Activation Of Apoptotic Process Research Articles

Anticancer Activity of Combretum fragrans F. Hoffm on Glioblastoma and Prostate Cancer Cell Lines.

Background:Cancer incidence has been growing in an alarming rate worldwide and new therapeutics are needed, particularly for intractable and chemoresistant cases. We evaluated the cytotoxic effects of Combretum fragrans F. Hoffm (Combretaceae) on glioblastoma (U87MG and C6) and prostate (PC-3) cancer cell lines. Methods:The cytotoxic effect of the methanolic extract of the stem bark of Combretum fragrans was assessed using XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) test. Expressions of Akt and ERK1/2 were determined using Western blot technique, while Caspase-3/7 kits were used to evaluate caspase-3/7 activity. Results: C. fragrans extract inhibited the proliferation of U87 (IC50 = 20.13 µg/mL), C6 (IC50 = 12.17 µg/mL), and PC-3 (IC50 = 11.50 µg/mL) cells. Treatment with the extract resulted in lower levels (p < 0.001) of phospho-ERK1/2 and phospho-Akt in U87 cells, and instead, higher levels of phospho-ERK1/2 (p < 0.001) in C6 and PC-3 cells. An increase in caspase-3/7 activity was observed, mainly after 24 hours of treatment, indicating the activation of apoptotic processes. Conclusion:Altogether, these results suggest that C. fragrans have potent anticancer properties. This plant should be further investigated for developing new anticancer drugs.

Сучасні уявлення про патогенез міокардиту

Діагностика та прогнозування перебігу міокардиту – одна з найскладніших і невирішених проблем сучасної кардіології не тільки в Україні, а й в розвинених країнах світу. Загальновідомо, що для розроблення адекватних методів діагностики, лікування та попередження ускладнень необхідними є фундаментальні знання, які стосуються патогенетичних механізмів розвитку і прогресування певного захворювання. В патогенезі порушення скоротливої функції серця і його дилатації як у гостру, так і в хронічну стадію міокардиту першочергову роль відіграють імунопатологічні реакції, які виявляються автоімунізацією та гіперреактивністю щодо структурних елементів серцевого м’яза. В основі патогенетичних механізмів вірусного міокардиту лежить комплекс чинників – пряма цитотоксична дія вірусу на кардіоміоцити, активація процесів апоптозу, а також реакцій первинного і вторинного імунітету, ураження мікросудинного русла, ремоделювання скоротливого апарату серцевого м’яза. Основними прозапальними цитокінами, які виробляються імунними клітинами у вогнищі запалення, є γ-інтерферон, фактор некрозу пухлини α, інтерлейкін (ІЛ) 1β, ІЛ-2, ІЛ-6, ІЛ-17А, ІЛ-23. Інший механізм ушкодження міокарда пов’язаний з активацією імунопатологічних реакцій гуморального типу із синтезом кардіоспецифічних антитіл, зокрема до β1-адренорецептора, кардіального міозину, актину, ламініну, віментину та інших структур серцевого м’яза. Суттєва роль у патогенезі міокардиту нині відводиться стимуляції toll-подібних рецепторів 2-го та 4-го типу й активації матриксних металопротеїназ, що має безпосередній зв’язок із продукцією прозапальних цитокінів. Перспективним для уточнення деяких патогенетичних мезханізмів запального ураження серцевого м’яза на сьогодні вважається і вивчення ролі різних типів мікроРНК. На цей час світовою кардіологічною спільнотою визнана актуальність подальшого вивчення різних механізмів патогенезу міокардиту з метою визначення тих патогенетичних ланок, вплив на які може зменшити патологічний ефект імунозапального ураження серця та тяжкість перебігу захворювання й поліпшити прогноз для пацієнтів з міокардитом.

Comparative Evaluation of the Expression of Apoptosis Proteins at the Influence of Metal Containing Polymeric Nanocomposites on the Brain

Background. The creation and the attempt to introduce into medicine new nanostructured materials dictate the need for careful study of their safety for human health and the environment. At the same time, the existing classical approaches to safety assessment do not always objectively reflect the real state of affairs; therefore, there is a need to introduce new approaches, including methods of intracellular safety assessment of nanoparticles and nanomaterials.The aim of this study was to evaluate expression level of apoptosis-regular proteins caspase-3 and bcl-2 in neurons of white outbred rats exposed to nanocomposites consisting of nanoparticles of metals (silver, bismuth and gadolinium) on a natural matrix of arabinogalactan isolated from Siberian larch wood.Materials and methods. Sixty outbred male rats were exposed to nanocomposites for 9 days at a dose of 500 μg of metal per 1 kg of animal body weight. The introduction of substances was carried out orally using atraumatic probe. Study of the activity of proteins of apoptosis modulators caspase-3 and bcl-2 and calculate the percentage of different types of reacted neurons was conducted. Statistics 6.1 was used to process results, p-values less than 0.01 were acknowledged as statistically significant.Results. It was found that the activation of apoptotic process occurs only when the nanocomposite is affected with the addition of silver nanoparticles, which are realized in a significant increase in the percentage of all types of neurons under study.Conclusion. Monitoring proteins – regulators of apoptosis can serve as a marker of early damage to nerve cells, since glial cells can form at the site of dead neurons that do not fulfill the main function of transmission of nerve impulses. The results obtained allow us to substantiate the need for a more thorough and prolonged study of silver nanoparticles encapsulated on the arabinogalactan matrix.

Transcriptional Regulation of Antioxidant Enzymes Activity and Modulation of Oxidative Stress by Melatonin in Rats Under Cerebral Ischemia / Reperfusion Conditions

The article studies the effect of melatonin on the intensity of free radical oxidation, the functioning of the enzymatic components of the antioxidant system and their transcriptional regulation in rats with experimental cerebral ischemia/reperfusion of the brain. The development of ischemia/reperfusion was characterized by the activation of apoptotic processes and the accumulation of mRNA of the genes Sod1, Cat, Gpx1, Gsr, Hif-1α, Nrf2, Nfkb2, and Foxo1 in the rats' brains. The use of melatonin in the presence of the pathological induction led to a change in these parameters towards the control values. In addition, the introduction of the hormone was accompanied by a decrease in lactate content, the level of lipoperoxidation products and oxidative modification of proteins, indicators of biochemiluminescence in the brain and blood serum. At the same time, there was a shift in the activity of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, which increased in the presence of a pathology, towards the control values. The revealed changes may be accounted for by antioxidant and neuroprotective properties of melatonin, which provided a decrease in the degree of mobilization of the protective systems in animal organism.

Single and repetitive microplastics exposures induce immune system modulation and homeostasis alteration in the edible mussel Mytilus galloprovincialis

Seashore invertebrates such as mussels are exposed to multiple bouts of pollution related to human activities. Plastic debris originating from land-based activities are a concerning issue as they may be fragmented in smaller pieces (microplastics, < 5 mm diameter) which have an excellent potential for uptake by a large variety of animals. Here, we set out to explore the whole transcriptome profiling of Mytilus galloprovincialis associated with temporal variability of microplastics concentrations. Mussels were submitted to (i) a single 18 days-exposure to a concentration of microplastics found during pollution events (4.6 E+5 polyethylene microbeads L−1), (ii) a recovery period to investigate the reversibility of microplastics effects and (iii) a repeated exposure to microplastics to evidence acclimation to microplastics pollution events. Overall, 18 days-exposure to microplastics was mostly associated with disruption of mussel global homeostasis resulting in the production of stress and immune-related proteins and as a consequence, a diminution of energy allocated to growth. During the recovery period, a contrasting response was observed with the activation of apoptotic processes and the up-regulation of immune-receptors and stress-related proteins (glutathione peroxidase, hsp70) in mussels previously exposed to microplastics. These divergent responses, suggest that the establishment of compensatory mechanism as an attempt to recover, is not sufficient to counteract physiological stress induced by the first exposure. Finally, the differences observed in gene expression between single and repeated exposures to microplastics suggest, under the experimental conditions tested, that mussels may be able to establish a stress-memory upon microplastics exposure.

ПОРІВНЯЛЬНА ОЦІНКА ВЕЛИЧИНИ АНТИАПОПТОТИЧНОГО ЕФЕКТУ СЕРЕД МОДУЛЯТОРІВ АКТИВНОСТІ NMDA-РЕЦЕПТОРІВ В УМОВАХ МОДЕЛЬНОЇ ПЕРЕХІДНОЇ ІШЕМІЇ РЕПЕРФУЗІЇ ОКА ЗА ДАНИМИ ПРОТОКОВО-ЦИТОМЕТРИЧНОГО АНАЛІЗУ

Модельна 60 хв. ішемія-реперфузія ока у щурів супроводжується активацією апоптотичних процесів в сітківці, про що достеменно свідчить збільшення в середньому в 20,22 раза пулу клітин, які перебувають у фазі SUB-G0G1, тобто мають ознаки фрагментації ядерної ДНК, що анонсує апоптоз. Однократна внутрішньовенна інфузія в лікувальному режимі через 30 хв після накладання лігатур розчинів амантадину або магнію сульфату, дозами 5 та 250 мг/кг відповідно, а також мемантин перорально з розрахунку 20 мг/кг, сприяють зменшенню відносної кількості ретиноцитів, які перебувають фазі SUB-G0G1 клітинного циклу, проявляючи тим самим антиапоптотичну дію. За спроможністю в умовах даної патології знижувати процес фрагментації ядерної ДНК, застосування розчину амантадину сульфату вірогідно переважає терапію розчином магнію сульфату або мемантином в середньому на 24,1 та 52,0 % відповідно.

The effects of the glial cell line-derived neurotrophic factor (GDNF) on the levels of mRNA of apoptotic genes Bax and Bcl-xl in the brain of mice genetically predisposed to pathological behavior

Similar to the other neurotrophic factors, the glial cell line-derived neurotrophic factor (GDNF) may inhibit apoptosis in vitro. However, the antiapoptotic GDNF effects in vivo are not known. We studied the effect of GDNF central administration of on the mRNA levels of genes encoding the proapoptotic protein Bax and the antiapoptotic protein Bcl-xl in the brain of ASC mice genetically predisposed to depressivelike behavior and mice of their nondepressive parental CBA strain. We found that the GDNF injection increased the Bcl-xl mRNA content in the hippocampi of mice of both strains (ρ < 0.05) and Bax mRNA (ρ < 0.05) in the hippocampus of ASC mice. Thus, we revealed both the antiand proapoptotic effects of GDNF in vivo. These effects substantially depended on the genotype of the animals. We also observed significant interstrain differences in the Bax and Bcl-xl mRNA levels. In ASC mice, the Bax mRNA levels were significantly higher (ρ < 0.001 and ρ < 0.01) in all investigated structures and the content of Bcl-xl mRNA was elevated in the midbrain. Our data demonstrate the activation of apoptotic processes in ASC mice and the substantial compensatory changes, probably directed to the rise of the threshold for neuronal apoptosis.

Spinal cord changes consequent to peripheral nerve injury: Neuroprotective role of thioctic acid enantiomers

Peripheral neuropathies are in general characterized by hyperalgesia and allodynia with alterations of muscular sensitivity and functions. Selected antioxidants have been proposed as a potential therapeutic agents in the treatment or prevention of several pathologies related to oxidative stress. Thioctic acid is an antioxidant existing in nature and expressed in two optical isomers. (+)-Thioctic acid is the naturally occurring enantiomer, whereas the most used formulation of the compound in clinical practice, synthetic thioctic acid, is the mixture of (+) and (-)-enantiomers. The present study assessed if compression of sciatic nerve, induced by loose ligation, is accompanied by a central nervous system (CNS) changes and if thioctic acid enantiomers treatment may have a possible neuroprotective role. Loose ligation of the right sciatic nerve was performed in spontaneously hypertensive rats (SHR), used as a model of increased oxidative stress, and in normotensive Wistar-Kyoto rats (WKY) used as a control group. Animals with sciatic nerve ligation were left untreated or were treated intraperitoneally for 14 days with (+/-)-(25 and 50 mg/Kg/day), (+)-(25 mg/Kg/day), (-)-(25 mg/Kg/day) thioctic acid. Analysis, centered on injury phenomena at level of dorsal root ganglia and spinal cord, consisted in an immunochemical and immunohistochemical analysis. In the spinal cord an increase of oxidative stress markers was observed in rats after sciatic nerve ligation. It was accompanied by astrogliosis and neuronal damage without activation of apoptotic process primarily in in the dorsal horn of spinal cord. Antioxidant treatment reduced oxidative stress and astrogliosis being (+)-thioctic acid was active than (+/-)- or (-)-enantiomers. The above results demonstrated the spinal cord changes induced by peripheral nerve injury and a neuroprotective effect elicited by thioctic acid. The demonstration of a more pronounced activity of (+)-thioctic acid suggests that appropriate antioxidant strategies may represent a suitable approach in the treatment of mechanical neuropathies.

Microglia Play a Role in Ethanol‐Induced Oxidative Stress and Apoptosis in Developing Hypothalamic Neurons

Animals exposed to alcohol during the developmental period develop many physiological and behavioral problems because of neuronal loss in various brain areas including the hypothalamus. Because alcohol exposure is known to induce oxidative stress in developing neurons, we tested whether hypothalamic cells from the fetal brain exposed to ethanol (EtOH) may alter the cell-cell communication between neurons and microglia, thereby leading to increased oxidative stress and the activation of apoptotic processes in the neuronal population in the hypothalamus. Using enriched neuronal and microglial cells from fetal rat hypothalami, we measured cellular levels of various oxidants (O2 -, reactive oxygen species, nitrite), antioxidants (glutathione [GSH]), antioxidative enzymes (glutathione peroxidase [GSH-Px], catalase, superoxide dismutase) and apoptotic death in neurons in the presence and absence of EtOH or EtOH-treated microglial culture medium. Additionally, we tested the effectiveness of antioxidative agents in preventing EtOH or EtOH-treated microglial conditioned medium actions on oxidative stress and apoptosis in neuronal cell cultures. Neuronal cell cultures showed increased oxidative stress, as demonstrated by higher cellular levels of oxidants but lower levels of antioxidant and antioxidative enzymes, as well as, increased apoptotic death following treatment with EtOH. These effects of EtOH on oxidative stress and cell death were enhanced by the presence of microglia. Antioxidative agents protected developing hypothalamic neurons from oxidative stress and cellular apoptosis which is caused by EtOH or EtOH-treated microglial culture medium. These data suggest that exposure of developing hypothalamic neurons to EtOH increases cellular apoptosis via the effects on oxidative stress of neurons directly and via increasing production of microglial-derived factor(s).

Mechanism of Alternariol monomethyl ether-induced mitochondrial apoptosis in human colon carcinoma cells

Alternariol monomethyl ether (AME) is a major mycotoxin produced by fungi of the genus Alternaria and a common contaminant of food products such as fruits and cereals worldwide. AME can cause serious health problems for animals as well as for humans. In this study, human colon carcinoma cells (HCT116) were used to explore the mechanisms of cell death induced by AME. Exposure of HCT116 cells to AME resulted in significant cytotoxicity manifested by a loss in cell viability mainly mediated by activation of apoptotic process. AME activated the mitochondrial apoptotic pathway evidenced by the opening of the mitochondrial permeability transition pore (PTP), loss of the mitochondrial transmembrane potential (ΔΨm) downstream generation of O 2 −, cytochrome c release and caspase 9 and 3 activation. Experiments conducted on isolated organelles indicated that AME does not directly target mitochondria to induce PTP-dependent permeabilization of mitochondrial membranes. Moreover, no difference was observed in Bax-KO cells in comparison to parental cells, suggesting that the pro-apoptotic protein Bax is not involved in AME-induced mitochondrial apoptosis. Our findings demonstrate for the first time that AME induces cell death in human colon carcinoma cells by activating the mitochondrial pathway of apoptosis.

Monocytic differentiation of leukemic HL‐60 cells induced by co‐treatment with TNF‐α and MK886 requires activation of pro‐apoptotic machinery

The block of hematopoietic differentiation program in acute myeloid leukemia cells can be overcome by differentiating agent like retinoic acid, but it has several side effects. A study of other differentiation signaling pathways is therefore useful to predict potential targets of anti-leukemic therapy. We demonstrated previously that the co-treatment of HL-60 cells with Tumor necrosis factor-alpha (TNF-alpha) (1 ng/mL) and inhibitor of 5-lipoxygenase MK886 (5 microm) potentiated both monocytic differentiation and apoptosis. In this study, we detected enhanced activation of three main types of mitogen-activated protein kinases (MAPKs) (p38, c-Jun amino-terminal kinase [JNK], extracellular signal-regulated kinase [ERK]), so we assessed their role in differentiation using appropriate pharmacologic inhibitors. The inhibition of pro-apoptotic MAPKs (p38 and JNK) suppressed the effect of MK886 + TNF-alpha co-treatment. On the other hand, down-regulation of pro-survival ERK pathway led to increased differentiation. Those effects were accompanied by increased activation of caspases in cells treated by MK886 + TNF-alpha. Pan-caspase inhibitor ZVAD-fmk significantly decreased both number of apoptotic and differentiated cells. The same effect was observed after inhibition of caspase 9, but not caspase 3 and 8. To conclude, we evidenced that the activation of apoptotic processes and pathways supporting apoptosis (p38 and JNK MAPKs) is required for the monocytic differentiation of HL-60 cells.

μ-Calpain mediated cleavage of the Na +/Ca 2+ exchanger in isolated mitochondria under A23187 induced Ca 2+ stimulation

Treatment of bovine pulmonary artery smooth muscle mitochondria with the calcium ionophore, A23187 (0.2 μM) stimulates μ-calpain activity and subsequently cleaves Na +/Ca 2+ exchanger (NCX). Pretreatment of the A23187 treated mitochondria with the calpain inhibitors, calpeptin or MDL28170 or with Ca 2+ chelator, EGTA does not cleave NCX. Treatment of the mitochondria with A23187 increases Ca 2+ level in the mitochondria, which subsequently dissociates μ-calpain–calpastatin association leading to the activation of μ-calpain. Immunoblot study of the A23187 treated mitochondria with the NCX polyclonal antibody indicates the degradation of mitochondrial inner membrane NCX (110 kDa) resulting in the doublet of ∼54–56 kDa NCX fragments. Moreover, in vitro cleavage of mitochondrial purified NCX by mitochondrial purified μ-calpain supports our conclusion. This cleavage of NCX may be interpreted as the main cause of Ca 2+ overload and could lay a key role in the activation of apoptotic process in pulmonary smooth muscle.

Activation of apoptotic processes during transition from hypertrophy to heart failure in guinea pigs

Changes in oxidative stress and apoptotic process were studied during the progression of a compensated hypertrophy to a decompensated heart failure in guinea pigs. Banding of the ascending aorta resulted in heart hypertrophy. At 10 wk, ventricle-to-body weight ratio and thickness of the interventricular septum as well as the left ventricular wall were increased significantly. Although fractional shortening and ejection fraction were decreased, there were no signs of heart failure. Furthermore, there was no increase in wet-to-dry weight ratios for the lungs and liver at this stage. However, at 20 wk, heart failure was characterized by a significant depression in heart function as indicated by a decrease in fractional shortening, and ejection fraction and a lesser increase in wall thickness from diastole to systole. Animals also showed clinical signs of heart failure, and the wet-to-dry weight ratios of the lungs and liver were significantly higher. Cardiomyocyte oxidative stress was significantly higher in the 20-wk aortic-banded group. The ratio of Bax to Bcl-xl showed an increase at 10 wk, and there was a further increase at 20 wk. Mitochondrial membrane potential in the aortic-banded animals was significantly decreased at 10 and 20 wk. Cytochrome c levels were higher in the cytosol compared with the mitochondria, leading to a considerable increase in the expression of p17 subunit of caspase-3. At 20 wk, both early and late stages of apoptosis were observed in isolated cardiomyocytes. It is suggested that an increase in oxidative stress initiates mitochondrial death pathway during the hypertrophic stage, leading to apoptosis and heart failure at a later stage.

Review article: mechanisms of action of mesalazine in preventing colorectal carcinoma in inflammatory bowel disease.

A series of epidemiological, experimental and preliminary clinical trials strongly suggest that mesalazine or 5-aminosalicyclic acid (5-ASA) may have antineoplastic and potentially prophylactic chemopreventive properties. It is assumed that mesalazine may have similar genetic and molecular targets as nonsteroidal anti-inflammatory drugs (NSAIDs), which is further supported by its close similarity with aspirin, differing only in its structure by the presence of an amino group at position 5 of the benzene ring. The putative chemopreventive actions include the inhibition of inflammatory cascades and/or reactions involved in cell growth and proliferation, such as cyclo-oxygenase (COX-1 and COX-2), which regulate cell proliferation through the formation of prostaglandins; lipoxygenase; nuclear factor kappaB (NFkappaB), responsible for the subsequent expression of pro-inflammatory molecules; MAP kinases and Bcl-2, as well as the activation of apoptotic processes, such as the stimulation of intestinal sphingomyelinase. The peroxisome-proliferator-activated receptor delta (PPARdelta), which also regulates gene transcription, is thought to play a role in both inflammatory and non-inflammatory driven carcinogenesis. This may be another significant target. It is hypothesized that 5-ASAs may prevent the enhancing effect of prostaglandins on PPARdelta binding to DNA by its COX inhibitory properties, decreasing proliferation of colorectal mucosal cells in non-inflammatory bowel disease patients with sporadic polyps of the large bowel.

Apoptosis in woman uterine cervix in pathologies associated with human papillomavirus.

The level of apoptosis in uterine cervical tissue was evaluated in healthy women and in patients with various pathologies. No signs of apoptosis were found in unchanged stratified epithelium, condyloma latum, and condyloma acuminatum. The level of apoptosis decreased with progression of neoplastic epithelial transformations, usually no apoptosis was observed in samples of stage III cervical intraepithelial neoplasms. The development of preinvasive carcinoma was accompanied by activation of apoptotic processes most pronounced in the upper third of the epithelium. In some stage I and stage I-II cervical intraepithelial neoplasms, apoptosis and elimination of the basal layer cells caused rejection of the epithelium which can explain regression of this pathology at the initial stages. The prevalence of human papillomavirus infection directly correlated with neoplastic changes in the cervical epithelium.

Subcellular distribution of calcium and ultrastructural changes after cerebral hypoxia-ischemia in immature rats

Recent data imply that mitochondrial regulation of calcium is critical in the process leading to hypoxic-ischemic brain injury. The aim was to study the subcellular distribution of calcium in correlation with ultrastructural changes after hypoxia-ischemia in neonatal rats. Seven-day-old rats were subjected to permanent unilateral carotid artery ligation and exposure to hypoxia (7.7% oxygen in nitrogen) for 90 min. Animals were perfusion-fixed after 30 min, 3 h or 24 h of reperfusion. Sections were sampled for light microscopy and electron microscopy combined with the oxalate–pyroantimonate technique. At 30 min and 3 h of reflow, a progressive accumulation of calcium was detected in the endoplasmic reticulum, cytoplasm, nucleus and, most markedly, in the mitochondrial matrix of neurons in the gray matter in the core area of injury. Some mitochondria developed a considerable degree of swelling reaching a diameter of several μm at 3 h of reflow whereas the majority of mitochondria appeared moderately affected. Chromatin condensation was observed in nuclei of many cells with severely swollen mitochondria with calcium deposits. A whole spectrum of morphological features ranging from necrosis to apoptosis was seen in degenerating cells. After 24 h, there was extensive injury in the cerebral cortex as judged by breaks of mitochondrial and plasma membranes, and a general decrease of cellular electron density. In the white matter of the core area of injury, the axonal elements exhibited varicosity-like swellings filled with calcium–pyroantimonate deposits. Furthermore, the thin myelin sheaths were loaded with calcium. Numerous oligodendroglia-like cells displayed apoptotic morphology with shrunken cytoplasm and chromatin condensation, whereas astroglial necrosis was not seen. In conclusion, markedly swollen ‘giant’ mitochondria with large amounts of calcium were found at 3 h of reperfusion often in neuronal cells with condensation of the nuclear chromatin. The results are discussed in relation to mitochondrial permeability transition and activation of apoptotic processes.

NMDA blockade attenuates caspase-3 activation and DNA fragmentation after neonatal hypoxia-ischemia.

The aim was to study the effects of an NMDA receptor antagonist on caspase-3 activation and DNA fragmentation after hypoxia-ischemia (HI) in 7-day-old rats. Animals were treated with vehicle or MK-801 (0.5 mg/kg) directly after HI and sacrificed 8, 24 or 72h later. MK-801 reduced injury (by 53%), cells positive for active caspase-3 (by 39%) and DNA fragmentation (by 79%) in the cerebral cortex. Furthermore, MK-801 significantly decreased caspase-3 activity, and Western blots revealed a tendency towards decreased proteolytic cleavage of the caspase-3 proform. The data imply that NMDA receptors are involved in the activation of apoptotic processes in the immature brain after HI.

Apoptosis of motor neurons with induction of caspases in the spinal cord after ischemia.

Some neuronal subpopulations are especially vulnerable to ischemic injury. In the spinal cord, large motor neurons are vulnerable to ischemia and are selectively lost after transient ischemia. However, the mechanisms of the neuronal loss have been uncertain. We hypothesized that spinal motor neurons might be lost by apoptosis and investigated a possible mechanism of neuronal death by detection of double-strand breaks in genomic DNA and immunohistochemical analysis for caspases, ie, interleukin-1beta converting enzyme (ICE), Nedd-2, and CPP32. We used a rabbit spinal cord ischemia model created with a balloon catheter. The spinal cord was removed at 8 hours, 1, 2, or 7 days after 15 minutes of transient ischemia, and histological changes were studied with hematoxylin-eosin staining. To detect double-strand breaks in DNA, a staining with terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) was performed. Furthermore, expression of ICE, Nedd-2, and CPP32 was investigated by Western blotting and immunohistochemical analysis. Motor neurons were selectively lost at 7 days after transient ischemia. TUNEL study demonstrated that no cells were positively labeled until 1 day after ischemia, but nuclei of some motor neurons were positively labeled at 2 days. Western blot analysis revealed no immunoreactivity for ICE and slight immunoreactivities for Nedd-2 and CPP32 in the sham-operated spinal cords. However, immunoreactivity became apparent at 8 hours after transient ischemia, decreased at 1 day, and returned to baseline level at 2 days. Immunohistochemical analysis demonstrated that motor neurons were responsible for induction of those caspases. Double-strand breaks in genomic DNA and induction of three caspases were demonstrated. These results indicate that motor neuron death in the spinal cord after transient ischemia is profoundly associated with activation of apoptotic processes.