Caspase-3 Immunopositive Cells Research Articles

Chronic administration of delta9-tetrahydrocannabinol protects hyperinsulinemic gastric tissue in rats.

Hyperinsulinemia (HI) can result from some reasons such as an increase in basal/fasting circulating insulin and/or potentiation of postprandial insulin production. Diabetes mellitus (DM) is indirectly related to HI since it both causes and results from insulin resistance. Understanding the causes of HI and treating this is crucial for preventing DM. Previous research has shown that delta9-tetrahydrocannabinol (THC) has medicinal benefits. In light of this, the relationship between THC and oxidative stress, DNA repair mechanism, apoptosis, and its regulatory impact on appetite hormones in the gastric tissue of hyperinsulinemic rats has been investigated for the first time. Male rats (Spraque-Dawley, total = 32) were used, and they were randomly divided into the following groups (n = 8 in each group): control (CTRL), HI, THC administered control (THC, 1.5 mg/kg/day, during 4 weeks), and THC administered HI (HI + THC) groups. The number of poly (ADP-ribose) polymerase-1 and proliferating cell nuclear antigen (PCNA) and caspase-3 immunopositive cells in the HI group was significantly reduced compared to the CTRL group. The number of PCNA and caspase-9 immunopositive cells was significantly increased in the HI + THC group compared to the HI group. Obestatin immunopositive cell numbers in the HI + THC group were higher than in the HI and CTRL groups. The results show that THC administration may affect the regulation of appetite hormones and regeneration in the fundus of rats with HI. Glutathione (GSH) levels were higher in the HI + THC group than in the HI group. Both immunohistochemical and biochemical analyses revealed that THC promotes regeneration and regulates appetite hormones in hyperinsulinemic gastric tissues.

Protective Effects of pomegranate peel extract on the diabetic–Induced Damage in Rat testis

Background: Diabetes mellitus (DM) is known to cause a state of general oxidative stress. The decreased fertility rates have been attributed to testicular inflammation, oxidative stress and apoptosis in male diabetic patients. Pomegranate peel extract (PPE), known for its high content of bioactive constituents, posses anti-inflammatory, antioxidant, antidiabetic properties. Aim of the work: To investigate the potential benefits of PPE against testicular injury-induced by diabetes.Material and Methods: Forty adult male albino rats were randomly allocated to four groups: control group, PPE group (500 mg/kg/day orally for 8 weeks), diabetic group (received single intraperitoneal injection of 60 mg/kg STZ) and diabetic treated with PPE group. At the end of the experiment, the testes were dissected out and processed for light microscopy, immunohistochemical stainning and transmission electron microscope. Serum testosterone hormone were investigated.Results: Examination of diabetic group revealed a significant reduced serum testosterone level, severely damaged testis with thickened tunica albuginea, thickening and discontinuity of the tubular basement membrane, shrunken tubules with occluded lumen, depleted germ cells, markedly lost elongated spermatids, congestion and significantly increased caspase-3 immunopositive cells area percent. TEM results revealed shrunken nuclei, ballooned mitochondria, cytoplasmic vacuolations and wide intercellular spaces. Diabetic treated with PPE group showed restoration of normal arrangement of the seminiferous tubules with lumen full with mature elongated spermatids and narrow interstitial spaces in-between.Conclusion: DM induced damaging effect on the testis, increased the expression of caspas-3 apoptotic marker, fibrosis and lowered the testosterone level. However, PPE administration counteracted most of these changes so it could be used as adjuvant therapy for DM.

Modulatory role of dietary Thymus vulgaris essential oil and Bacillus subtilis against thiamethoxam-induced hepatorenal damage, oxidative stress, and immunotoxicity in African catfish (Clarias garipenus).

Thiamethoxam (TMX) is a widely used neonicotinoid insecticide for its effective potential for controlling insects from the agricultural field, which might induce toxicity to the aquatic biota. In this study, the role of the probiotic Bacillus subtilis (BS) and a phytogenic oil extract of Thymus vulgaris essential oil (TVEO) in the modulation of thiamethoxam (TMX)-induced hepatorenal damage, oxidative stress, and immunotoxicity in African catfish (Clarias garipenus) has been evaluated. Fish were subjected to TMX (5mgL-1) and fed with a diet either supplemented with BS (1000ppm) or TVEO (500ppm). The experiment lasted for 1month. By the end of the experiment, blood was sampled for biochemical analysis and fish organs and tissues were collected for histopathological and immunohistochemical examinations. Results showed a substantial increase of serum markers of hepatorenal damage such as the activities of aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) and levels of blood urea nitrogen (BUN) and creatinine with an obvious decrease of serum protein levels in the TMX-intoxicated group. Also, there was a considerable increase in malondialdehyde (MDA) levels and glutathione-S-transferase (GST) activity. TMX remarkably suppressed serum lysozyme activity, respiratory burst activity, and phagocytosis with a conspicuous elevation of the levels of interleukins (interleukin-1 beta (IL-1β) andinterleukin-6 IL-6). The histopathological findings showed that TMX induced degenerative changes and necrosis in the gills, liver, head kidneys, and spleen of the intoxicated fish. Significant alterations of frequency, size, and area percentage of melanomacrophage centers (MMCs), decreased splenocyte proliferation, and increased number of caspase-3 immunopositive cells were also observed. Contrariwise, the concurrent supplementation of either BS or TVEO in the diets of catfish partially mitigated both the histopathological and histomorphometric lesions of the examined tissues. Correspondingly, they improved the counts of proliferating cell nuclear antigen (PCNA) and caspase-3 immunopositive splenocytes. In conclusion, the co-administration of either BS or TVEO in catfish diets partially diminished the toxic impacts of TMX. Nonetheless, the inclusion of TVEO in the diets of catfish elicited better protection than BS against TMX-induced toxicity in response to its potential anti-inflammatory, antioxidant, anti-apoptotic, and immune-stimulant effects.

Does Omega-3 Ameliorate Olanzapine-Induced Testicular Toxicity in Adult Albino Rats? A Histo-morphometric Study

Introduction: Olanzapine is one of the most frequently used antipsychotics that had undesirable effects on male sexual function. Aim of the work: To explore possible protective effect of omega-3 on olanzapine - induced testicular toxicity. Material and methods: Forty adult albino rats were used and alienated into four equal groups: I (control), II (omega-3 treated), III (olanzapine treated) and IV (olanzapine +omega-3 treated).Group I received vehicles of olanzapine and omega-3 orally once daily. Group II received 400 mg/kg/day of omega-3 orally. Group III received oral olanzapine 0.5 mg/kg/day. Group IV received a combination of the same mentioned doses of both drugs. After 14 weeks, testicular specimens were taken and processed for light and transmission electron microscopic study. Results: Group III showed shrunken degenerated seminiferous tubules (ST) with corrugated basement membrane whose lumina were devoid of spermatozoa. Few degenerated spermatogonia, primary spermatocytes, elongated spermatids and many Sertoli cells (SCs) were seen. The tubules were separated with wide interstitial spaces and congested blood vessels. Ultrastructural results revealed spermatogonia with degenerated nucleus, loss of nuclear membranes and cell boundaries of primary spermatocytes. Elongated spermatids appeared abnormal in shape with defects in acrosomal cap. A high significant decrease in mean ST diameter, height of germinal epithelium while a high significant increase in SCs count and number of caspase-3 immunopositive cells were observed. Group IV showed improvement of the previously mentioned results. Conclusion: Omega-3 could protect against olanzapine induced testicular toxicity therefore; it can be used as an adjuvant therapy with antipsychotic drugs.

Protective Effects of Zinc on 2.45GHz Electromagnetic Radiation-Induced Oxidative Stress and Apoptosis in HEK293 Cells.

Several epidemiological studies have shown that exposure to electromagnetic radiation (EMR) can be harmful to human health. The purpose of this study was to examine oxidative parameters and apoptosis induced by EMR in human kidney embryonic cells (HEK293) and to investigate whether zinc (Zn) has protective effect on EMR-induced apoptosis in HEK293 cells. For our experiment, HEK293 cells were divided into four main groups, control, EMR, 50μM Zn + EMR, and 100μM Zn + EMR. HEK293 cells of EMR groups were exposed to 2.45GHz EMR for 1h. In Zn groups, HEK293 cells were incubated with different concentrations of Zn for 48h before EMR exposure. Oxidative stress parameters were determined by spectrophotometric method; bcl-2 and caspase-3 were assessed immunohistochemically and TUNEL method was performed for apoptotic activity. EMR group had higher malondialdehyde (MDA) level and lower superoxide dismutase (SOD) activity compared with control group. In Zn-applied groups, MDA was decreased and SOD activity was increased compared with EMR group. The number of the apoptotic cells and caspase-3 immunopositive cells at EMR group was increased significantly compared with the control group, whereas bcl-2 was decreased. Besides, Zn-treated groups showed a significant reduction in the number of apoptotic cells and caspase-3 from that of EMR group, whereas there was an increase in bcl-2 immunopositivity. Our findings show that EMR caused oxidative stress and apoptotic activation in HEK293 cells. Zn seems to have protective effects on the EMR by increasing SOD activity and bcl-2 immunopositivity, decreasing lipid peroxidation and caspas-3 immunopositivity.

Intranasally Administered S100A9 Amyloids Induced Cellular Stress, Amyloid Seeding, and Behavioral Impairment in Aged Mice.

Amyloid formation and neuroinflammation are major features of Alzheimer's disease pathology. Proinflammatory mediator S100A9 was shown to act as a link between the amyloid and neuroinflammatory cascades in Alzheimer's disease, leading together with Aβ to plaque formation, neuronal loss and memory impairment. In order to examine if S100A9 alone in its native and amyloid states can induce neuronal stress and memory impairment, we have administered S100A9 species intranasally to aged mice. Single and sequential immunohistochemistry and passive avoidance behavioral test were conducted to evaluate the consequences. Administered S100A9 species induced widespread cellular stress responses in cerebral structures, including frontal lobe, hippocampus and cerebellum. These were manifested by increased levels of S100A9, Bax, and to a lesser extent activated caspase-3 immunopositive cells. Upon administration of S100A9 fibrils, the amyloid oligomerization was observed in the brain tissues, which can further exacerbate cellular stress. The cellular stress responses correlated with significantly increased training and decreased retention latencies measured in the passive avoidance test for the S100A9 treated animal groups. Remarkably, the effect size in the behavioral tests was moderate already in the group treated with native S100A9, while the effect sizes were large in the groups administered S100A9 amyloid oligomers or fibrils. The findings demonstrate the brain susceptibility to neurotoxic damage of S100A9 species leading to behavioral and memory impairments. Intranasal administration of S100A9 species proved to be an effective method to study amyloid induced brain dysfunctions, and S100A9 itself may be postulated as a target to allay early stage neurodegenerative and neuroinflammatory processes.

Ameliorative effect of selenium in cisplatin-induced testicular damage in rats

In this study, we investigated the protective effect of selenium (Se) on cisplatin (Cis) induced testicular damage using histopathological, immunohistochemical and biochemical approaches. Twenty-one male Wistar rats were equally divided into three groups of seven rats each: control (C), Cis, and Cis+Se. Cis and Cis+Se group rats received Cis at a dose of 12mg/kg b.w./day, intraperitoneally for 3 consecutive days. Cis+Se group rats received selenium via oral gavage 3mg/kg/day (twice-a day as 1.5mg/kg) until 11th consecutive days starting at 5 days before cisplatin injection. C group received only 0.9% NaCl intraperitoneally and orally at same time and at equal volume. After the treatment, the histopathological, immunohistochemical and biochemical examinations were performed. In seminiferous tubules of Cis treated rats were observed the most consistent findings characterized with vacuolization, desquamation, disorganization, and also was a considerable reduction in elongated spermatids, however the Cis+Se group exhibited improved histopathologic changes. In the immunohistochemical examinations, caspase-3 immunopositive cells displayed higher in the Cis group according to C and Cis+Se groups. Bcl-2 and NF-κB staining revealed a moderate number in the C group and significantly fewer in the Cis group compared to the Cis+Se groups. Additionally, MDA levels were also significantly increased in the Cis group in comparison to Control group, but pretreatment with selenium prevented elevation of MDA levels significantly in Cis+Se group rats. This study indicates that Cis-treatment induced testicular apoptosis and lipid peroxidation, and combined treatment with selenium prevented severity of the toxicity in rats.

Hypoxia inducible factor-1α expression is associated with hippocampal apoptosis during epileptogenesis

Cell apoptosis can cause hippocampal neuronal loss after epileptic seizures. Hypoxia inducible factor (HIF)-1α is an important factor mediating apoptosis after brain injuries, such as cerebral ischemia and traumatic brain injures, but little research has been done on its role in the lithium chloride–pilocarpine induced epileptic model. Here, we used a rat model of pilocarpine-induced status epilepticus (SE) to investigate HIF-1α expression and apoptosis in the hippocampus, and to explore their relationship during epileptogenesis. 120 male Sprague Dawley (SD) rats were treated with lithium chloride–pilocarpine injections and divided into an experimental group (administered by MK-801) and a positive control group (administered by saline). Then the HIF-1α expression and hippocampal apoptosis were investigated by histological confirmation and western blotting at 24h, 3d, 7d and 14d, respectively. The results showed that the administration of MK-801 significantly reduced (P<0.05) HIF-1α expression and hippocampal apoptosis during epileptogenesis in comparison with the positive control. Moreover, the expression of HIF-1α and hippocampal apoptosis presented significant time-dependent changes (P<0.01) within 2 weeks, and their positive correlation (P<0.05) analyzed by Pearson׳s correlation analysis. Meanwhile, the HIF-1α immunostained cells were distributed in accord with TUNEL immunostained cells and Caspase-3 immunopositive cells in the hippocampus. These results indicate that the HIF-1α expression is associated with hippocampal apoptosis, and suggest that HIF-1α is an important factor during epileptogenesis.

EFFECT OF BASIC FIBROBLAST GROWTH FACTOR ON BEHAVIOR ABILITY AND NEURONS OF THE HIPPOCAMPAL CA3 AREA IN ALZHEIMER'S DISEASE MODEL RATS

The study aimed to examine the behavior ability and morphological changes in neuron in the hippocampal CA3 area of Alzheimer's disease (AD) model rats induced by β-amyloid protein (Aβ1–42) and observe the potentiality of the neuroprotective effect of basic fibroblast growth factor (bFGF) on the AD model rats. A total of 70 Wistar rats were randomly divided into the normal control group, the AD model group and the bFGF treatment group. The AD model rats were established by microinjection of Aβ1–42 solution into right hippocampal CA1 area. The bFGF was injected into the abdominal cavity of rats in the bFGF treatment group, and identical volume physiological saline was given for the other two groups. The colorimetric method was used to detect the choline acetyltransferase (ChAT) and acetylcholinesterase (AchE) activity after the behavior capability was determined. Real time-PCR (RT-PCR) was used to evaluate the expression of VEGF mRNA of the rat hippocampal CA3 area. Caspase-3 immunopositive cells in the hippocampal CA3 area were observed under a light microscopy and quantitative analysis were performed by cell morphometric technique. The ultra-microstructure of the neurons was also observed by a transmission electron microscopy (TEM). The results indicated that compared with the AD model group, the learning and memory abilities of the bFGF treatment group were obviously improved and the ChAT activity significantly increased (p &lt; 0.05), whereas the AChE activity, expression of VEGF mRNA and quantity of Caspase-3 immunopositive cells notably decreased (p &lt; 0.05). Under TEM, the neurons in the hippocampal CA3 area of the normal control group had moderate electron density, rule nucleus, integrity perinuclear membrane, evenly distributed chromoplasm and abundant cell organelle, however the neurons of the AD model group showed severely damaged, exhibiting cell body pyknosis, irregular nuclear membranes concentrated, intracytoplasm content concentrated, decreased or unclear organelles. The neuronic pathological lesion of the bFGF treatment group had lessened than that of the AD model group; some of them had distinct neuronal structure and abundant cell organelle. BFGF could efficiently improve the behavior ability and decrease the pathological lesion of hippocampus of the AD model rats, which might promote the neuroprotective effect in the AD.

The anti-oxidant and anti-apoptotic effects of nebivolol and zofenopril in a model of cerebral ischemia/reperfusion in rats

The aim of this experiment was to investigate whether nebivolol and zofenopril have protective effects against oxidative damage and apoptosis induced by cerebral ischemia/reperfusion (I/R). There were seven groups of rats, with each containing eight rats. The groups were: the control group, I/R group, I/R plus zofenopril, I/R plus nebivolol, I/R plus nebivolol and zofenopril, zofenopril only and nebivolol only. Cerebral I/R was induced by clamping the bilateral common carotid artery and through hypotension. The rats were sacrificed 1h after ischemia, and histopathological and biochemical analyses were carried out on their brains. The total antioxidant capacity was evaluated by using an automated and colorimetric measurement method developed by Erel. I/R produced a significant increase in the levels of total oxidant status and malondialdehyde levels, the number of caspase-3 immunopositive cells and activities of prolidase and paraoxonase in brain when compared with the control group (p<0.05). A significant decrease in brain total antioxidant capacity and nitric oxide levels were found in I/R group when compared with the control group (p<0.05). Both nebivolol and zofenopril treatment prevented decreasing of the total antioxidant capacity and nitric oxide levels, produced by I/R in the brain (p<0.05). Both nebivolol and zofenopril treatment prevented the total oxidant status, malondialdehyde levels, activities of paraoxonase and prolidase from increasing in brains of rats exposed to I/R (p<0.05). In conclusion, both nebivolol and zofenopril protected rats from ischemia-induced brain injury. The protection may be due to the indirect prevention of oxidative stress and apoptosis.

Atrazine exposure affects growth, body condition and liver health in Xenopus laevis tadpoles

Six studies were performed regarding the effects of atrazine, the most frequently detected pesticide in fresh water in the US, on developing Xenopus laevis tadpoles exposed 5 days post-hatch through Nieuwkoop Faber Stage 62. The levels of atrazine tested included those potentially found in puddles, vernal ponds and runoff soon after application (200 and 400 μg/L) and a low level studied by a number of other investigators (25 μg/L). One study tested 0, 25 and 200 μg/L, another tested 0, 200 and 400 μg/L, while the remaining four studies tested 0 and 400 μg/L. During all exposures, mortality, growth, metamorphosis, sex ratio, fat body (a lipid storage organ) size and liver weights, both relative to body weight, were evaluated. In selected studies, feeding behavior was recorded, livers and fat bodies were histologically evaluated, liver glycogen and lipid content were determined by image analysis, and immunohistochemical detection of activated caspase-3 in hepatocytes was performed. The NOEC was 25 μg/L. None of these exposure levels changed sex ratios nor were intersex gonads noted, however, no definitive histological evaluation of the gonads was performed. Although a marginal increase in mortality at the 200 μg/L level was noted, this was not statistically significant. Nor was there an increase in mortality at 400 μg/L versus controls. At the 400 μg/L level, tadpoles were smaller than controls by 72 h of exposure and remained smaller throughout the entire exposure. Appetite was not decreased at any exposure level. Slowed metamorphosis was noted only at 400 μg/L in two of five studies. Livers were significantly smaller in the study that tested both 200 and 400 μg/L, yet no pathological changes or differences in glycogen or lipid stores were noted. However, livers from 400 μg/L exposed tadpoles had higher numbers of activated caspase-3 immunopositive cells suggesting increased rates of apoptosis. Fat body size decreased significantly after exposure to 200 and 400 μg/L although these organs still contained some lipid and lacked any pathology. Since this was noted across all studies, it was considered the most sensitive indicator of atrazine exposure measured. The changes noted in body and organ size at 200 and 400 μg/L atrazine indicated exposure throughout development compromised the tadpoles. Significant reductions in fat body size could potentially decrease their ability to survive the stresses of metamorphosis or reduce reproductive fitness as frogs rely on stored lipids for these processes.

Antiapoptotic effect of angiotensin-II type-1 receptor blockade in renal tubular cells of hyperoxaluric rats

In this study, we investigated the protective effect of losartan as an AT1 receptor antagonist by evaluating the expression of apoptosis-regulatory genes that contribute to the progressive damage in the renal tubules of hyperoxaluric rats. Rats were divided into 4 groups of 10 each; control (C), ethylene glycol (EG), ethylene glycol + losartan (EG + L) and Losartan (L). For 4 weeks 0.8% EG, as a precursor for oxalate, was administered to EG and EG + L and losartan (300 mg/l) was administered to groups EG + L and L. Urine and blood samples were collected for biochemical determination. Bcl-2, bax, caspase-3 and TGF-beta 1 antibodies were used for immunohistochemistry. Apoptosis was determined by TUNEL method. A marked increase in urinary oxalate levels of the rats in EG and EG + L groups was found. In the EG group a diffuse amount of oxalate crystals into the tubular lumina and interstitium in the cortex was observed. In the EG group GBM thickening, interstitial fibrosis and tubular atrophy with infiltration of mononuclear cell findings reduced in the EG + L group were presented as well. In the EG group, immunoreactivity of TGF-beta 1 was increased in glomeruli and tubuli. In the EG + L group, immunoreactivity of TGF-beta 1 was decreased compared to the EG group. Bax expression increased in the renal tubules of EG group and reduced in the EG + L group comparing to the control. In the EG + L group, the immunoreactivity of bcl-2 was increased in glomeruli. In EG + L treated group, number of caspase-3 immunopositive cells were decreased compared to all groups (P < 0.01). Apoptotic cells were increased in the EG-treated group compared to the other groups. Decreased apoptotic cell number was observed in the EG + L compared to the EG group (P < 0.01). Our findings suggest that losartan may provide a beneficial effect against tubulointerstitial damage and decrease renal tubular apoptosis caused by hyperoxaluria.

Protection by Polaprezinc Against Radiation-Induced Apoptosis in Rat Jejunal Crypt Cells

Polaprezinc, an anti-ulcer drug, is a chelate compound consisting of zinc and L-carnosine. Polaprezinc has been shown to prevent gastric mucosal injury. The anti ulcer effects of polaprezinc have been ascribed to its antioxidative property. The effect of polaprezinc on ionizing radiation-induced apoptosis was studied in the jejunal epithelial crypt cells of rats. Seven-to eight week-old Wistar rats, which were treated with 100 mg/kg of polaprezinc orally 1h before irradiation or 2% carboxymethyl cellulose sodium in controls, were exposed to whole body X-ray irradiation at 2 Gy. The number of apoptotic cells per jejunum crypt was counted in haematoxylin and eosin stained sections at 0-6 h after irradiation. TUNEL positive cells and immunopositive cells for active caspase-3 per crypt were also counted. Accumulation of p53, p21(WAF1/CIP1) and Bax expression in the jejunum after irradiation were examined by Western blot analyses. Polaprezinc treatment given prior to radiation resulted in a significant reduction in numbers of apoptotic cells, TUNEL positive cells and active caspase-3 immunopositive cells in jejunal crypt cells. Polaprezinc treatment resulted in decreases of p53 accumulation, p21(WAF1/CIP1) and Bax expression after irradiation. Polaprezinc has a protective effect against ionizing radiation induced apoptosis in rat jejunal crypt cells.

Peroxynitrite and caspase-3 expression after ischemia/reperfusion in mouse cardiac arrest model.

NO is a putative neurotransmitter and neuromodulator in the brain. NO is not functioning as a direct neurotoxin. NO with the superoxide radical product peroxynitrite (ONOO-) is much more cytotoxic under tissue impairment conditions. Caspase-3, a potent effector of apoptosis that is triggered via several different signaling pathways, may play a very important role in neuronal cell death caused by various brain injuries. The relationship between mouse caspase-3 and peroxynitrite remains unclear. In the present study, we examined the in vivo expression of 3-nitrotyrosine (a metabolite of peroxinitrite) and caspase-3 after cerebral ischemia produced in a global ischemia model using mice (i.e., a cardiac arrest model). 3-nitrotyrosine immunoreactivity was detected in neuronal cells in the hippocampal dentate nucleus, and cortical regions starting at 12 hrs after ischemia. In particular, numerous neuronal cells were highly immunoreactive for 3-nitrotyrosine in the cortical regions. In hippocampal CA1 pyramidal neurons, 3-nitrotyrosine immunoreactivity was detected from 24 hrs. Caspase-3 immunopositive cells were observed in approximately the same area in which the positive reaction to the anti-nitrotyrosine antibody was observed. These results provide direct evidence for the induction of 3-nitrotyrosine and caspase-3 expression in vivo in an ischemia model using mice. The present findings suggest that peroxynitrite generated by cerebral ischemia/ reperfusion was strongly cytotoxic and induced neuronal cell death (apoptosis) mediated by caspase-3.

Temporal and spatial profile of caspase 8 expression and proteolysis after experimental traumatic brain injury.

Recent studies have demonstrated that the downstream caspases, such as caspase 3, act as executors of the apoptotic cascade after traumatic brain injury (TBI) in vivo. However, little is known about the involvement of caspases in the initiation phase of apoptosis, and the interaction between these initiator caspases (e.g. caspase 8) and executor caspases after experimental brain injuries in vitro and in vivo. This study investigated the temporal expression and cell subtype distribution of procaspase 8 and cleaved caspase 8 p20 from 1 h to 14 days after cortical impact-induced TBI in rats. Caspase 8 messenger RNA levels, estimated by semiquantitaive RT-PCR, were elevated from 1 h to 72 h in the traumatized cortex. Western blotting revealed increased immunoreactivity for procaspase 8 and the proteolytically active subunit of caspase 8, p20, in the ipsilateral cortex from 6 to 72 h after injury, with a peak at 24 h after TBI. Similar to our previous studies, immunoreactivity for the p18 fragment of activated caspase 3 also increased in the current study from 6 to 72 h after TBI, but peaked at a later timepoint (48 h) as compared with proteolyzed caspase 8 p20. Immunohistologic examinations revealed increased expression of caspase 8 in neurons, astrocytes and oligodendrocytes. Assessment of DNA damage using TUNEL identified caspase 8- and caspase 3-immunopositive cells with apoptotic-like morphology in the cortex ipsilateral to the injury site, and immunohistochemical investigations of caspase 8 and activated caspase 3 revealed expression of both proteases in cortical layers 2-5 after TBI. Quantitative analysis revealed that the number of caspase 8 positive cells exceeds the number of caspase 3 expressing cells up to 24 h after impact injury. In contrast, no evidence of caspase 8 and caspase 3 activation was seen in the ipsilateral hippocampus, contralateral cortex and hippocampus up to 14 days after the impact. Our results provide the first evidence of caspase 8 activation after experimental TBI and suggest that this may occur in neurons, astrocytes and oligodendrocytes. Our findings also suggest a contributory role of caspase 8 activation to caspase 3 mediated apoptotic cell death after experimental TBI in vivo.