Transferase-mediated dUTP-biotin Nick End Labeling Positive Cells Research Articles

Naringin protects against inflammation and apoptosis induced by intestinal ischemia-reperfusion injury through deactivation of cGAS-STING signaling pathway.

Effective amelioration of ischemia/reperfusion (I/R)-induced intestinal injury and revealing its mechanisms remain the challenges in both preclinic and clinic. Potential mechanisms of naringin in ameliorating I/R-induced intestinal injury remain unknown. Based on pre-experiments, I/R-injured rat intestine in vivo and hypoxia-reoxygenation (H/R)-injured IEC-6 cells in vitro were used to verify that naringin-alleviated I/R-induced intestinal injury was mediated via deactivating cGAS-STING signaling pathway. Naringin improved intestinal damage using hematoxylin and eosin staining and decreased alanine aminotransferase and aspartate aminotransferase contents in plasma. Naringin decreased inflammation characterized by reducing IL-6, IL-1β, TNF-α, and IFN-β contents in both plasma and IEC-6 cells. Naringin mitigated oxidative stress via recovering superoxide dismutase, glutathione, and malondialdehyde levels in the I/R-injured intestine. Naringin reduced the expression of apoptotic proteins, including Bax, caspase-3, and Bcl-2, and reduced terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling-positive cells both in vivo and in vitro, and decreased Hoechst 33342 signals in vitro. cGAS, STING, p-TBK1, p-IRF3, and NF-κB expressions were up-regulated both in vivo and in vitro respectively and the up-regulated indexes were reversed by naringin. Transfection of cGAS-siRNA and cGAS-cDNA significantly down-regulated and up-regulated cGAS-STING signaling-related protein expressions, respectively, and partially weakened naringin-induced amelioration on these indexes, suggesting that deactivation of cGAS-STING signaling is the crucial target for naringin-induced amelioration on I/R-injured intestine.

Protective effects of histone deacetylase 6 specific inhibitor tubastatin A on subarachnoid hemorrhage in rats and the underlying mechanisms.

Subarachnoid hemorrhage (SAH) is a serious cerebrovascular disease. Early brain injury (EBI) and cerebral vasospasm are the main reasons for poor prognosis of SAH patients. The specific inhibitor of histone deacetylase 6 (HDAC6), tubastatin A (TubA), has been proved to have a definite neuroprotective effect on a variety of animal models of acute and chronic central nervous system diseases. However, the neuroprotective effect of TubA on SAH remains unclear. This study aims to investigate the expression and localization of HDAC6 in the early stage of SAH, and to evaluate the protective effects of TubA on EBI and cerebral vasospasm after SAH and the underlying mechanisms. Adult male SD rats were treated with modified internal carotid artery puncture to establish SAH model. In the first part of the experiment, rats were randomly divided into 6 groups: a sham group, a SAH-3 h group, a SAH-6 h group, a SAH-12 h group, a SAH-24 h group, and a SAH-48 h group. At 3, 6, 12, and 24 h after SAH modeling, the injured cerebral cortex of rats in each group was taken for Western blotting to detect the expression of HDAC6. In addition, the distribution of HDAC6 in the cerebral cortex of the injured side was measured by immunofluorescence double staining in SAH-24 h group rats. In the second part, rats were randomly divided into 4 groups: a sham group, a SAH group, a SAH+TubAL group (giving 25 mg/kg TubA), and a SAH+TubAH group (giving 40 mg/kg TubA). At 24 h after modeling, the injured cerebral cortex tissue was taken for Western blotting to detect the expression levels of HDAC6, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining to detect apoptosis, and hematoxylin and eosin (HE) staining to detect the diameter of middle cerebral artery. The protein expression of HDAC6 began to increase at 6 h after SAH (P<0.05), peaked at 24 h (P<0.001), and decreased at 48 h, but there was still a difference compared with the sham group (P<0.05). HDAC6 is mainly expressed in the cytoplasm of the neurons. Compared with the sham group, the neurological score was decreased significantly and brain water content was increased significantly in the SAH group (both P<0.01). Compared with the SAH group, the neurological score was increased significantly and brain water content was decreased significantly in the SAH+TubAH group (both P<0.05), while the improvement of the above indexes was not significant in the SAH+TubAL group (both P>0.05). Compared with the sham group, the expression of eNOS was significantly decreased (P<0.01) and the expressions of iNOS and HDAC6 were significantly increased (P<0.05 and P<0.01, respectively) in the SAH group. Compared with the SAH group, the expression of eNOS was significantly increased, and iNOS and HDAC6 were significantly decreased in the SAH+TubA group (all P<0.05). Compared with the SAH group, the number of TUNEL positive cells was significantly decreased and the diameter of middle cerebral artery was significantly increased in the SAH+TubA group (both P<0.05) . HDAC6 is mainly expressed in neurons and is up-regulated in the cerebral cortex at the early stage of SAH. TubA has protective effects on EBI and cerebral vasospasm in SAH rats by reducing brain edema and cell apoptosis in the early stage of SAH. In addition, its effect of reducing cerebral vasospasm may be related to regulating the expression of eNOS and iNOS.

Evaluation of the possible protective role of naringenin on gentamicin-induced ototoxicity: A preliminary study

ObjectivesThe purpose of this study is to evaluate the possible protective role of naringenin in gentamicin-induced ototoxicity through an audiological, biochemical and histopathological evaluation. MethodsThis study was conducted on 32 adult male rats that were randomized into 4 groups(control, gentamicin, naringenin + gentamicin, and naringenin). Naringenin was given to the rats via oral gavage in a dose of 50 mg/kg/day during the 14 day study period. Gentamicin was given by the intraperitoneal route in a dose of 120 mg/kg/day. Audiological assessment was performed by the distortion product otoacoustic emission (DPOAE) and auditory brainstem response (ABR) measurements, applied to all rats at the beginning of the study, and also on day 14. Biochemical parameters were calculated on day 14 to evaluate the oxidative and antioxidative status. Their cochleae were removed and examined histopathologically, also on day 14. The cochlea of animals were evaluated with the terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end labeling (TUNEL) method for apoptosis. ResultsOn days 14, DPOAE values and ABR thresholds were preserved in group 3(naringenin + gentamicin) when compared with group 2(gentamicin)(p < 0.008). The total oxidant status values and oxidative stress index values were significantly higher in group 2(gentamicin) than in other groups (p < 0.008). The total antioxidant status value was significantly higher in group 3(naringenin + gentamicin) and group 4(naringenin) than in group 2(gentamicin)(p < 0.008). The number of TUNEL positive cells in both the organ of Corti and the stria vascularis were found to be statistically lower in group 3(naringenin + gentamicin) than in group 2(gentamicin)(p < 0.05). ConclusionOur study has demonstrated that the ototoxic effect generated by gentamicin could be ameliorated with the concurrent use of naringenin.

Preliminary morphological and immunohistochemical changes in rat hippocampus following postnatal exposure to sodium arsenite

The effects of arsenic exposure during rapid brain growth period (RBGP) (postnatal period 4-11) on pyramidal neurons of cornu ammonis (specifically CA1 and CA3 regions) and granule cells of dentate gyrus (DG) of rat hippocampus were studied. Wistar rat pups, subdivided into the control (group I) and the experimental groups (group II, III, and IV), received distilled water and sodium arsenite (aqueous solution of 1.0, 1.5, and 2.0 mg/kg body weight, respectively) by intraperitoneal (i.p.) route. On postnatal day (PND) 12, the animals were sacrificed and brain tissue obtained. Paraffin sections (8 μm thick) stained with Cresyl Violet (CV) were observed for morphological and morphometric parameters. Arsenic induced programmed cell death (apoptosis) was studied using Terminal deoxyribonucleotidyl transferase mediated dUTP biotin Nick End Labeling (TUNEL) technique on the paraffin sections. Microscopy revealed decreased number and isolation of pyramidal neurons in superficial layers, misalignments of pyramidal cells in stratum pyramidale (SP) of CA1 and CA3 in experimental group III and IV, and presence of polymorphic cells in subgranular zone of ectal limb of dentate gyrus (suggestive of arsenic induced proliferation and migration of granule cells in the dentate gyrus). Morphometric assessments quantified and confirmed the microscopic findings. The mean nuclear area of pyramidal cells was increased and cell density was decreased in the CA1, CA3, and DG of experimental groups in comparison to the control group. Increase in the TUNEL positive cells in DG was observed in the experimental group IV, suggestive of increased apoptosis. These observations confirm vulnerability of pyramidal (CA1, CA3) and granule cells (DG) of hippocampus during RBGP.

Multimodal In Vivo Imaging and Blood Monitoring of Intrinsic and Extrinsic Apoptosis

Noninvasive detection and in vivo imaging of apoptosis plays a critical role in the development of therapeutics in many different fields including cancer. We have developed an apoptosis biosensor by fusing green fluorescent protein (GFP) to the N-terminus of the naturally secreted Gaussia luciferase separated by a caspase-3 cleavage peptide consisting of aspartic acid (D), glutamic acid (E), valine (V), and aspartic acid (D) or DEVD. We showed that this fusion is retained in the cytoplasm of cells in an inactive form. Upon apoptosis, the DEVD peptide is cleaved in response to caspase-3 activation, freeing ssGluc, which can now enter the secretory pathway where it is folded properly and is released from the cells and can be detected in the conditioned medium in culture or in blood of live animals ex vivo over time. Because Gluc is secreted from cells via conventional pathway through the endoplasmic reticulum (ER), Golgi and vesicles, we showed that the presence of Gluc in these compartments in response to apoptosis can be visualized in vivo using bioluminescence imaging. This reporter provides a valuable tool for imaging and real-time monitoring of apoptosis and is compatible with high-throughput functional screening application in cultured cells and animal models.

Intracerebroventricular administration of riluzole prevents morphine-induced apoptosis in the lumbar region of the rat spinal cord

Opiates are the most effective drugs for pain relief. However, the repeated use of opiates induces tolerance to their analgesic effects. It has been shown that this morphine-induced tolerance is associated with apoptosis in the central nervous system. The aim of this study is to evaluate the effects of intracerebroventricular (icv) administration of riluzole, an anti-glutamatergic drug, on morphine-induced apoptosis in the lumbar region of the rat spinal cord. Animals were given daily injections of morphine and vehicle, morphine and riluzole, or riluzole alone. Nociception was assessed using a hot plate apparatus, and apoptosis was assessed using the in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The levels of anti-apoptotic factors Bcl-2 and HSP 70 and the pro-apoptotic agent caspase-3 were evaluated using immunoblotting. The glutamate concentration in the lumbar spinal cord was measured with high performance liquid chromatography (HPLC). The results indicate that the icv administration of riluzole attenuated morphine tolerance and reduced the number of TUNEL positive cells. Immunoblotting revealed that the levels of the selected anti-apoptotic agents were greater in the treatment groups compared to the controls. Furthermore, the results demonstrated that the administration of riluzole can attenuate the morphine-induced elevation of glutamate in the lumbar spinal cord. In conclusion, icv administration of riluzole attenuated morphine-induced tolerance to analgesia and apoptosis in addition to preventing the morphine-induced increase of glutamate in the lumbar spinal cord of rats.

Exogenous thioredoxin prevents ethanol-induced oxidative damage and apoptosis in mouse liver

Ethanol-induced liver injury is characterized by increased formation of reactive oxygen species (ROS) and inflammatory cytokines, resulting in the development of hepatic steatosis, injury, and cell death by necrosis and apoptosis. Thioredoxin (Trx), a potent antioxidant and antiinflammatory molecule with antiapoptotic properties, protects animals from a number of inflammatory diseases. However, the effects of ethanol on Trx or its role in ethanol-induced liver injury are not known. Female C57BL/6 mice were allowed ad libitum access to a Lieber-deCarli ethanol diet with 5.4% of calories as ethanol for 2 days to acclimate them to the diet, followed by 2 days with 32.4% of calories as ethanol or pair-fed control diet. Hepatic Trx-1 was decreased by ethanol feeding; daily supplementation with recombinant human Trx (rhTrx) prevented this ethanol-induced decrease. Therefore, we tested the hypothesis that administration of rhTrx during ethanol exposure would attenuate ethanol-induced oxidative stress, inflammatory cytokine production, and apoptosis. Mice were treated with a daily intraperitoneal injection of either 5 g/kg of rhTrx or phosphate-buffered saline (PBS). Ethanol feeding increased accumulation of hepatic 4-hydroxynonenal protein adducts, expression of hepatic tumor necrosis factor alpha, and resulted in hepatic steatosis and increased plasma aspartate aminotransferase and alanine aminotransferase. In ethanol-fed mice, treatment with rhTrx reduced 4-hydroxynonenal adduct accumulation, inflammatory cytokine expression, decreased hepatic triglyceride, and improved liver enzyme profiles. Ethanol feeding also increased transferase-mediated dUTP-biotin nick-end labeling-positive cells, caspase-3 activity, and cytokeratin-18 staining in the liver. rhTrx treatment prevented these increases. In summary, rhTrx attenuated ethanol-induced increases in markers of oxidative stress, inflammatory cytokine expression, and apoptosis.

The Effects of Dantrolene on Hypoxic-Ischemic Injury in the Neonatal Rat Brain

The pathophysiology of brain damage from hypoxia or ischemia has been ascribed to various mechanisms and cascades. Intracellular calcium overload and a calcium excitotoxic cascade have been implicated. It has been suggested that disturbances of endoplasmic reticulum calcium homeostasis are involved in the induction of neuronal cell injury. Two types of intracellular Ca2+-release channels, involving the ryanodyne receptor and the inositol (1,4,5)-triphosphate receptor, are essential for Ca2+ signaling in cells. Dantrolene, which is used for the treatment of malignant hyperthermia syndrome, has been reported to inhibit Ca2+ release through ryanodyne receptors from the endoplasmic reticulum into the cytosol. We designed this study to investigate the neuroprotective effects of dantrolene on hypoxic-ischemic brain damage in the neonatal rat brain. Seven-day-old Sprague-Dawley rats were assigned into two groups; control group (n = 69) and dantrolene group (n = 60). Dimethyl sulfoxide was administered intracerebroventricularly in the control group, and dantrolene in dimethyl sulfoxide was similarly administered to the dantrolene group, before hypoxic-ischemic brain injury (HII). HII was induced by the ligation of the common carotid artery under isoflurane anesthesia, followed by exposure to about 2.5 h of hypoxia (oxygen concentration was maintained at 7%-8%). 1H magnetic resonance spectroscopy was performed 1 day after HII. This noninvasive method evaluated apoptotic processes in the brain after HII. Morphologic score analyses and the calculated percentage of infarct areas after 2,3,5-triphenyltetrazolium chloride staining 14 days after HII were also used to evaluate the effects of dantrolene on HII. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining was performed 1 day after HII using 24 more rats. The lipid/creatine ratios in the right hemispheres in the dantrolene group 1 day after HII were significantly lower than those of the control group (P < 0.05). There was no significant difference between the two groups in the N-acetylaspartate/creatine ratios. The gross morphologic scores were lower in the dantrolene group than in the control group (P < 0.05), and infarct area (%) after 2,3,5-triphenyltetrazolium chloride staining was less in the dantrolene group than in the control group (P < 0.05) 14 days after HII. Further work with 24 rats showed no significant difference, however, in the number of TUNEL positive cells on the two groups. Our results show that dantrolene, administered intracerebroventricularly before HII, had a neuroprotective effect in HII model of the neonatal rat brain.

Neuroprotective effects of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), an antioxidant in middle cerebral artery occlusion induced focal cerebral ischemia in rats

Objectives: In the present study, we have investigated the neuroprotective potential of 6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), in middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia.Methods: Sprague–Dawley rats were subjected to 2 hours of MCAO followed by 22 or 70 hours of reperfusion. After reperfusion, rats were evaluated for neurological deficits and cerebral infarction. Brain malondialdehyde (MDA) level and in situ terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) were also estimated.Results: Focal cerebral ischemia produced a significant infarct volume and neurological scores as compared with sham-operated animals. Cerebral ischemia reperfusion injury was associated with an increase in lipid peroxidation in ipsilateral and contralateral hemisphere of brain along with an increase in TUNEL positive cells in ipsilateral hemisphere of brain sections indicating oxidative stress and DNA fragmentation, respectively. Trolox (10 and 30 mg/kg, i.p.) treatment significantly decreased neurological damage which was evident from the reduction in infarct volume and neurological score. Trolox (30 mg/kg) also attenuated oxidative stress and DNA fragmentation.Discussion: Oxidative stress-induced neuronal damage is implicated in the pathophysiology of cerebral ischemia. Our study suggests that Trolox is a potent neuroprotective agent in focal cerebral ischemia and its neuroprotective effects may be attributed to the reduction of lipid peroxidation and DNA fragmentation.

Deletion of Bid Impedes Cell Proliferation and Hepatic Carcinogenesis

Mechanisms that control the proliferation capability of the initiated cells during hepatocarcinogenesis are still largely unclear. We investigated the role of a pro-death Bcl-2 family protein, Bid, in liver tumor development using a neonatal diethylnitrosamine model. Diethylnitrosamine was administrated to 15-day-old wild-type and bid-null mice. The development of microfoci at the early stage and of gross tumors at the later stage was compared between the two groups of mice. Both microfoci and gross tumor development were significantly retarded in the bid-null mice, despite reduced cell death as measured by TUNEL staining. Further studies indicated that there were significantly less proliferating cells in diethylnitrosamine-treated bid-null livers. The regulation of cell proliferation by Bid was confirmed in two other systems not involving carcinogenesis. Hepatocyte proliferation following partial hepatectomy and T lymphocyte proliferation following anti-CD3 stimulation were both retarded in bid-null mice. Thus, these studies revealed a previously undisclosed function of Bid in regulating cell proliferation, which can be important to tumor development. Furthermore, the role of Bid in promoting hepatocarcinogenesis is in contrast to its reported role in suppressing myeloid leukemia and thus suggests an organ- and/or etiology-specific role of the Bcl-2 family proteins in regulating oncogenesis.

Neuroprotective effect of 4-amino-1,8-napthalimide, a poly(ADP ribose) polymerase inhibitor in middle cerebral artery occlusion-induced focal cerebral ischemia in rat

In the present study, neuroprotective effect of 4-amino-1,8-napthalimide (4-ANI), a poly(ADP-ribose) polymerase (PARP) inhibitor was investigated in middle cerebral artery occlusion (MCAo)-induced focal ischemia. Sprague–Dawley rats were subjected to 2 h of middle cerebral artery occlusion followed by 22 h of reperfusion. After 22 h of reperfusion rats were evaluated for cerebral infarction, neurological deficits, brain NAD levels, and in situ terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL). Focal ischemia produced significant infarct volume (201±14 mm 3), neurological scores (2±0.5) and 28±4.5% brain NAD depletion. Ischemia was associated with increased in TUNEL positive cells in brain sections indicating DNA fragmentation. 4-ANI treatment (1 and 3 mg/kg, i.p.) significantly decreased infarct volume to 35±7% and 70±6%, respectively. Neurological functions were also significantly improved at these doses. 4-ANI (3 mg/kg) completely reversed brain NAD depletion and significantly reduced the increase in the number of TUNEL positive cells. Nevertheless, 4-ANI treatment did not alter cerebral blood flow and blood pressure. Our study suggests 4-ANI is a potent neuroprotective agent in focal cerebral ischemia and its neuroprotective effects may be attributed to reduction of NAD depletion and DNA fragmentation.

Perinatal development of the rat kidney: apoptosis and epidermal growth factor.

Localization of apoptotic cells in the kidney of perinatal rats was examined by the terminal deoxynucleotidyl transferase-mediated d-UTP-biotin nick end labeling (TUNEL) method and electron microscopy. Perinatal changes in the percentage of kidney cells with DNA fragmentation were determined by flow cytometric analysis. Through observation of two successive sections, the relationship between the localization of the epidermal growth factor receptor (EGFR) positive cells and TUNEL positive cells in the kidney was determined. From fetal day 18 to neonatal day 5, TUNEL positive cells were noted in immature glomeruli, collecting ducts and interstitium. Electron microscopically, chromatin condensed nuclei and apoptotic bodies were seen in the same tissue component as the TUNEL positive cells. The percentage of DNA fragmented cells significantly increased from fetal days 18 to 20 and significantly decreased from fetal days 20 to 22, while they still remained low in the neonatal period. The TUNEL positive cells in immature glomeruli and collecting ducts were not reactive to the EGFR antibody. The TUNEL positive cells were not observed in the proximal tubular cells, which were positive to EGFR antibody. These results indicate that apoptotic cells are present in the kidney throughout the perinatal period in the rat and that EGF plays an important role in perinatal development of the rat kidney.

Massive cell death of cerebellar granule neurons accompanied with caspase-3-like protease activation and subsequent motor discoordination after intracerebroventricular injection of vincristine in mice

Vincristine, a microtubule-depolymerizing agent, is known to induce neuronal cell damage. Biochemical, histological and behavioral alterations were investigated after intracerebroventricular injection of vincristine in mice. Intracerebroventricular injection of vincristine caused caspase-3-like protease activation followed by nucleosomal release in the cerebellum. Histological examinations showed that vincristine-induced damage was relatively specific to granule cells in the cerebellum, and terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick-end labeling-positive cells were observed among these cells. Chromatin condensation, one of the criteria for apoptosis, was seen on electron microscopy. Behavioral changes, namely head movements, pivoting and backward walking, were observed in parallel with the increase of caspase-3-like protease activity and nucleosomal release. Furthermore, motor function tests (bulb balance test and rotating rod test) showed deficits of motor coordination ability. These observations suggest that intracerebroventricular vincristine causes massive apoptosis of cerebellar granule cells accompanied with caspase-3-like protease activation, leading to motor dysfunction, in this model. These vincristine-treated mice should be a useful in vivo model for examination of neuronal apoptosis, which might be involved in a variety of neurodegenerative diseases.

Adenovirus Mediated Gene Transfer of Antiapoptotic Protein in Hepatic Ischemia–Reperfusion Injury: The Paradoxical Effect of Bcl-2 Expression in the Reperfused Liver

Background. Although Bcl-2 is well known to have antiapoptotic activities in vitro and in vivo, the role of Bcl-2 remains controversial. In the present study, we evaluated whether the adenovirus mediated gene transfer of hBcl-2 could exert an antiapoptotic effect in a rat model of hepatic ischemia–reperfusion (I/R) injury.Materials and methods. Each 6 × 109 plaque forming unit adenovirus vector encoding LacZ (AxCAilacZ) or hBcl-2 (AxCAhbcl2) was intravenously administered 48 h before I/R injury, in groups 1 and 2, respectively. In group 3, 1 ml of normal saline was injected instead of the virus vectors. Hepatic I/R injury was induced by the temporal occlusion of all hepatic influent vessels for 30 min under a portosystemic shunt. The animals were sacrificed at 6 h, 1, 3, and 14 days after reperfusion (each n = 12 in groups 1 and 2, and n = 8 in group 3). The expressions of hBcl-2 and Bax were evaluated at both the mRNA level by reverse transcription polymerase chain reaction and the protein level by immunohistochemistry. To assess the hepatocyte and sinusoidal endothelial cell damage after I/R injury, the serum asparate aminotransferase (AST), alanine aminotransferase, and hyaluronic acid were all evaluated. The number of apoptotic cells was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL).Results. To evaluate the antiapoptotic activities of the hBcl-2 sequence encoded into AxCAhbcl2, rat hepatocarcinoma cells were transfected with AxCAhbcl2 (103 moi) or AxCAilacZ (103 moi) and then challenged with TGF-β1 protein (5 ng/ml) to induce apoptosis. Apoptotic cells were counted by TUNEL staining in about 2500 cells, and it was found that adenovirus mediated gene transfer of hBcl-2 significantly protected rat hepatocarcinoma cells from TGF-β1 induced apoptosis (14.2 ± 1.2%) in comparison to those of LacZ (21.9 ± 1.4%). In the reperfused liver in vivo, the mRNA expression of hBcl-2 was detected only in the hBcl-2 transfected group 2. In group 2, a strong degree of Bcl-2 immunoreactivity was recognized as early as 6 h after reperfusion, while it was not recognized in groups 1 and 3 at 6 h after reperfusion. The AST levels were significantly higher in group 2 (AST: 356 ± 100.1 IU/L) than those in group 1 (AST: 102.7 ± 15 IU/L) at 1 day after reperfusion (P < 0.05). The number of TUNEL positive cells was significantly higher in group 2 than in groups 1 and 3 at 1 day after reperfusion.Conclusions. These results indicated that an overexpression of antiapoptotic protein Bcl-2 paradoxically exerted a proapoptotic effect in the reperfused liver. The in vivo role of Bcl-2 should thus be carefully evaluated, depending on the levels of expression and the target organ.

Evaluation of cell proliferation, apoptosis, and angiogenesis in transitional cell carcinoma of the renal pelvis and ureter

Objectives. To investigate cell proliferation, apoptosis, and angiogenesis and their roles in transitional cell carcinoma (TCC) of the renal pelvis and ureter. Methods. Formalin-fixed and paraffin-embedded tissue blocks from 42 patients with TCC of the renal pelvis and ureter were studied. Cell proliferation was assessed by Ki-67 immunostaining, and the proliferation index (PI) was expressed as a percentage of Ki-67-positive cells. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and the apoptotic index (AI) was expressed as a percentage of TUNEL positive cells. Angiogenesis was evaluated by CD31 immunostaining, and microvessel density (MVD) was expressed as the average of the microvessel count. Results. The PI ranged from 5.9% to 48.0% (median 20.03%), AI from 1.0% to 4.2% (median 2.26%), and MVD from 16.0 to 146.0 (median 56.88) in TCC of the renal pelvis and ureter. Statistical analysis revealed close associations of both PI and MVD with tumor stage and of AI with tumor grade. Our study demonstrated a strong relationship between PI and MVD, but did not show associations of AI with PI or MVD in TCC of the renal pelvis and ureter. Conclusions. It is suggested that the high activity of tumor cell proliferation with rich neovascularization may be related to the high malignant potential of the cancer, and evaluation of cell proliferation combined with angiogenesis may be useful in predicting the progression of the renal pelvic and ureteral TCC.

Endonuclease cleavage of DNA in the aged cochlea of Mongolian gerbil

Apoptosis is the most common form of physiological cell death, plays an important role in a variety of physiological and pathological situations, and is characterized by an endonuclease activation. Aged gerbils (24-month-old) showed a significant elevation of the distortion product otoacoustic emission (DPOAE) threshold and a significant decrease of the DPOAE level as compared to young gerbils (3- to 6-month-old), suggesting a disturbance of the outer hair cell in the aged cochlea. Furthermore, the endocochlear potential in the aged (15–75 mV) was apparently lower than that in the young (80–90 mV), indicating strial damage with aging. A terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method demonstrated the presence of the DNA fragmentation predominantly in the outer hair cells and spiral ganglion cells. These changes were sporadically observed in the inner hair cells and supporting cells. TUNEL positive cells were markedly observed in some aged gerbils showing deterioration in cochlear function, which may suggest the individual variability of presbycusis. On the other hand, the strial cells showed a marked atrophy, but no DNA fragmentation. These findings suggest that the aged change in the outer hair cells and spiral ganglion cells involves an apoptotic death pathway. However, strial atrophy with aging seems to be unrelated to apoptosis.

Endothelial Cell Apoptosis in Lipopolysaccharide–Induced Lung Injury in Mice

Background: Studies have shown the importance of apoptosis in vascular injury in vitro. We postulated that apoptosis of the endothelium contributes to vascular injury in vivo and may be involved in acute lung injury. Methods: To test this hypothesis, we investigated the incidence of endothelial cell apoptosis in acute lung injury induced in mice by the administration of lipopolysaccharide (LPS). Male ICR mice were administered LPS (20 mg/kg body weight) intravenously and sacrificed at specified times thereafter. Results: Histologic findings were consistent with acute lung injury which increased with time from 3 to 48 h after injection. Electrophoretic analysis of DNA that was extracted from lung tissue and 3'–end–labeled with digoxigenin demonstrated a fragmentation of DNA starting at 6 h. In situ terminal deoxynucleotidyl transferase–mediated dUTP biotin nick end–labeling (TUNEL) demonstrated DNA strand breaks in the endothelial cells. TUNEL also revealed DNA strand breaks in bronchial and alveolar epithelial cells as well as inflammatory cells in the interstitium. These TUNEL–positive cells appeared 6 h after injection. Electron–microscopic examination of the endothelium strongly suggested the morphological characteristics of apoptosis. Conclusion: Apoptosis was induced by LPS administration in endothelial cells in vivo. A role for such apoptosis is suggested in acute lung injury.

Apoptosis as a mechanism of 2-chloroethylethyl sulfide-induced cytotoxicity

Apoptosis is a mode of active cell death. We have examined whether 2-chloroethylethyl sulfide (CEES), a sulfur vesicating agent, triggers apoptosis as a cytotoxic mechanism. Incubation of thymocytes with CEES, resulted in an induction of apoptotic features of cell death. Treatment of cells with 100 μM CEES for 5 h increased DNA fragmentation to ∼40% of control. The fragmentation of DNA was visualized by agarose gel electrophoresis. It showed ladder pattern of DNA fragmentation, which indicates internucleosomal cleavage of DNA. Further evidence of apoptosis was observed in morphological changes of nuclei by using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. The percentage of TUNEL positive cells was dependent upon CEES concentrations. CEES induced the classical morphological features of apoptosis in nucleus. These features were accompanied by condensation of chromatin, which arranged in sharply declined clumps and fragmentation of nucleus. To study requirement for synthesis of new protein in CEES-induced apoptosis, we studied the effect of cycloheximide for apoptotic activity. This protein synthesis inhibitor did not suppress the CEES-induced apoptotic activity. Taken together, these results suggest that CEES-induced apoptosis as a cytotoxic mechanism and this process occurs independent of synthesis of new protein.

Studies on DNA fragmentation and disruption of the blood‐brain barrier in delayed radiation injuries in the rat brain

Delayed effect of radiation injury to the central nervous system often results in improper prognosis for the patients with malignant brain tumors. Two types of cell, the endothelial cell and the oligodendrocyte, are considered to be causative lesions for delayed radiation injury. Recently, morphological studies with both light and electron microscope have also indicated involvement of apoptosis at the acute stage of radiation injury. In the rat radiation injury model, we first examined the capillary endothelial cells when they were damaged after irradiation, using immunohistochemical analysis of endothelial blood antigen (EBA) as one of the indicators of normal function of the blood‐brain barrier (BBB). Secondly, we examined the types and location of DNA‐fragmented cells using in situ end‐labeling using the TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling) method. A single dose of 10, 20 or 30 Gy was irradiated to the hemispheres and the brains were analyzed at 3, 6 and 12 months later. Although damage of endothelial cells was seen in the brains at 6 months after 20 Gy irradiation, disruption of BBB, as indicated by no immunoreactivity of EBA, occurred only in the brains at 12 months after 30 Gy irradiation. DNA fragmentation, indicated by TUNEL positive cells, were shown in the oligodendrocyte, astrocytes and endothelial cells in the brains at 6 months after 20 Gy irradiation. The results suggest that both disruption of BBB and apoptosis in the glial cells and endothelial cells are involved in the delayed radiation injury in the rat brain.

Apoptosis Identified by DNA Fragmentation in Epidermal Neoplasms

In the previous study, we demonstrated apoptotic cells in basal cell carcinomas by means of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). Positive staining cells were present at the peripheries and centers of the nests. The number of positive cells increased in cases showing deep infiltration. We also stained other epidermal neoplasms using this modified TUNEL method. In squamous cell carcinomas, many positive cells were observed in poorly differentiated types, but only a few cells were stained in well-differentiated types. TUNEL positive cells were rare in Bowen's disease. No positive cells were seen in seborrheic keratosis or verruca vulgaris. Our studies showed that apoptotic cells were more common in rapid growth neoplasms than in slow growth neoplasms.