Apoptosis In Adult Rats Research Articles

Hypericum perforatum L. Nanoemulsion Mitigates Cisplatin-Induced Chemobrain via Reducing Neurobehavioral Alterations, Oxidative Stress, Neuroinflammation, and Apoptosis in Adult Rats.

Cisplatin (Cis) is a potent chemotherapeutic agent; however, it is linked with oxidative stress, inflammation, and apoptosis, which may harmfully affect the brain. Hypericum perforatum L. (HP L.) is a strong medicinal plant, but its hydrophobic polyphenolic compounds limit its activity. Therefore, our study aimed to investigate the neuroprotective action of HP L. and its nanoemulsion (NE) against Cis-induced neurotoxicity. The prepared HP.NE was subjected to characterization. The droplet size distribution, surface charge, and morphology were evaluated. In addition, an in vitro dissolution study was conducted. Compared to Cis-intoxicated rats, HP L. and HP.NE-treated rats displayed improved motor activity and spatial working memory. They also showed an increase in their antioxidant defense system and a reduction in the levels of pro-inflammatory cytokines in the brain. Moreover, they showed an increase in the expression levels of the PON-3 and GPX genes, which are associated with a reduction in the brain levels of COX-2 and TP-53. These findings were confirmed by reducing the immunohistochemical expression of nuclear factor kappa (NF-ƘB) and enhanced Ki-67 levels. In conclusion, HP L. is a promising herb and could be used as an adjuvant candidate to ameliorate chemotherapeutic-induced neurotoxicity. Moreover, HP.NE has superior activity in lessening Cis-induced oxidative stress, inflammation, and apoptosis in brain tissue.

Resveratrol ameliorates neuronal apoptosis and cognitive impairment by activating the SIRT1/RhoA pathway in rats after anesthesia with sevoflurane.

Studies have shown that long-term exposure to sevoflurane (SEV) may cause post-operative cognitive dysfunction. This study aimed to investigate the effects of resveratrol (RES) treatment on the changes in the cognitive function of rats after prolonged anesthesia with SEV. Seventy-six adult male rats were used in this study. The SEV model was established under continuous anesthesia for 6 hours. Rats were randomly classified into four groups as follows: Control, SEV+vehicle, SEV+pre-RES (RES was administered 24 hours before establishing the SEV model), and SEV+post-RES (RES was administered 1 hour after establishing the SEV model) groups. Neurobehavioral outcomes and the potential mechanism underlying RES-mediated neuroprotection through the silent information regulator 1 (SIRT1)/RhoA signaling pathway were evaluated. The water maze test showed that long-term exposure to SEV may lead to loss of learning and memory ability in rats (p < 0.05). Compared with the SEV+vehicle group, the RES treatment groups showed significantly improved neurobehavioral scores (p < 0.05). In addition, the SEV+pre-RES group had a better outcome than the SEV+vehicle group on days 1 or 2 (p < 0.05), unlike the SEV+post-RES group (p > 0.05). Western blotting showed that SIRT1, RhoA, and cleaved Caspase-3 (CC3) expression significantly increased in the SEV+vehicle group (p < 0.05), while Bcl2 expression decreased (p < 0.05). RES treatment further upregulated SIRT1 and Bcl2 expression and downregulated the expression of RhoA and CC3 (p < 0.05). In conclusion, RES treatment improved cognitive dysfunction by reducing neuronal apoptosis in adult rats exposed to SEV. RES partly exerted a neuroprotective effect through the activation of the SIRT1/RhoA signaling pathway.

Effects of nitrite graded doses on hepatotoxicity and nephrotoxicity, histopathological alterations, and activation of apoptosis in adult rats.

Nitrites are found in several forms; they are widely found in water resources and used as additives and preservatives for food and as a color source. We investigated the hazardous effects of exposing rats to different doses of nitrites. Moreover, we examined such impacts, after acute ingestion, on liver and renal tissues in rats and to what extent this affects the organs' functions. Animals were divided into five groups: one control group 1 (group C) and four sodium nitrite (NaNO2)-treated group (8 rats per group). The four NaNO2-treated groups include group 2 (N20), group 3 (N40), group 4 (N60), and group 5 (N75). NaNO2 was dissolved in distilled water, and single acute dose was orally given by gavage at 20, 40, 60, and 75mg/kg body weight, respectively. Our results revealed significant increase of liver enzymes activity-aspartate transaminase (AST), alanine aminotransferase (ALT), and creatinine between different groups with increasing doses of nitrite ingestion. The results of hepatic and renal oxidative stress showed significant increase in the malondialdehyde (MDA) levels and significant decrease in the antioxidant parameters, such as reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT), as the dose of nitrite increases. Further, the methemoglobin percent showed significant increase with increasing nitrite doses. Abnormal morphological alterations in the liver and kidney tissues were obviously proportional to the administered nitrite doses. The expression of caspase 3 and Bax level showed enhanced induction of immunoexpression, especially in the high doses of nitrites. On the other hand, the maximal immunoexpression level of anti-apoptotic marker Bcl2 was found in lower doses of nitrites, whereas marked decrease of Bcl2 levels was observed in the higher doses. In conclusion, administration of sodium nitrite in a dose-dependent manner is capable of inducing cellular and genetic toxicities and causes disturbance in biochemical analysis, oxidative and anti-oxidative balance, and methemoglobinemia. It also makes histopathological alterations and leads to the activation of apoptosis-related Bax, Bcl2, and caspase 3 genes of liver and kidney tissues in rats.

Promotion of mitochondrial fusion protects against developmental PBDE-47 neurotoxicity by restoring mitochondrial homeostasis and suppressing excessive apoptosis.

Polybrominated diphenyl ethers (PBDEs)-induced neurotoxicity is closely associated with mitochondrial abnormalities. Mitochondrial fusion and fission dynamics are required for the maintenance of mitochondrial homeostasis. However, little is known about how PBDEs disrupt this dynamics and whether such disruption contributes to impaired neurodevelopment.Methods: We investigated the effects of 2, 2', 4, 4'-tetrabromodiphenyl ether (PBDE-47), the dominant congener in human samples, on mitochondrial fusion and fission dynamics using PC12 cells, a well-defined in vitro neurodevelopmental model. We also evaluated the effects of perinatal low-dose PBDE-47 exposure on hippocampal mitochondrial dynamics and its association with neurobehavioral changes in adult Sprague-Dawley rats.Results: In vitro, PBDE-47 disrupted mitochondrial dynamics by inhibiting mitochondrial fusion and fission simultaneously, accompanied by mitochondrial fragmentation, membrane potential dissipation, ATP loss, and apoptosis activation. Specifically, enhancing mitochondrial fusion by the chemical promoter M1 or adenovirus-mediated mitofusin 2 (Mfn2) overexpression rescued PBDE-47-caused mitochondrial dynamic, morphological and functional impairments, prevented the resultant apoptosis and promoted neuronal survival. Unexpectedly, either stimulating mitochondrial fission by adenovirus-mediated fission protein 1 (Fis1) overexpression or suppressing mitochondrial fission by the mitochondrial division inhibitor-1 (Mdivi-1) failed to reverse whereas aggravated PBDE-47-induced mitochondrial damage and neuronal death. Importantly, promoting mitochondrial fusion by Mfn2 overexpression neutralized the detrimental effects elicited by Fis1 overexpression after PBDE-47 treatment. Finally, perinatal oral administration of PBDE-47 elicited neurobehavioral deficits and hippocampal neuronal loss via apoptosis in adult rats, which were associated with mitochondrial dynamics alterations manifested as a fragmented phenotype.Conclusion: Our results suggest that PBDE-47 disrupts mitochondrial dynamics to induce mitochondrial abnormalities, triggering apoptosis and thus contributing to neuronal loss and subsequent neurobehavioral deficits. Targeting mitochondrial fusion may be a promising therapeutic intervention against PBDE-47 neurotoxicity.

P,p\u2019-DDT induces testicular oxidative stress-induced apoptosis in adult rats

BackgroundThe 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p’-DDT) is a known persistent organic pollutant and male reproductive toxicant. The present study is designed to test the hypothesis that oxidative stress mediates p,p’-DDT-induced apoptosis in testis.MethodsMale Wistar rats received an intraperitoneal (ip) injection of the pesticide at doses of 50 and 100mg/kg for 10 consecutive days. The oxidative stress was evaluated by biomarkers such lipid peroxidation (LPO) and metallothioneins (MTs) levels. Antioxidant enzymes activities was assessed by determination of superoxide dismutase (SOD), catalase (CAT) and hydrogen peroxide (H2O2) production. In addition, glutathione-dependent enzymes and reducing power in testis was evaluated by glutathione peroxidase (Gpx), glutathione reductase (GR), glutathione S-transferase (GST) activities and reduced and oxidized glutathione (GSH - GSSG) levels. Apoptosis was evaluated by DNA fragmentation detected by agarose gel electrophoresis. Germinal cells apoptosis and the apoptotic index was assessed through the TUNEL assay.ResultsAfter 10 days of treatment, an increase in LPO level and H2O2 production occurred, while MTs level, SOD and CAT activities were decreased. Also, the Gpx, GR, GST, and GSH activities were decreased, whereas GSSG activity was increased. Testicular tissues of treated rats showed pronounced degradation of the DNA into oligonucleotides as seen in the typical electrophoretic DNA ladder pattern. Intense apoptosis was observed in germinal cells of DDT-exposed rats. In addition, the apoptotic index was significantly increased in testis of DDT-treated rats.ConclusionsThese results clearly suggest that DDT sub-acute treatment causes oxidative stress in rat testis leading to apoptosis.

Influence of mild traumatic brain injury during pediatric stage on short-term memory and hippocampal apoptosis in adult rats.

Traumatic brain injury (TBI) is a leading cause of neurological deficit in the brain, which induces short- and long-term brain damage, cognitive impairment with/without structural alteration, motor deficits, emotional problems, and death both in children and adults. In the present study, we evaluated whether mild TBI in childhood causes persisting memory impairment until adulthood. Moreover, we investigated the influence of mild TBI on memory impairment in relation with hippocampal apoptosis. For this, step-down avoidance task, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and immunohistochemistry for caspase-3 were performed. Male Sprague-Dawley rats were used in the experiments. The animals were randomly divided into two groups: sham-operation group and TBI-induction group. The mild TBI model was created with an electromagnetic contusion device activated at a velocity of 3.0 m/sec. The results showed that mild TBI during the pediatric stage significantly decreased memory retention. The numbers of TUNEL-positive and caspase-3-positive cells were increased in the TBI-induction group compared to those in the sham-operation group. Defective memory retention and apoptosis sustained up to the adult stage. The present results shows that mild TBI induces long-lasting cognitive impairment from pediatric to adult stages in rats through the high level of apoptosis. The finding of this study suggests that children with mild TBI may need intensive treatments for the reduction of long-lasting cognitive impairment by secondary neuronal damage.

Effects of isoflurane anesthesia on NR1 expression and neuronal apoptosis in hippocampus and cortex of adult rats

Objective To evaluate the effects of isoflurane anesthesia on NR1 expression and neuronal apoptosis in the hippocampus and cortex of adult rats .Methods Thirty-six adult male Sprague-Dawley rats , weighing 250-280 g ,were randomly assigned into 3 groups using a random number table ：control group （group C , n=6 ） ,O2 inhalation group （group O , n=6 ） and isoflurane anesthesia group （group I , n=24 ） .The rats were exposed to 2% isoflurane （group I） ,to pure oxygen （group O） ,or to air （group C） for 2 h .At 2 h ,and 1 ,7 and 14 days after the rats were awake （T1-4 ） ,Morris water maze test was performed .The rats were then sacrificed and brains were removed for isolation of the hippocampus and cortex .NR1 expression was detected using SABC immuno-histochemical technique and neuronal apoptosis was determined using TUNEL .Results Compared with group C , the escape latency at T2 and total swimming distance at T1 ,2 were significantly prolonged , and the expression of NR1 in hippocampi was down-regulated at T1 ,2 in group I ,and the expression of NR1 in the cortex was down-regulated in O and I groups （ P 0.05 ） .Conclusion Isoflurane anesthesia can decrease the cognitive function transiently ,which is related to inhibition of up-regulation of NR1 expression in the hippocampi ,but not related to neuronal apoptosis in adult rats . Key words: Isoflurane; Cognition disorders; Hippocampus; Receptors,N-methyl-D-aspartate; Apoptosis

Nigerian bonny‐light crude oil induces alteration in testicular stress response proteins and caspase‐3 dependent apoptosis in albino wistar rats

In the past few decades, there has been much concern about the adverse health effects of environmental contaminants in general and Crude Oil in particular around the Niger Delta region of Nigeria where all the crude Oil exploration is taking place. Studies have shown the repro-toxic effects of Bonny-light crude oil (BLCO). However, the insight into the mechanisms of gonadal toxicity induced by BLCO is not well known. In this study, we sought to elucidate the mechanism(s) underpinning the gonadal effects within hours of exposure to BLCO. Experimental rats were divided into five groups of four each. Animals were orally administered with a single dose of BLCO (800 mg/kg body weight) and killed at 0, 6, 12, 24, and 72 h post-treatment. The levels and time-course of induction of stress response proteins and apoptosis-related proteins like cytochorome C, caspase 3 and procaspase 9, Fas-FasL, NF-kB and TNF-α were determined to assess sequential induction of apoptosis in the rat testis. DNA damage was assessed by TUNEL assay. Administration of BLCO resulted in a significant increase in the levels of stress response proteins and apoptotis- related proteins as early as 6 h following exposure. Time-dependent elevations in the levels of the proteins were observed. The DNA damage was measured and showed time-dependent increase in the TUNEL positive cells of testicular cells. The study demonstrates induction of testicular apoptosis in adult rats following exposure to a single dose of BLCO.

Pharmacological Inhibition of Caspase-2 Protects Axotomised Retinal Ganglion Cells from Apoptosis in Adult Rats

Severing the axons of retinal ganglion cells (RGC) by crushing the optic nerve (ONC) causes the majority of RGC to degenerate and die, primarily by apoptosis. We showed recently that after ONC in adult rats, caspase-2 activation occurred specifically in RGC while no localisation of caspase-3 was observed in ganglion cells but in cells of the inner nuclear layer. We further showed that inhibition of caspase-2 using a single injection of stably modified siRNA to caspase-2 protected almost all RGC from death at 7 days, offering significant protection for up to 1 month after ONC. In the present study, we confirmed that cleaved caspase-2 was localised and activated in RGC (and occasional neurons in the inner nuclear layer), while TUNEL+ RGC were also observed after ONC. We then investigated if suppression of caspase-2 using serial intravitreal injections of the pharmacological inhibitor z-VDVAD-fmk (z-VDVAD) protected RGC from death for 15 days after ONC. Treatment of eyes with z-VDVAD suppressed cleaved caspase-2 activation by >85% at 3–4 days after ONC. Increasing concentrations of z-VDVAD protected greater numbers of RGC from death at 15 days after ONC, up to a maximum of 60% using 4000 ng/ml of z-VDVAD, compared to PBS treated controls. The 15-day treatment with 4000 ng/ml of z-VDVAD after ONC suppressed levels of cleaved caspase-2 but no significant changes in levels of cleaved caspase-3, -6, -7 or -8 were detected. Although suppression of caspase-2 protected 60% of RGC from death, RGC axon regeneration was not promoted. These results suggest that caspase-2 specifically mediates death of RGC after ONC and that suppression of caspase-2 may be a useful therapeutic strategy to enhance RGC survival not only after axotomy but also in diseases where RGC death occurs such as glaucoma and optic neuritis.

Low dose bisphenol A impairs spermatogenesis by suppressing reproductive hormone production and promoting germ cell apoptosis in adult rats

Bisphenol A (BPA), an estrogenic chemical, has been shown to reduce sperm count; however, the underlying mechanisms remain unknown. Herein, we show that oral administration of BPA (2 µg/kg) for consecutive 14 days in adult rats (BPA rats) significantly reduced the sperm count and the number of germ cells compared to controls. The serum levels of testosterone and follicle-stimulating hormone (FSH), as well as the level of GnRH mRNA in BPA rats were lower than those of control rats. Testosterone treatment could partially rescue the reduction of germ cells in BPA rats. Notably, the number of apoptotic germ cells was significantly increased in BPA rats, which was insensitive to testosterone. Furthermore, the levels of Fas, FasL and caspase-3 mRNA in the testicle of BPA rats were increased in comparison with controls. These results indicate that exposure to a low dose of BPA impairs spermatogenesis through decreasing reproductive hormones and activating the Fas/FasL signaling pathway.

Methoxychlor induces apoptosis via mitochondria- and FasL-mediated pathways in adult rat testis

In the past few years, there has been much concern about the adverse health effects of environmental contaminants in general and organochlorine in particular. Studies have shown the repro-toxic effects of long-term exposure to methoxychlor, a member of the organochlorine family. However, the insight into the mechanisms of gonadal toxicity induced by methoxychlor is not well known. In the present study we sought to elucidate the mechanism(s) underpinning the gonadal effects within hours of exposure to methoxychlor. Experimental rats were divided into six groups of four each. Animals were orally administered with a single dose of methoxychlor (50 mg/kg body weight) and killed at 0, 3, 6, 12, 24, and 72 h post-treatment. The levels and time-course of induction of apoptosis-related proteins like cytochorome C, caspase 3 and procaspase 9, Fas–FasL and NF-κB were determined to assess sequential induction of apoptosis in the rat testis. DNA damage was assessed by TUNEL assay and flowcytometry. Administration of methoxychlor resulted in a significant increase in the levels of cytosolic cytochrome c and procaspase 9 as early as 6 h following exposure. Time-dependent elevations in the levels of Fas, FasL, pro- and cleaved caspase 3 were observed. The DNA damage was measured and showed time-dependent increase in the TUNEL positive cells, and also by flowcytometry of testicular cells. The study demonstrates induction of testicular apoptosis in adult rats following exposure to a single dose of methoxychlor.

Lactational coumestrol exposure increases ovarian apoptosis in adult rats.

This study is the first to examine the increased apoptosis in the adult rat ovary after lactational exposure to coumestrol (COU), a potent phytoestrogen. Lactating dams were gavaged at doses of 0.01, 0.1, 1, and 10 mg/kg COU during the lactation period and the reproductive effects of female pups were investigated in young adults. Rats were sacrificed at postnatal days (PND) 81–84. Ovarian weights were reduced significantly at 0.1 and 1.0 mg/kg COU. The reduction in the ovarian weight occurred in parallel with an increase in the apoptosis at PND 135–140. A marked dose-dependent increase in the expressions of active caspase-3 and -7 was observed in ovarian granulosa cells. Immunostaining for active caspase-3 and the TUNEL staining of apoptotic cells were also increased in ovaries exposed to COU in a dose-dependent manner. These results suggest new sights into the effect of lactational exposure to COU on the female reproductive health.

Lindane induces testicular apoptosis in adult Wistar rats through the involvement of Fas–FasL and mitochondria-dependent pathways

Lindane, an organochlorine pesticide, is known to impair testicular functions and fertility. To elucidate the mechanism(s) underpinning the gonadal effects of lindane, we sought to investigate the levels of apoptosis-related proteins, namely cytochrome c, caspase-3 and-9, Fas and FasL in the testis of adult rats. Furthermore, the study aims to delineate whether nuclear factor kappa B (NF-κB) is involved in meditating the testicular effects of lindane. Animals were administered with a single dose of lindane (5 mg/kg body weight) and sacrificed at specific post-treatment intervals (0, 3, 6, 12, 24 and 72 h). Significant elevations in the levels of cytosolic cytochrome c with a parallel increase in pro-caspase-9 were observed as early as 6 h following exposure. Time-dependent elevations in the levels of Fas, FasL and caspase-3 were observed. Immunofluorescence studies revealed increased colocalization of Fas and caspase-3 in peritubular germ cells. FasL levels were increased in Sertoli and peritubular germ cells. The cytoplasmic levels of NF-κB p65 decreased from 3 h following exposure with a maximal decline at 12 and 24 h. Changes in the localization of NF-κB were observed with maximal nuclear translocation in germ cells at 12 and 24 h. Terminal deoxynucleotidyl transferase-mediated dUTP nickend-labeling (TUNEL) assay revealed a time-dependent increase in the number of apoptotic cells. Taken together, the data illustrate induction of testicular apoptosis in adult rats following exposure to a single dose of lindane. Early activation of NF-κB in contrast to late increase in Fas expression suggests a pro-apoptotic role of NF-κB in testicular response to lindane.

Repeated estradiol administration alters different aspects of neurogenesis and cell death in the hippocampus of female, but not male, rats

Estradiol has been shown to have neuroprotective effects, and acute estradiol treatment enhances hippocampal neurogenesis in the female brain. However, little is known about the effects of repeated administration of estradiol on the female brain, or about the effects of estradiol on the male brain. Gonadectomized male and female adult rats were injected with 5-bromo-2-deoxyuridine (BrdU) (200 mg/kg), and then 24 h later were given subcutaneous injections of either estradiol benzoate (33 μg/kg) or vehicle daily for 15 days. On day 16, animals were perfused and the brains processed to examine cells expressing Ki-67 (cell proliferation), BrdU (cell survival), doublecortin (young neuron production), pyknotic morphology (cell death), activated caspase-3 (apoptosis), and Fluoro-Jade B (degenerating neurons) in the dentate gyrus. In female rats, repeated administration of estradiol decreased the survival of new neurons (independent of any effects on initial cell proliferation), slightly increased cell proliferation, and decreased overall cell death in the dentate gyrus. In male rats, repeated administration of estradiol had no significant effect on neurogenesis or cell death. We therefore demonstrate a clear sex difference in the response to estradiol of hippocampal neurogenesis and apoptosis in adult rats, with adult females being more responsive to the effects of estradiol than males.

Increased expression of estrogen receptor beta in pachytene spermatocytes after short-term methoxyacetic acid administration.

Degeneration of primary spermatocytes by apoptosis occurs during normal spermatogenesis, as well as in several pathological conditions, including exposure to specific testicular toxicants. The mechanisms that regulate the death and survival of primary spermatocytes, however, are still not well understood. The recent localization of estrogen receptor beta (ERbeta) and P450 aromatase in pachytene spermatocytes suggests a role for estrogens in this step of spermatogenesis. Using a well-known model of pachytene spermatocyte apoptosis in adult rats consisting of the administration of methoxyacetic acid (MAA), we investigated the participation of ERbeta during the initial phase of apoptosis, prior to germ cell loss. Adult rats were treated with a single intraperitoneal dose of MAA, and DNA laddering analysis confirmed apoptotic cell death in the testis. In enriched germ cell fractions and testis from MAA-treated animals, ERbeta mRNA increased significantly at 3 and 6 hours, respectively. Next, stage-specific induction of ERbeta mRNA was demonstrated by use of laser capture microdissection of seminiferous tubules in combination with semiquantitative reverse transcription-polymerase chain reaction. The ERbeta protein also increased significantly after 6 hours and was mainly immunolocalized in the cytoplasm of pachytene spermatocytes of afflicted tubules. The cytoplasmic localization was confirmed by Western blot analysis of isolated cytoplasmic and nuclear fractions of testicular extracts. Finally, the MAA activation of ERbeta was tested in vitro in HepG2 cells cotransfected with ERbeta and a reporter construct that contained a consensus estrogen responsive element. Addition of MAA at similar doses used in vivo elicited a similar estrogenic activation as did estradiol at 1 nmol/L concentration. The present results raise the possibility that cytoplasmic ERbeta participates in the apoptotic process of pachytene spermatocytes induced by MAA. Whether MAA interacts with ERbeta in the cytoplasm of primary spermatocytes, preventing the progression of the first meiotic division, however, remains to be determined.

Kainic acid-induced seizures produce necrotic, not apoptotic, neurons with internucleosomal DNA cleavage: implications for programmed cell death mechanisms

Prolonged seizures (status epilepticus) induced by kainic acid activate programmed cell death mechanisms, and it is believed that kainic acid-induced status epilepticus induces neuronal apoptosis. In order to test this hypothesis, adult rats were subjected to 3-h kainic acid-induced seizures, with 24- or 72-h recovery periods. Neuronal death was assessed by light microscopy with the Hematoxylin and Eosin stain and with in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL stain), by electron microscopy, and by agarose gel electrophoresis of DNA extracted from five vulnerable brain regions. Spontaneous and MK-801-induced apoptotic neurons from retrosplenial cortex of neonatal rats, evaluated by light and electron microscopy, were used as positive controls for apoptosis. Surprisingly, the large chromatin clumps of apoptotic neurons were TUNEL negative, whereas the cytoplasm showed light-to-moderate TUNEL staining, consistent with a lack of identifiable nuclear membranes ultrastructurally, and with intermingling of nuclear and cytoplasmic contents. Ultrastructurally, the acidophilic neurons produced by kainic acid-induced status epilepticus, identified with Hematoxylin and Eosin stain, were dark, shrunken and necrotic, with pyknotic nuclei containing small, dispersed chromatin clumps, and with cytoplasmic vacuoles, some of which were swollen, disrupted mitochondria. No apoptotic cells were seen. Acidophilic neurons were found in up to 20 of 23 brain regions examined and comprised 10–25% of the total number of neurons examined. A subset of these neurons (<10% of the total number of neurons in five of 23 regions) had TUNEL-positive nuclei 72 h but not 24 h after status epilepticus. Internucleosomal DNA cleavage (DNA “laddering”) occurred in the four most damaged brain regions examined by electron microscopy 24 h after SE and the three most damaged regions 72 h after status epilepticus. Our results demonstrate that kainic acid-induced status epilepticus produces neuronal necrosis and not apoptosis in adult rats. The necrotic neurons show nuclear pyknosis, chromatin condensation and DNA laddering. Programmed cell death mechanisms activated by kainic acid-induced status epilepticus occur in neurons which become necrotic and could contribute to necrotic, as well as apoptotic, neuronal death.

Neonatal primary neuronal death induced by capsaicin and axotomy involves an apoptotic mechanism

To clarify the mechanism of capsaicin-induced primary neuronal cell death, newborn and adult rats were given a subcutaneous injection of capsaicin (50 mg/kg). Neonatal capsaicin injection induced neuronal apoptosis in the trigeminal ganglion. Apoptotic neurons had peripheral stacks of long parallel endoplasmic reticulum that are characteristic to primary neurons of the B-type, and exhibited nucleoplasmic condensation, nuclear shrinkage and cytoplasmic fragmentation. Light microscopically, apoptotic neurons exhibited a sign of DNA fragmentation as revealed by a nick end labelling method. The proportion of apoptotic cells was quite low during the first 12 h after capsaicin injection (<1%), rapidly increase to 10.44% by 24 h, and decreased to 0.29% by 48 h. Normal and vehicle control levels of apoptosis were <1%. Nerve growth factor (NGF, 0.5 mg/kg) simultaneously administered with capsaicin reduced the incidence of apoptosis by about 35% at 24 h post-injection. Neonatal transection of the infraorbital nerve induced neuronal apoptosis similar to that produced by the neonatal capsaicin in the maxillary division of the trigeminal ganglion. Unlike capsaicin, however, the neurotomy-induced apoptosis was seen in neurons of both the A- and B-types. Neither the capsaicin injection nor the neurotomy induced apoptosis in adult rats, though mitochondrial swelling similar to that seen at 0.5 h after neonatal capsaicin was observed after capsaicin injection in adults. The results indicate that the capsaicin-induced and nerve injury-induced primary neuronal damages in newborn rats share a common final pathway, apoptosis.

Significance of apoptosis in the temporal and stage-specific loss of germ cells in the adult rat after gonadotropin deprivation.

The major objectives of the present study were to document the temporal and stage-specific acceleration of germ cell apoptosis in adult rats after selective suppression of pituitary gonadotropins by GnRH antagonist (GnRH-A) treatment, and to examine the possibility that apoptosis is the sole mechanism of germ cell death in response to hormonal deprivation. Groups of adult male rats were given a daily injection of a vehicle for 14 days or GnRH-A (1.25 mg/kg BW) for 2, 5, 7, and 14 days. Analysis of testicular apoptotic DNA fragmentation revealed a detectable increase at Day 5 and a maximal increase at 14 days after treatment. In situ analysis of germ cell apoptosis fully corroborated the observed increase in the degree of DNA fragmentation with time and also revealed a stage-related activation of apoptosis of specific germ cells. A low incidence (0.06-0.09) of germ cell apoptosis (expressed as numbers per Sertoli cell) was detectable at stages I, IX-XI, and XII-XIV in control rats. Mean incidence of apoptotic germ cells specifically at stages VII-VIII increased significantly (0.40 +/- 0.06) by Day 5 and increased another 2.2-fold (over the 5-day treatment values) on Day 7 after GnRH-A treatment as compared to values in controls, where no apoptosis was detected. Significantly increased incidence of apoptosis at stages IX-XI (0.37 +/- 0.05) over control values (0.07 +/- 0.01) was noted by Day 7. Within the study paradigm, the highest number of dying cells occurred by Day 14, at which time a modest but significant (p < 0.05) increase in the incidence of apoptosis was also noted at stages I, II-IV, V-VI, and XII-XIV in comparison with control values. Stages VII-VIII and IX-XI still exhibited the higher number of cells undergoing apoptosis (0.97 +/- 0.22, and 1.03 +/- 0.22, respectively). Comparison between rates of apoptosis and cell degeneration measured at stages VII-VIII demonstrated an intimate association (r = 0.94; p < 0.001) between apoptosis and germ cell loss, strongly supporting the concept that germ cell death (at these stages) after removal of hormonal support in the adult rat occurs almost exclusively via apoptosis.