Differential time course of neuronal and glial apoptosis in neonatal rat dorsal root ganglia after sciatic nerve axotomy

Sensory neurons in neonatal rat lumbar dorsal root ganglia die after sciatic nerve axotomy, and previous studies have estimated the total cell loss to be 40–95%. We have used the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) technique, combined with immunohistochemistry, to investigate the contribution of apoptosis to the cell loss that occurs after unilaterally transecting the sciatic nerve of new-born rats. TUNEL-positive cells were detected 1 day post-lesion, and their number peaked 3 days after the injury. Combining TUNEL labelling with immunohistochemistry, for neuron-specific neurofilament 150 kDa, or glial-specific S-100β, enabled us to identify dying neurons and dying glia. One day after axotomy, most of the TUNEL-positive cells (58%) were neurons, whereas 3 days post-injury, only a small number of dying cells (6%) were neuronal. This lower incidence was due to a decrease in neuronal death and an increase in glial death. The glia in the dorsal root ganglia therefore die subsequent to the neurons. The apoptotic nature of the cell death was confirmed by electron microscopy, with fine structural features of apoptotic cell death, e.g. chromatin compaction and membrane blebbing, being observed in both glia and neurons. Our results confirm that extensive apoptosis occurs in the neonatal lumbar dorsal root ganglia after sciatic nerve section, and show that neurons and glial cells die with different time-courses. The results suggest a neuron-glia trophic interdependence in the dorsal root ganglia.     

整合素α_5、β_1抗体对睾丸生精细胞凋亡的影响

目的观察整合素α_5、β_1抗体体内干预试验对Wistar大鼠睾丸生精细胞凋亡的作用。方法腹腔内注射α_5、β_1抗体,采用原位末端脱氧核糖核酸转移酶标记技术,分析Wistar大鼠生精细胞凋亡情况。结果α_5、β_1试验组大鼠睾丸生精细胞明显减少(P0.001)。结论α_5、β_1抗体具有阻止Wistar大鼠生精细胞凋亡的作用。     

Identification of metabolic pattern and bioactive form of resveratrol in human medulloblastoma cells

Cancer preventive reagent trans-resveratrol is intracellularly biotransformed to different metabolites. However, it is still unclear whether trans-resveratrol exerts its biological effects directly or through its metabolite(s). This issue was addressed here by identifying the metabolic pattern and the bioactive form of resveratrol in a resveratrol-sensitive human medulloblastoma cell line, UW228-3. The cell lysates and condition media of UW228-3 cells with or without 100 μM resveratrol treatment were analyzed by HPLC and LC/MS which revealed (1) that resveratrol was chemically unstable and the spontaneous generation of cis-resveratrol reduced resveratrol's anti-medulloblastoma efficacy and (2) that resveratrol monosulfate was the major metabolite of the cells. To identify the bioactive form of resveratrol, a mixture-containing approximately half fraction of resveratrol monosulfate was prepared by incubating trans-resveratrol with freshly prepared rat brain lysates. Medulloblastoma cells treated by 100 μM of this mixture showed attenuated cell crisis. The overall levels of the three brain-associated sulfotransferases (SULT1A1, 1C2 and 4A1) were low in medulloblastoma cells in vivo and in vitro in comparison with that in human noncancerous and rat normal cerebella; resveratrol could more or less up-regulate the production of these enzymes in UW228-3 cells but their overall level was still lower than that in normal cerebellum tissue. Our study thus demonstrated for the first time that trans-resveratrol is the bioactive form in medulloblastoma cells in which the expression of brain-associated SULTs was down-regulated, resulting in the increased intracellular bioavailability and anti-medulloblastoma efficacy of trans-resveratrol.     

A cytotoxic hydroperoxy sterol from the brown alga,Nizamuddinia zanardinii

Background

The marine environment is a unique source of bioactive natural products, of which Nizamuddinia zanardinii is an important brown algae distributed in Oman Sea. Literature revealed that there is no report on phytochemistry and pharmacology of this valuable algae.

Methods

Bioguided fractionation of the methanolic extract of Nizamuddinia zanardinii, collected from Oman Sea, led to the isolation of a hydroperoxy sterol. Its structure was determined by analysis of the spectroscopic data as 24-hydroperoxy-24-vinyl cholesterol (HVC). In vitro cytotoxic activity of this compound was evaluated against HT29, MCF7, A549, HepG2 and MDBK cell lines.

Results

Although 24(R)-hydroproxy-24-vinylcholesterol has been previously reported from Sargassum and Padina species, it is the first report on the presence of this compound from N. zanardinii. This compound exhibited cytotoxicity in all cell lines (IC₅₀, 3.62, 9.09, 17.96, 32.31 and 37.31 μg/mL respectively). HVC was also evaluated for apoptotic activity and demonstrated positive results in terminal deoxynucleotidyl transferase dUTP Nick End labeling (TUNEL) assay suggesting it a candidate for further apoptotic studies.

Conclusions

Nizamuddinia zanardinii, a remarkable brown algae of Oman Sea, is a good source of hydroproxy sterols with promising cytotoxic on various cell lines particularly human colon adenocarcinoma.     

The neuroprotective effects of intraperitoneal injection of hydrogen in rabbits with cardiac arrest

Objective

The purpose of this study was to investigate the neuroprotective effects of intraperitoneal injection of hydrogen (H₂) in rabbits with cardiac arrest (CA).

Methods

A rabbit model of CA was established by the delivery of alternating current between the esophagus and chest wall to induce ventricular fibrillation. Before CA, the animals were randomly divided into four groups: a sham group (no CA), a CA group, a CA + low dose (10 ml/kg) H₂ group (CA + H₂ group 1), and a CA + high dose (20 ml/kg) H₂ group (CA + H₂ group 2). In the first experiment, animals were observed for 72 h after the restoration of spontaneous circulation (ROSC). The neurological scores were assessed at 24, 48 and 72 h after ROSC. The rabbits that survived until 72 h were sacrificed using an overdose of anesthetic, and the brain tissues were collected and Nissl-stained to observe nerve cell damage in the hippocampal CA1 area. In addition, TUNEL assay was performed to detect apoptosis. In the second experiment, animals were observed for 6 h after ROSC. Blood samples and brain hippocampal tissues were collected, and differences in oxidative stress indicators were compared among the four groups.

Results

Intraperitoneal injection of H₂ improved the 72-h survival rate and neurological scores, reduced neuronal injury and inhibited neuronal apoptosis. Intraperitoneal injection of H₂ reduced oxidative stress indicators in the plasma and hippocampal tissues and enhanced antioxidant enzyme activity. No significant difference was observed between the two CA groups treated with different doses of H₂.

Conclusions

Intraperitoneal injection of H₂ is a novel hydrogen administration method and can reduce cerebral ischemia-reperfusion injury and improve the prognosis of cardiopulmonary cerebral resuscitation in a rabbit model of CA.     

Anatomic distribution of apoptosis in medulla oblongata of infants and adults

The aim of the study was to evaluate the distribution of apoptosis in the medullary nuclei of infants and adults who died of hypoxic-ischaemic injury. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) in brainstems from 22 adults (7 subjects who died of opiate intoxication, 15 who died of other hypoxic-ischaemic injury) and 10 infants. The nuclei examined included the hypoglossal, dorsal motor nucleus of the vagus, nucleus tractus solitarii, nucleus of the spinal trigeminal tract, cuneate, vestibular and inferior olivary nuclei. A morphometric analysis with the optical disector method was performed to calculate the mean percentages (+/- standard deviation) of TUNEL-positive neuronal and glial cells for the sample populations. Opiate deaths did not have higher apoptotic indices than other adult hypoxic-ischaemic deaths. Statistically significant differences between adults and infants were found in the neuronal apoptotic indices of the cuneate (28.2 +/- 16.3% vs. 6.9 +/- 8.7%), vestibular (24.7 +/- 15.0% vs. 11.3 +/- 11.4%), nucleus tractus solitarii (11.2 +/- 11.2% vs. 2.3 +/- 2.4%), dorsal motor nucleus of the vagus (6.8 +/- 8.5% vs. 0.1 +/- 0.2%) and hypoglossal (6.6 +/- 5.7% vs. 0.1 +/- 0.2%), indicating higher resistance of the neuronal populations of these infant medullary nuclei to terminal hypoxic-ischaemic injury or post-mortem changes. Differences in neuronal apoptotic index were also statistically significant among nuclei, suggesting differential characteristics of survival. Nuclei with higher neuronal apoptotic indices were the cuneate, vestibular and nucleus of the spinal trigeminal tract, which are located in the lateral medullary tegmentum and share the same vascular supply from the posterior inferior cerebellar artery.     

Expressional evaluation of vascular endothelial growth factor (VEGF) protein in urinary bladder carcinoma patients exposed to cigarette smoke

Angiogenesis is a fundamental process in tumor growth and metastasis. Expression of vascular endothelial growth factor (VEGF) as prognostic indicator has been documented in many types of human tumors. However, the mechanisms responsible for angiogenesis in urinary bladder carcinoma patients are not well defined. Certain carcinogens in tobacco cause DNA damage and may produce specific mutations. In order to investigate the relationship between tobacco smoking, altered patterns of VEGF expression and apoptosis, we have analyzed a group of 125 incident patients with transitional cell carcinoma and 100 cases of control with inflammatory lesions of the bladder. We assessed VEGF overexpression by the use of anti- VEGF antibody through immunohistochemistry, and apoptosis by TUNEL Assay. Expression of VEGF and apoptosis was noticed in 43.2% and 52.8% cases respectively. Both VEGF and apoptosis increased with increasing tumor grade. Apoptosis was seen to be significantly higher in both sexes in the age group of ≥ 50 years (p<0.05) but expression of VEGF was significantly higher among males in the age group of ≥ 50 years (p<0.05). We observed an insignificant association between cigarettes smoking and VEGF overexpression (p>0.05) and significant association with apoptosis. These data support the hypothesis that certain carcinogens derived from cigarette smoking may induce VEGF mutations and apoptosis which in turn are involved in early steps of bladder carcinogenesis.