Activated Caspase-3 Immunoreactivity Research Articles

Telmisartan neuroprotective effects in 3-nitropropionic acid Huntington's disease model in rats: Cross talk between PPAR-γ and PI3K/Akt/GSK-3β pathway

Huntington's disease (HD) is an inherited devastating neurodegenerative disorder with disabling motor and cognitive derangements that hinder the patients from performing their daily activities. AimsThe present study was carried out to investigate telmisartan-induced neuroprotection against 3-nitropropionic acid (3-NP) model of HD in rats. Main methodsTelmisartan was administered orally with a dose of 10 mg/kg/day, 1 h prior to 3-NP (10 mg/kg/day, i.p) for 14 days. Key findings3-NP-injected animals which were treated with telmisartan showed marked improvement in muscle strength and motor functions evaluated by rotarod, grip strength, and open field tests. Moreover, administration of telmisartan attenuated 3-NP-induced oxidative stress, neuro-inflammation, and apoptosis with prominent decline in malondialdehyde striatal content in addition to NADPH oxidase reduced expression contrary to noticeable increment in reduced glutathione content. Additionally, the pro-inflammatory markers; tumor necrosis factor-α, interleukin-1β, prostaglandin E2, and cyclooxygenase-2 contents were significantly reduced along with decreased active caspase-3 immunoreactivity. Telmisartan was also implicated in the modulation of phosphatidyl inositol 3-kinase/protein kinase B/glycogen synthase kinase-3β (PI3K/Akt/GSK-3β) and extracellular signal-regulated kinase (ERK) 1/2 cascades with consequent anti-oxidative, anti-inflammatory, and anti-apoptotic effects. Photomicrographs of telmisartan-treated animals confirmed its neuroprotective effects showing dismounted neuronal death and obvious increase in neuronal survival. These beneficial effects could be attributed to telmisartan's ability to induce peroxisome proliferator activated receptor-γ expression as well as its well-known blocking effect of angiotensin-II receptors type 1. SignificanceSubsequently, telmisartan is deemed as a promising candidate for HD management.

Involvement of caspase-3 in apoptosis of human lymphocytes exposed to cadmium chloride.

Lymphocytes are a group of white blood cells with a variety of roles their integrity is crucial for the body's immune responses. Cadmium, a heavy metal and environmental pollutant, is known as a toxicant to exert its adverse effects on some sort of cells including blood cells. In this study, human lymphocytes were divided into 3 groups: (1) lymphocytes at 0-h, (2) lymphocytes at 24h (control), (3) lymphocytes treated with cadmium chloride (15μM). Lymphocyte viability and plasma membrane integrity were assessed in these groups. In addition, the occurrence of apoptosis was investigated by assessment of nucleus diameter and flow cytometry. Activation of caspase-3 was also detected by immunocytochemistry. Result showed that lymphocyte's viability and plasma membrane integrity decreased in lymphocytes treated with cadmium as compared with the control group. Decreased nucleus diameter and result of flow cytometry demonstrated cadmium-induced apoptosis in human lymphocytes. Furthermore, lymphocytes treated with cadmium displayed intensely activated caspase-3 immunoreactivity in their cytoplasm. In conclusion, cadmium not only negatively effect on viability and plasma membrane, but also induces caspase-dependent apoptosis in human lymphocytes.

Mechanistic role of cAMP and hepatocyte growth factor signaling in thioacetamide-induced nephrotoxicity: Unraveling the role of platelet rich plasma

Chronic kidney diseases occur as result of exposure to wide range of deleterious agents as environmental pollutants, toxins and drug. Currently, there is no effective protective therapy against renal damage, fibrosis and its sequel of end stage renal disease. Platelet-rich plasma (PRP) has a progressively gained consideration in wound healing, repair/regeneration of damaged tissues and conservation of organ function. However, its impact on thioacetamide (TAA) induced chronic renal damage has not been elucidated yet. So, the present study was carried out to evaluate the possible protective and regenerative effect of PRP against TAA induced renal damage and their potential underlying mechanism. PRP treatment improved redox state, renal function disturbed histologicl features; decreased monocyte chemo-attractant protein-1 (MCP-1) level; increased Peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) marker of mitochondrial biogenesis and metabolism; cyclic adenosine monophosphate (cAMP); hepatocyte growth factor (HGF) and autophagy protein beclin-1 level. In addition, PRP treatment decreased apoptosis and fibrosis as evidenced by decreased active caspase3 and α-SMA expression and immunoreactivity, respectively. In conclusion, PRP could potentially protect against TTA-induced chronic kidney damage by alleviating oxidative stress, improving, mitochondrial biogenesis, autophagy, disruption of the inflammatory, apoptotic and fibrotic response induced by TTA.

Partial Loss of Presenilin Impairs Age-Dependent Neuronal Survival in the Cerebral Cortex

Mutations in the presenilin (PSEN1 and PSEN2) genes are linked to familial Alzheimer's disease (AD) and cause loss of its essential function. Complete inactivation of presenilins in excitatory neurons of the adult mouse cerebral cortex results in progressive memory impairment and age-dependent neurodegeneration, recapitulating key features of AD. In this study, we examine the effects of varying presenilin dosage on cortical neuron survival by generating presenilin-1 conditional knock-out (PS1 cKO) mice carrying two, one, or zero copies of the PS2 gene. We found that PS1 cKO;PS2(+/-) mice at 16 months exhibit marked neurodegeneration in the cerebral cortex with ∼17% reduction of cortical volume and neuron number, as well as astrogliosis and microgliosis compared with ∼50% reduction of cortical volume and neuron number in PS1 cKO;PS2(-/-) mice. Moreover, there are more apoptotic neurons labeled by activated caspase-3 immunoreactivity and TUNEL assay in PS1 cKO;PS2(+/-) mice at 16 months, whereas apoptotic neurons are increased in the PS1 cKO;PS2(-/-) cerebral cortex at 4 months. The accumulation of the C-terminal fragments of the amyloid precursor protein is inversely correlated with PS dosage. Interestingly, levels of PS2 are higher in the cerebral cortex of PS1 cKO mice, suggesting a compensatory upregulation that may provide protection against neurodegeneration in these mice. Together, our findings show that partial to complete loss of presenilin activity causes progressively more severe neurodegeneration in the mouse cerebral cortex during aging, suggesting that impaired presenilin function by PSEN mutations may lead to neurodegeneration and dementia in AD.

Efectos de la dexmedetomidina en conjunto con el precondicionamiento isquémico remoto en la lesión de isquemia-reperfusión renal en ratones

Background and objectivesThe aim of this study was to evaluate the effects of remote ischemic preconditioning by brief ischemia of unilateral hind limb when combined with dexmedetomidine on renal ischemia-reperfusion injury by histopathology and active caspase-3 immunoreactivity in rats. Methods28 Wistar albino male rats were divided into 4 groups. Group I (Sham, n=7): laparotomy and renal pedicle dissection were performed at 65th minute of anesthesia and the rats were observed under anesthesia for 130min. Group II (ischemia-reperfusion, n=7): at 65th minute of anesthesia bilateral renal pedicles were clamped. After 60min ischemia 24h of reperfusion was performed. Group III (ischemia-reperfusion+dexmedetomidine, n=7): at the fifth minute of reperfusion (100μg/kg intra-peritoneal) dexmedetomidine was administered with ischemia-reperfusion group; reperfusion lasted 24h. Group IV (ischemia-reperfusion+remote ischemic preconditioning+dexmedetomidine, n=7): after laparotomy, three cycles of ischemic preconditioning (10min ischemia and 10min reperfusion) were applied to the left hind limb and after 5min with group iii. ResultsHistopathological injury scores and active caspase-3 immunoreactivity were significantly lower in the Sham group compared to the other groups. Histopathological injury scores in groups iii and iv were significantly lower than group ii (P=.03 and P=.05). Active caspase-3 immunoreactivity was significantly lower in the group iv than group ii (P=.01) and there was no significant difference between group ii and group iii (P=.06). ConclusionsPharmacologic conditioning with dexmedetomidine and remote ischemic preconditioning when combined with dexmedetomidine significantly decreases renal ischemia-reperfusion injury histomorphologically. Combined use of two methods prevents apoptosis via active caspase-3.

Thrombin Mediates Vagal Apoptosis and Dysfunction in Inflammatory Bowel Disease

BackgroundIn inflammatory bowel disease, autonomic dysfunction contributes to symptoms, morbidity, and health care resource utilization. Efferent vagal neurons, which provide the primary parasympathetic input to the gastrointestinal tract, are housed in the dorsal motor nucleus of the vagus (DMV) in the brainstem. This study seeks to characterize the effects of IBD on DMV neuronal survival and function. MethodsTNBS (picrylsulfonic acid) was administered by enema to induce colitis in rats. Brain sections through the DMV were examined for neuronal apoptosis using TUNEL labeling, and for glial cell activation by immunofluorescence. Prothrombin production was evaluated via quantitative RT-PCR from DMV tissue, as well as by double immunofluorescence in DMV sections. To investigate the effects of thrombin in the DMV, thrombin or thrombin and an antagonist to its receptor were administered into the fourth ventricle via a stereotactically placed cannula. DMV sections were then examined for apoptosis by TUNEL assay. To evaluate the effect of thrombin on DMV neuronal function, we examined calcium signaling in primary DMV neuron cultures following exposure to thrombin and other neurotransmitters. ResultsTNBS colitis is associated with significantly increased rates of DMV neuronal apoptosis, affecting 12.7 % of DMV neurons in animals with colitis, compared to 3.4 % in controls. There was a corresponding increase in DMV neuron activated caspase-3 immunoreactivity (14.8 vs. 2.6 % of DMV neurons). TNBS-treated animals also demonstrated significantly increased DMV astrocyte and microglial immunoreactivity, indicating glial cell activation. DMV prothrombin production was significantly increased in TNBS colitis, with a close anatomic relationship between prothrombin and microglia. Direct DMV exposure to thrombin replicated the apoptosis and activation of caspase-3 seen in TNBS colitis; these effects were prevented by coadministration of the PAR-1 inhibitor FR171113. Cultured DMV neurons exhibited impaired calcium signaling in response to neurotransmitters following exposure to thrombin. Glutamate-induced calcium transients decreased by 59 %, and those triggered by GABA were reduced by 61 %. PAR-1 antagonism prevented these thrombin-induced changes in calcium signaling. ConclusionsIBD is associated with DMV microglial activation and production of prothrombin. Thrombin in the DMV causes vagal neuron apoptosis and decreased sensitivity to neurotransmitters.

Effects of dexmedetomidine in conjunction with remote ischemic preconditioning on renal ischemia–reperfusion injury in rats

Background and objectivesThe aim of this study was to evaluate the effects of remote ischemic preconditioning by brief ischemia of unilateral hind limb when combined with dexmedetomidine on renal ischemia–reperfusion injury by histopathology and active caspase-3 immunoreactivity in rats. Methods28 Wistar albino male rats were divided into 4 groups. Group I (Sham, n=7): Laparotomy and renal pedicle dissection were performed at 65th minute of anesthesia and the rats were observed under anesthesia for 130min. Group II (ischemia–reperfusion, n=7): At 65th minute of anesthesia bilateral renal pedicles were clamped. After 60min ischemia 24h of reperfusion was performed. Group III (ischemia–reperfusion+dexmedetomidine, n=7): At the fifth minute of reperfusion (100μg/kg intra-peritoneal) dexmedetomidine was administered with ischemia–reperfusion group. Reperfusion lasted 24h. Group IV (ischemia–reperfusion+remote ischemic preconditioning+dexmedetomidine, n=7): After laparotomy, three cycles of ischemic preconditioning (10min ischemia and 10min reperfusion) were applied to the left hind limb and after 5min with group III. ResultsHistopathological injury scores and active caspase-3 immunoreactivity were significantly lower in the Sham group compared to the other groups. Histopathological injury scores in groups III and IV were significantly lower than group II (p=0.03 and p=0.05). Active caspase-3 immunoreactivity was significantly lower in the group IV than group II (p=0.01) and there was no significant difference between group II and group III (p=0.06). ConclusionsPharmacologic conditioning with dexmedetomidine and remote ischemic preconditioning when combined with dexmedetomidine significantly decreases renal ischemia–reperfusion injury histomorphologically. Combined use of two methods prevents apoptosis via active caspase-3.

Quercetin postconditioning attenuates myocardial ischemia/reperfusion injury in rats through the PI3K/Akt pathway

Quercetin (Que), a plant-derived flavonoid, has multiple benefical actions on the cardiovascular system. The current study investigated whether Que postconditioning has any protective effects on myocardial ischemia/reperfusion (I/R) injury in vivo and its potential cardioprotective mechanisms. Male Sprague-Dawley rats were randomly allocated to 5 groups (20 animals/group): sham, I/R, Que postconditioning, Que+LY294002 [a phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway inhibitor], and LY294002+I/R. I/R was produced by 30-min coronary occlusion followed by 2-h reperfusion. At the end of reperfusion, myocardial infarct size and biochemical changes were compared. Apoptosis was evaluated by both TUNEL staining and measurement of activated caspase-3 immunoreactivity. The phosphorylation of Akt and protein expression of Bcl-2 and Bax were determined by Western blotting. Que postconditioning significantly reduced infarct size and serum levels of creatine kinase and lactate dehydrogenase compared with the I/R group (all P<0.05). Apoptotic cardiomyocytes and caspase-3 immunoreactivity were also suppressed in the Que postconditioning group compared with the I/R group (both P<0.05). Akt phosphorylation and Bcl-2 expression increased after Que postconditioning, but Bax expression decreased. These effects were inhibited by LY294002. The data indicate that Que postconditioning can induce cardioprotection by activating the PI3K/Akt signaling pathway and modulating the expression of Bcl-2 and Bax proteins.

Effects of dietary soy on ethanol-impaired placentation and fetal growth

s / Placenta 34 (2013) A1–A99 A37 multivitamin supplementation; however, the safety of niacin in pregnancy has not been established. In vitro studies demonstrate the potential for teratogenicity with high doses of niacin. Moreover, excessive niacin levels consumed by the non-pregnant population demonstrated induced apoptosis within hepatocytes. However, no studies to date have analysed the effects of high doses of niacin on placental health and function. Thus, it was hypothesized that increasing doses of niacin past the normal physiological concentrationwill initiate a stress response in an in vitro placental explant culture model. Methodology: Human placental explants were collected following elective Caesarean section from term healthy pregnancies at the Ottawa General Hospital. Placental explants were exposed to increasing concentrations (2-1000 ug/mL) of niacin for a 24-hour incubation period at 5% oxygen (N1⁄44). Placental cell viability and function were assessed through measurement of lactate dehydrogenase (LDH) release, activated caspase-3 immunoreactivity, and human chorionic gonadotropin (hCG) production. Results: Across all doses of niacin tested (2-1000 mg/mL) no changes in LDH release, immunoreactivity for activated caspase-3 and hCG production were observed. Conclusion: There is no evidence to suggest that excessive niacin induces aberrant changes in placental cell viability or function. Additional in vitro experiments are needed to confirm that niacin intake past the currently established UL for pregnancy does not harm the feto-placental unit. http://dx.doi.org/10.1016/j.placenta.2013.06.109 P1.73. EFFECTS OF DIETARY SOY ON ETHANOL-IMPAIRED PLACENTATION AND FETAL GROWTH Fusun Gundogan , Wei Qi , Jeffrey Gilligan , Suzanne de la Monte 2,3 Women and Infants Hospital, Providence, RI, USA; Rhode Island Hospital, Providence, RI, USA; Warren Alpert Medical School at Brown University,

Effect of Ethyl Pyruvate on Paclitaxel-Induced Neuropathic Pain in Rats

BackgroundAlthough paclitaxel is a widely used chemotherapeutic agent for the treatment of solid cancers, side effects such as neuropathic pain lead to poor compliance and discontinuation of the therapy. Ethyl pyruvate (EP) is known to have analgesic effects in several pain models and may inhibit apoptosis. The present study was designed to investigate the analgesic effects of EP on mechanical allodynia and apoptosis in dorsal root ganglion (DRG) cells after paclitaxel administration.MethodsRats were randomly divided into 3 groups: 1) a control group, which received only vehicle; 2) a paclitaxel group, which received paclitaxel; and 3) an EP group, which received EP after paclitaxel administration. Mechanical allodynia was tested before and at 7 and 14 days after final paclitaxel administration. Fourteen days after paclitaxel treatment, DRG apoptosis was determined by activated caspase-3 immunoreactivity (IR).ResultsPost-treatment with EP did not significantly affect paclitaxel-induced allodynia, although it tended to slightly reduce sensitivities to mechanical stimuli after paclitaxel administration. After paclitaxel administration, an increase in caspase-3 IR in DRG cells was observed, which was co-localized with NF200-positive myelinated neurons. Post-treatment with EP decreased the paclitaxel-induced caspase-3 IR. Paclitaxel administration or post-treatment with EP did not alter the glial fibrillary acidic protein IRs in DRG cells.ConclusionsInhibition of apoptosis in DRG neurons by EP may not be critical in paclitaxel-induced mechanical allodynia.

Neuroprotective effects of uridine in a rat model of neonatal hypoxic–ischemic encephalopathy

Neonatal hypoxic–ischemic encephalopathy (HIE) is a major cause of neurological disability requiring newer therapeutic strategies. Uridine is the principal circulating pyrimidine in humans and a substrate for nucleotides and membrane phospholipids. The objective of this study was to investigate the effects of uridine in a neonatal rat model of HIE. Rat pups subjected to hypoxic–ischemic insult on postnatal day 7 were injected intraperitoneally with either saline or uridine (100, 300 or 500mg/kg) for three consecutive days and brains were collected for evaluation of brain infarct volume and apoptosis. Compared with Control group, uridine at 300 and 500mg/kg doses significantly reduced percent infarct volume, TUNEL(+) cell ratio and active Caspase-3 immunoreactivity in the cortex, as well as in CA1 and CA3 regions of the hippocampus. Uridine (300 and 500mg/kg) also decreased active Caspase-3 expression in the ipsilateral hemisphere. These data indicate that uridine dose-dependently reduces brain injury in a rat model of neonatal HIE by decreasing apoptosis.

Mild hypothermia reduces activated caspase-3 up to 1 week after a focal cerebral ischemia induced by endothelin-1 in rats

Hypothermia is a promising neuroprotective therapy that has been shown to reduce apoptosis after an ischemic insult. This study evaluated the effect of mild hypothermia on activated caspase-3 up to 1 week after the induction of a stroke.Endothelin-1 (Et-1) was used to elicit transient focal cerebral ischemia in rats. Twenty minutes after the ischemic insult, a state of mild hypothermia (33°C) was imposed for a duration of 2h. The functional outcome, infarct volume and activated caspase-3 immunoreactivity (IR) were assessed at 8, 24 and 72h, and one week after the insult. During the experiment the cerebral blood flow (CBF) was measured via Laser Doppler Flowmetry.Hypothermia improved the neurological outcome at all of the time points studied compared to the normothermic group, and was associated with a reduction in infarct volume. In both groups, activated caspase-3 IR peaked 24h after the Et-1 induced insult and hypothermia significantly reduced the number of apoptotic cells at 8h, 24h and 1 week after ischemia. Furthermore, the hypothermic treatment did not affect the CBF in the Et-1 model.These findings indicate that in the Et-1 model, hypothermia exerts a long lasting effect on stroke-induced apoptosis.

Different pattern of changes in calcium binding proteins immunoreactivity in the medial prefrontal cortex of rats exposed to stress models of depression

Reductions in the number and size of neurons in the medial prefrontal cortex (mPFC) have been documented in many post-mortem studies of depressed patients and animals exposed to stress. Here, we examined the effect of chronic unpredictable stress (CUS) and chronic mild stress (CMS) on specific populations of neurons in the rat mPFC. Antibodies directed against parvalbumin (PV), calbindin D-28K (CB) and active caspase-3 have been used to quantify the numerical density of PV-immunoreactive (PV-ir), CB-ir and active caspase-3-ir cells, and to measure the relative optical density of neuropil. CUS decreased the density of CB-ir neurons and the optical density of CB-ir neuropil. In turn, CMS increased the densities of both CB-ir neurons and neuropil, while PV-ir neurons and PV-ir neuropil were not changed. The frequency distribution of neuronal surface areas was significantly different only for PV-ir neurons, and only between the control and CUS group. CMS reduced the density of active caspase-3-ir cells while CUS did not. We concluded that the mPFC reveals a different pattern of changes in neurons containing calcium binding proteins and active caspase-3 immunoreactivity in response to CUS and CMS.

Anesthetic Effects on the Developing Nervous System

Anesthetic Effects on the Developing Nervous System

335. X-LINKED INHIBITOR OF APOPTOSIS PROTEIN (XIAP) EXPRESSION PATTERNS IN THE SHEEP OVARY

At reproductive age, the ovary undergoes continual cyclicity of follicles due to multiple positive and negative signals that promote follicle growth and development, selection for ovulation, or atresia. The majority of follicles undergo atresia, a degenerative process involving granulosa cell apoptosis. This process is executed by caspases, which are cysteine proteases. Caspases are potently inhibited by XIAP providing a potential mechanism to control follicular atresia. The study objective was to test the hypothesis that XIAP will show elevated expression in healthy antral follicles compared to atretic antral follicles and will show an inverse relationship with active caspase-3 immunoreactivity in the same antral follicle during the sheep estrous cycle. Reproductively mature Romney ewes (n = 9) had estrous cycles synchronized with a prostaglandin F2α analogue, then tissue was collected on days 14, 15 and 16 of the subsequent natural cycle. Analysis involved determining the presence and localization of XIAP using in situ hybridization histochemistry and immunohistochemistry then comparing XIAP expression patterns with distribution of active caspase-3 protein. XIAP mRNA was not detected in primordial, primary and secondary follicles. In contrast, XIAP protein was present from the primary stage onwards. Antral follicles showed positive XIAP mRNA and protein expression in both granulosa and thecal cell layers and antral follicles on the same tissue section showed variable expression. All day 14 antral follicles were positive for XIAP mRNA expression irrespective of the level of active caspase-3 immunoreactivity, whereas an inverse relationship between active caspase-3 and XIAP was apparent in the majority of day 15 and 16 antral follicles. XIAP protein was widely expressed in active caspase-3 negative antral follicles and indicated a negative correlation with the onset of active caspase-3 expression in the majority of follicles. These results indicate that XIAP may regulate follicular atresia and act as an indicator of follicular health in the sheep ovary.

Mild as well as severe insults produce necrotic, not apoptotic, cells: Evidence from 60-min seizures

We tested the hypothesis that mild insults produce apoptotic, and severe insults necrotic, cells by subjecting adult Wistar rats to 60-min instead of 3-h generalized seizures. Rats' brains were evaluated 6 and 24h later for evidence of neuronal necrosis by light and electron microscopy, the presence of TUNEL staining and active caspase-3 immunoreactivity, and for evidence of DNA laddering 24h after seizures. Apoptotic neurons from the retrosplenial cortex of postnatal day 8 rat pups served as positive controls. Six and 24h after seizures, 16 and 15 brain regions respectively out of 24 showed significant numbers of acidophilic neurons by hematoxylin and eosin stain. Three brain regions had significant numbers of TUNEL-positive neurons 24h after seizures. No neurons showed active caspase-3 immunoreactivity. Acidophilic neurons were necrotic by electron-microscopic examination. Ultrastructurally, they were shrunken and electron-dense, with shrunken, pyknotic nuclei and swollen mitochondria with disrupted cristae. Nuclei did not contain the irregular chromatin clumps found after 3-h seizures. None of the six brain regions studied ultrastructurally that show DNA laddering 24h after 3-h seizures showed DNA laddering 24h after 60-min seizures, probably because there were too few damaged neurons, although the lack of chromatin clumping might have been a contributing factor. Following seizures, a mild as well as a severe insult produces caspase-3-negative necrotic neurons. These results do not support the hypothesis that mild insults produce apoptotic, and severe insults, necrotic, cells.

Inhibition of cell death results in hyperganglionosis: implications for enteric nervous system development

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC) that delaminate from the neural tube and undergo extensive migration and proliferation in order to colonize the entire length of the gut and differentiate into many millions of neurons and glial cells. Although apoptotic programmed cell death is an essential physiological process during development of the majority of the vertebrate nervous system, apoptosis within early ENS development has not been comprehensively investigated. The aim of this study was to determine the presence and extent of apoptosis within the vagal NCC population that gives rise to most of the ENS in the chick embryo. We demonstrated that apoptotic cells, as shown by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling and active caspase-3 immunoreactivity, are present within an electroporated green fluorescent protein (GFP) and human natural killer-1 (HNK-1) immunopositive NCC population migrating from the vagal region of the neural tube to the developing foregut. Inhibition of caspase activity in vagal NCC, by electroporation with a dominant-negative form of caspase-9, increased the number of vagal NCC available for ENS formation, as shown by 3-dimensional reconstruction of serial GFP or HNK-1 labelled sections, and resulted in hyperganglionosis within the proximal foregut, as shown by NADPH-diaphorase whole gut staining. These findings suggest that apoptotic cell death may be a normal process within the precursor pool of pre-enteric NCC that migrates to the gut, and as such it may play a role in the control of ENS formation.

Effects of memantine and galantamine given separately or in association, on memory and hippocampal neuronal loss after transient global cerebral ischemia in gerbils

Galantamine is an acetylcholinesterase inhibitor and memantine is a non competitive antagonist of NMDA receptors that are being used to treat Alzheimer's disease (AD) patients. The fact that drugs with different mechanisms of action are available to treat AD introduces the prospect of prescribing drug combinations to amplify drug efficacy. This study was planed to evaluate the potential neuroprotective effects of galantamine combined with memantine in a transient global cerebral ischemia model in gerbils. Animal groups included in the study were: sham, ischemia, and ischemia plus galantamine (1 mg/kg and 10 mg/kg), memantine (10 mg/kg and 20 mg/kg), 1 mg/kg galantamine plus 10 mg/kg memantine, and 10 mg/kg galantamine plus 10 mg/kg memantine, respectively. Surviving pyramidal neurons in the CA1 subfield of the hippocampus, TUNEL, caspase-3 and SOD-2 immunohistochemistries, and the object placement test were evaluated 72 h after reperfusion. Memantine did not exert a clear neuroprotective effect, nor did it prevent spatial memory loss. In a previous study using the same experimental model, galantamine was neuroprotective and improved spatial memory. In this study, the association of 10 mg/kg memantine with 10 mg/kg galantamine increased the number of living pyramidal neurons, reduced TUNEL, active caspase-3 and SOD-2 immunoreactivity, and preserved spatial memory after ischemia-reperfusion injury; however, the effects of the combination were not statistically different from those observed in animals treated with galantamine alone. We believe these results are of interest from a clinical point of view because the association of both drugs is being used in clinical practice and in clinical trials to treat Alzheimer's disease and vascular dementia.

AMPA-induced dark cell degeneration is associated with activation of caspases in pyramidal neurons of the rat hippocampus

Various neurons in the central nervous system (CNS) exhibit selective vulnerability to AMPA-induced delayed neurotoxicity known as dark cell degeneration. Hippocampal pyramidal neurons in the CA1 and CA3 regions display such vulnerability that encompasses morphological changes including cytoplasmic and nuclear condensation, neuronal shrinkage, formation of cytoplasmic vacuoles, and general failure of physiology. The present study was undertaken to ascertain the potential involvement of initiator (caspase-9) and executor (caspase-3) caspases in AMPA-receptor-induced dark cell degeneration in pyramidal neurons. Immunohistochemical analyses revealed that immunoreactivity of the active form of caspase-9 and -3 was increased in pyramidal neurons in CA1 and CA3 regions of the hippocampus following AMPA (100 microM). Elevated levels of active caspase-9 immunoreactivity generally preceded elevations in active caspase-3 immunoreactivity. The pan caspase inhibitor FK011 effectively attenuated AMPA-induced dark cell degeneration in both CA1 and CA3 regions. Collectively, the data suggest a role for these caspases in mediating AMPA-induced toxicity in pyramidal neurons of the rat hippocampus.

Tissue Fixation Effects on Immunohistochemical Staining of Caspase-3 in Brain Tissue

Fixation methods for tissue often vary amongst clinical and research laboratories. To evaluate the effects of fixation method on studies of brain tissue, we examined immunohistochemical outcomes amongst 2 fixatives, 4 caspase-3 antibodies, and 2 species (human infants and piglets). Fixatives were 10% neutral buffered formalin (NBF) or 10% NBF and glacial acetic acid (FAA). Antibodies for caspase-3 were commercially obtained and included 2 for active caspase-3, and 2 for procaspase-3 (CASP3 and CPP32). Immunohistochemical staining of caspase-3 varied with fixation method, with the greatest effect of fixation method observed for the active caspase-3 antibodies and this effect was present in both species. In NBF-fixed tissue, active caspase-3 immunoreactivity was only visible microscopically, and was specific to neuronal cell bodies. In FAA-fixed tissue, active caspase-3 immunoreactivity was visible macroscopically, and predominantly present in fiber tracts and fasciculi compared with neuronal bodies. Fixation and species differences were also identified for the procaspase-3 antibodies, CASP3 and CPP32, where FAA-fixed pig tissue showed abundant staining of blood vessels that were not observed in the NBF-fixed pig tissue or in the human tissue. This study characterizes differences in immunohistochemical outcomes using commercially available antibodies for caspase-3, according to tissue fixation method and species.