl-2 protein levels were increased during maturation while Bax expression remained unchanged. Maximum Bcl-2 expression was observed following 5 days of differentiation. Examination of Bcl-2 and Bax following glutamate treatment revealed that the expression of these proteins was inversely regulated. Exposure to glutamate (0.001-10 mM) for 20+/-2 h resulted in a dose-dependent decrease in cell survival (as measured by MTT analysis) that was maximal at 10 mM. These results further support the role of apoptosis in glutamate-mediated cell death. Furthermore, a significant decrease in Bcl-2 levels was observed at 1 mM and 10 mM glutamate (32.1%+/-4.8 and 33.7+/-12.8%, respectively) while a significant upregulation of Bax expression (88.2+/-17.9%) was observed at 10 mM glutamate. Interestingly, Bcl-2 and Bax levels in cells treated with glutamate from 12-24 h were not significantly different from those of control. Taken together, these findings provide additional evidence for the reciprocal regulation of Bcl-2 and Bax expression by glutamate and suggest that neuronal excitotoxicity may, in part, result from the inverse regulation of these proteins.     

Melatonin attenuates neuronal NADPH-d/NOS expression in the hypoglossal nucleus of adult rats following peripheral nerve injury

Oxidative stress and massive production of nitric oxide (NO) have been implicated in the neuropathogenesis following peripheral nerve injury. This study was aimed to ascertain whether melatonin would exert its neuroprotective effect on the lesioned hypoglossal neurons after peripheral axotomy, since it is known to reduce the oxidative damage in a variety of experimental neuropathologies in which NO is involved. Right-sided hypoglossal nerve transection was performed in adult rats following which the animals were given two different doses of melatonin administered intraperitoneally for 3, 7, 14, 21 and 30 successive days. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry were carried out to detect the neuronal NADPH-d/NOS expression in the hypoglossal nucleus (HN). At various time intervals following axotomy, the neurons in the affected HN were induced to express NADPH-d/NOS reactivity on the lesioned side peaking at 14 days. However, the enzyme expression was markedly depressed by melatonin treatment in a dose-dependent manner in terms of frequency of labelled neurons and staining intensity. It is suggested that the suppressive effect of melatonin on NADPH-d/NOS expression may be attributed to its antioxidant properties. Hence, in consideration of therapeutic strategies for reducing the oxidative stress following peripheral nerve injury, melatonin may prove to be beneficial.     

Red nucleus neurons of Bcl-2 over-expressing mice are protected from cell death induced by axotomy

The Bcl-2 proto-oncogene regulates apoptosis and prevents cell death. We studied the effect of Bcl-2 gene over-expression on the survival of axotomized red nucleus (RN) neurons after unilateral hemisection at cervical segment 4/5 (C4/5) in mice. Seventy-five percent of RN neurons survived in Bcl-2 over-expressing mice 1 or 2 months after surgery compared with only 55% of RN neurons in wild-type mice. However, Bcl-2 gene over-expression does not prevent lesion-induced shrinkage of RN neurons.     

大鼠局灶性脑缺血再灌注后海马Bcl-2、Bax蛋白的表达

目的 探讨大鼠局灶性脑缺血再灌注后Bcl-2、Bax蛋白在海马表达的变化.方法 线栓法制作大鼠局灶性脑缺血再灌注模型,应用免疫组化染色检测Bcl-2、Bax蛋白表达,应用TUNEL法检测海马区细胞凋亡.结果 缺血再灌注2h后海马神经元Bcl-2、Bax蛋白开始表达,Bcl-2蛋白12h达高峰,Bax蛋白12h～24h达高峰,之后开始下降.再灌注2h后海马凋亡细胞开始表达,随着再灌注时间的延长,其表达不断增加.Bcl-2/ Bax的比率在再灌注开始时升高,再灌注12h达高峰,随后开始下降.结论 凋亡是脑缺血再灌注损伤的重要形式之一,Bcl-2/ Bax的改变与缺血再灌注后海马的神经元存亡有关,缺血再灌注可导致海马神经元凋亡.     

大鼠局灶性脑缺血Bcl-2和Bax的表达与细胞凋亡

目的:研究大鼠局灶性脑缺血再灌注后凋亡相关基因Bcl-2和Bax在缺血皮层表达的变化及其与神经元凋亡的关系。方法:线栓法制作大鼠局灶性脑缺血再灌注模型,免疫组化法观察Bcl-2和Bax的表达变化,TUNEL法观察神经元凋亡的情况。结果:再灌注2h后皮层神经元Bcl-2表达开始明显上调,6h为高峰,之后开始下降。再灌注早期 Bax在皮层神经元的表达即明显增强,24～48h达高峰。Bcl-2／Bax的比率在再灌注开始时升高,6h达高峰,随后开始下降。TUNEL阳性细胞主要分布在缺血中心的边缘,再灌注48h之内,随时间的延长而不断增加。结论:Bcl-2／Bax的比率改变与缺血再灌注后的神经元存亡相关。     

大鼠脑缺血再灌注后Caspase-3、Bcl-2和Bax的表达

目的探讨大鼠脑缺血再灌注后caspase-3、Bcl-2和Bax在脑皮质神经元中的表达。方法将动物随机分为假手术组及缺血组,参照zea longa线栓法建立大鼠左侧大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)局灶性脑缺血再灌注模型,各组大鼠分别在左侧MCAO2h再灌注不同时间点断头取脑,脑皮质神经元中caspase-3、Bcl-2和Bax的表达通过免疫组化法来测定。结果缺血组大鼠脑皮质caspase-3的表达较假手术组显著增强(P<0.01),缺血组大鼠脑皮质Bcl-2的表达较假手术组显著增强(P<0.01),缺血组大鼠脑皮质Bax的表达较假手术组显著增强(P<0.01)。结论短暂性脑缺血再灌注上调脑皮质神经元中caspase-3和Bax的表达促细胞凋亡,上调脑皮质神经元中Bcl-2的表达抗细胞凋亡。     

Angiotensin II attenuates NMDA receptor-mediated neuronal cell death and prevents the associated reduction in Bcl-2 expression

While angiotensin II (Ang II) plays a major role in the regulation of blood pressure, fluid homeostasis and neuroendocrine function, recent studies have also implicated the peptide hormone in cell growth, differentiation and apoptosis. In support of this, we have previously demonstrated that Ang II attenuates N-methyl-D-aspartate (NMDA) receptor signaling [Molec. Brain Res. 48 (1997) 197]. To further examine the modulatory role of Ang II on NMDA receptor function, we investigated the effect of angiotensin receptor (AT) activation on NMDA-mediated cell death and the accompanying decrease in Bcl-2 expression. The viability of differentiated N1E-115 and NG108-15 neuronal cell lines was reduced following exposure to NMDA in a dose-dependent manner. MTT analysis (mitochondrial integrity) revealed a decrease in cell survival of 49.4+/-12.3% in NG108 cells and 79.9+/-6.8% in N1E cells following treatment with 10 mM NMDA for 20 h. Cytotoxicity in N1E cells was inhibited by the noncompetitive NMDA receptor antagonist, MK-801. Further, NMDA receptor-mediated cell death in NG108 cells was attenuated by treatment with Ang II. The Ang II effect was inhibited by both AT1 and AT2 receptor antagonists, losartan and PD123319, respectively, suggesting that both receptor subtypes may play a role in the survival effect of Ang II. Since it has been shown that activation of NMDA receptors alters the expression of Bcl-2 family proteins, Western blot analysis was performed in N1E cells to determine whether Ang II alters the NMDA-induced changes in Bcl-2 expression. A concentration-dependent decrease of intracellular Bcl-2 protein levels was observed following treatment with NMDA, and this reduction was inhibited by MK801. Addition of Ang II suppressed the NMDA receptor-mediated reduction in Bcl-2. The Ang II effect on NMDA-mediated changes in Bcl-2 levels was blocked by PD123319, but was not significantly changed by losartan, suggesting AT2 receptor specificity. Taken together, these results suggest that Ang II attenuates NMDA receptor-mediated neurotoxicity and that this effect may be due, in part, to an alteration in Bcl-2 expression.     

Effects of genistein on neuronal apoptosis, and expression of Bcl-2 and Bax proteins in the hippocampus of ovariectomized rats

Genistein is one of several isoflavones that has a structure similar to 17β-estradiol, has a strong antioxidant effect, and a high affinity to estrogen receptors. At 15 weeks after ovariectomy, the expression of Bcl-2 in the hippocampus of rats decreased and Bax expression increased, with an obvious upregulation of apoptosis. However, intraperitoneal injection of genistein or 17β-estradiol for 15 consecutive weeks from the second day after operation upregulated Bcl-2 protein expression downregulated Bax protein expression, and attenuated hippocampal neuron apoptosis. Our experimental findings indicate that long-term intervention with genistein can lead to a decrease in apoptosis in hippocampal neurons following ovariectomy, upregulate the expression of Bcl-2, and downregulate the expression of Bax. In addition, genistein and 17β-estradiol play equal anti-apoptotic and neuroprotective roles.     

亚低温治疗对大鼠脑梗死后Bax和Bcl-2表达的影响

目的探讨亚低温对大鼠急性脑梗死Bax、Bcl-2表达的影响。方法清洁级（SPF）雄性健康SD大鼠,采用线栓法建立局灶性脑梗死模型,分别于缺血3、6、12 h给予亚低温治疗,缺血24 h取材观察。实验分常温组（大鼠10只）、亚低温3、6、12 h组（每组大鼠各10只）,假手术组（大鼠6只）,用TTC染色测定脑梗死体积,用免疫组化的方法检测Bax、Bcl-2的表达水平。结果与常温组比较,亚低温组脑梗死体积明显减少（P〈0.01）、Bcl-2表达上调、Bax表达下调（P〈0.05）。亚低温组脑梗死体积3 h〈6 h〈12 h组,Bcl-2表达3 h〉6 h〉12 h、Bax表达3 h〈6 h〈12 h（P〈0.05）。结论亚低温治疗可能通过抑制Bax和Bcl-2表达,从而抑制神经元凋亡、降低脑梗死体积,脑缺血后越早期给予亚低温治疗效果越好。     

Bcl-2 and Bax protein expression in human myopathies.

Expression of the apoptosis-related proteins Bcl-2 and Bax was analysed by means of immunohistochemistry in muscle biopsies from 13 patients suffering from different muscular diseases (inclusion body myositis n=4, polymyositis n=3, Becker muscular dystrophy n=4, necrotizing myopathy n=2, and controls n=4), in an attempt to learn about the role of these proteins in human muscle diseases associated with muscle fiber necrosis and regeneration. Increased Bcl-2 and Bax immunoreactivity was observed as fine granular precipitates in the cytoplasm or as subsarcolemmal aggregates in pathological cases. Increased Bcl-2 and Bax immunoreactivity was detected in some necrotic fibers, regenerating fibers, ring fibers and some apparently normal muscle fibers. In addition, increased Bcl-2 and Bax immunoreactivity was observed in the periphery of rimmed vacuoles and in muscle fibers with abnormal mitochondria in patients suffering from inclusion body myositis. Double-labelling immunohistochemistry disclosed co-localization of both proteins in about 50% of Bcl-2-immunoreactive fibers. Finally, double-labelling immunohistochemistry using N-CAM antibodies revealed that some Bax-positive fibers were regenerating fibers. Since increased Bax immunoreactivity was not restricted to necrotic muscle fibers, the present results suggest that over-expression of this protein in human myopathies is probably independent of the process of cell death.     

The neuronal excitability time-dependently changes after lipopolysaccharide administration in mice: possible role of cyclooxygenase-2 induction

The parameters of pentylenetetrazol (PTZ)-induced seizures have been evaluated at various time intervals after lipopolysaccharide (LPS; Escherichia coli O111:B4, 100 microg/kg, i.p.) administration in mice. A proconvulsant effect occurred 4h after LPS injection with decreased seizure latency and enhanced seizure intensity. In contrast, the incidence of seizures was reduced 18 h after LPS injection. There were no significant alterations on seizure parameters 2, 8, 12, and 24h after LPS treatment. SC-58236, a selective cyclooxygenase (COX)-2 inhibitor (20 or 40 mg/kg, s.c.) treatment alone had no effect on PTZ-induced seizures, but reversed the antiseizure activity observed 18 h after LPS injection. However, SC-58236 treatment partially restored the proconvulsant changes that were observed 4h after LPS administration. On the other hand, COX-1-selective inhibitor valeryl salicylate (20 or 40 mg/kg, s.c.) itself facilitated PTZ-induced seizures. Thus, it was not possible to evaluate the effects of valeryl salicylate on the excitability changes after LPS injection. These results indicate that the parameters of PTZ-induced seizures change time-dependently after LPS treatment, in which proconvulsant and anticonvulsant states could be seen in a sequence. It seems that COX-2 isoenzyme may be involved in the neuronal excitability changes due to LPS.     

钙拮抗剂对大鼠缺血性脑损伤后Bcl-2和Bax基因表达的作用

目的探讨大鼠实验性脑缺血后海马组织Bcl-2和Bax基因的表达及尼莫地平预处理对Bcl-2和Bax基因表达的影响。方法采用线栓法建立大鼠脑缺血模型；尼莫地平预处理；逆转录聚合酶链式反应(RT-PCR)法测定其海马组织中Bcl-2和Bax mRNA。结果大鼠大脑中动脉阻断(MCAO)后，海马组织Bcl-2和Bax基因均被诱导表达，Bcl-2表达量持续升高，而Bax表达量在脑缺血24h达高峰，随后逐渐下降。在脑缺血后6h和24h，经尼莫地平预处理7d的大鼠，海马组织Bcl-2基因的表达水平较未经预处理的大鼠明显上调，而Bax基因表达水平则较未经预处理的大鼠明显下调。结论钙拮抗剂尼莫地平能有效地调控大鼠缺血性脑损伤后海马组织Bcl-2和Bax基因表达的水平，为干预脑卒中后基因表达提供依据。     

促红细胞生成素与大鼠面神经损伤后运动神经元TNF-a及Bax的表达

This study sought to evaluate the effect of high-dose erythropoietin (EPO; 5 000 IU/kg) on the expression of tumor necrosis factor-alpha (TNF-α) and Bax in the facial nucleus after facial nerve transection in rats. A total of 42 Wistar rats of both genders were used in this study, and 40 rats were randomly divided into 2 groups: EPO group and model group. The EPO group was treated with EPO once a day for 5 days at a dose of 5 000 IU/kg body weight. The model group was treated with saline of the same amount. At day 3 after EPO (or saline) treatment, the right facial nerves of the 40 rats were transected at the level of the stylomastoid foramen, with the left sides untreated. The remaining 2 rats that did not undergo axotomy served as the control group. The surviving motor neurons in operated rats were counted in coronal paraffin sections of the facial nucleus. The expression of TNF-α and Bax in the facial nucleus was detected by immunohistochemical staining at days 3, 7, 14, 21, and 28 after axotomy. At days 14, 21, and 28 after facial nerve axotomy, a significantly greater proportion of facial motor neurons survived in the EPO group than in the model group. After axotomy, the expression of TNF-α and Bax increased in motor neurons in both the EPO and the model groups. TNF-α expression reached its peak level at day 14 after axotomy, while Bax expression reached its peak level at day 21. TNF-α expression was much lower in the EPO group than in the model group at all time points. No significant difference in Bax expression was found between the EPO and the model groups. These results indicate that high-dose EPO treatment attenuates the increase in TNF-α expression in the facial nucleus and reduces the loss of motor neurons after facial nerve transection in rats. However, high-dose EPO treatment has little effect on Bax expression.