Early administration of methylprednisolone decreases apoptotic cell death after spinal cord injury

The purpose of this study is to evaluate, in an experimental model of spinal cord injury (SCI), the presence of apoptotic cell death after trauma and if early administration of a single bolus of methylprednisolone (MP) influences apoptosis in the zone of trauma and in adjacent spinal cord segments. For this study, a total of 96 adult female Wistar rats were subjected to spinal contusion at the T6-T8 level, producing immediate paraplegia. Forty-eight animals (treated group) received a single intraperitoneal injection of MP, at a dose of 30 mg/kg body weight, 10 minutes later. Cells undergoing apoptosis were detected by means of immunohistochemical labeling with the monoclonal antibody Apostain (anti-ssDNA MAb F7-26), in the injured spinal cord tissue, both in the zone of the lesion and in the adjacent spinal segments (rostral and caudal zones), 1, 4, 8, 24 and 72 hours and 1 week after injury. Apoptosis was detected in neurons and glial cells in the zone of the lesion 1 hour after trauma, with a pattern that showed no changes 4 hours later. Between 4 and 8 hours postinjury, the number of apoptotic cells increased, after which it decreased over the following days. In the adjacent spinal segments, apoptotic cells were detected 4 hours after trauma, and increased progressively over the remainder of the study, the number of apoptotic cells being similar in the lesion zone and in rostral and caudal zones one week after injury. When the group of MP-treated animals was considered, significant decreases in the number of apoptotic cells were detected in the lesion zone 24 hours after injury, and in the rostral and caudal zones, at 72 hours and at 1 week after trauma. These findings show that early administration of a single bolus of MP decreases apoptotic cell death after SCI, supporting the utility of MP in reducing secondary damage in injured spinal cord tissue.     

Zebrafish anti-apoptotic protein zfBcl-xL can block betanodavirus protein alpha-induced mitochondria-mediated secondary necrosis cell death

Betanodavirus protein alpha induces cell apoptosis or secondary necrosis by a poorly understood process. In the present work, red spotted grouper nervous necrosis virus (RGNNV) RNA 2 was cloned and transfected into tissue culture cells (GF-1) which then underwent apoptosis or post-apoptotic necrosis. In the early apoptotic stage, progressive phosphatidylserine externalization was evident at 24h post-transfection (p.t.) by Annexin V-FLUOS staining. TUNEL assay revealed apoptotic cells at 24-72 h p.t, after which post-apoptotic necrotic cells were identified by acridine orange/ethidium bromide dual dye staining from 48 to 72 h p.t. Protein alpha induced progressive loss of mitochondrial membrane potential (MMP) which was detected in RNA2-transfected GF-1 cells at 24, 48, and 72 h p.t., which correlated with cytochrome c release, especially at 72 h p.t. To assess the effect of zfBcl-xL on cell death, RNA2-transfected cells were co-transfected with zfBcl-x(L). Co-transfection of GF-1 cells prevented loss of MMP at 24 h and 48 h p.t. and blocked initiator caspase-8 and effector caspase-3 activation at 48 h p.t. We conclude that RGNNV protein alpha induces apoptosis followed by secondary necrotic cell death through a mitochondria-mediated death pathway and activation of caspases-8 and -3.     

Self-assembling Molecular Medicine for the Subacute Phase of Ischemic Stroke

Neurochemical Research - Ischemic stroke leads to acute neuron death and forms an injured core, triggering delayed cell death at the penumbra. The impaired brain functions after ischemic stroke are...     

Sequential induction of mitotic catastrophe followed by apoptosis in human leukemia MOLT4 cells by imidazoacridinone C-1311

Imidazoacridinone C-1311 is a DNA-targeting antitumor intercalator/alkylator currently undergoing Phase II clinical trials. Here, we elucidated the sequence of death responses to C-1311 in human leukemia MOLT4 cells using drug concentration (30 nM) that causes near complete cell growth inhibition at 48 h. Early (6-12 h) responses included transient accumulation of cells at the G2/M border followed by also transient rise in several mitotic markers. Mitotic attempts were largely abnormal, resulting in numerous multinucleated cells (peaking at 24-39 h and declining markedly at later times). These events, indicative of mitotic catastrophe, were not associated with immediate cell death. The fraction of necrotic cells did not exceed 3%. Also, the classical manifestations of apoptosis were marginal at 24 h and their progression clearly followed the decline in the fraction of mitotic and multinucleated cells. Quantification of several apoptotic markers (including phosphatidylserine externalization, apoptotic DNA breaks, mitochondrial dysfunction, caspase activation, and cell membrane integrity) showed a considerable progression and the shift from early to late apoptosis at later times. At 72 h, >80% of cells were apoptotic. Collectively, these findings show that C-1311-induced mitotic catastrophe is not the ultimate death event but rather a step precipitating delayed, albeit massive, apoptotic responses.     

光化学法脑缺血后细胞凋亡及其相关基因bcl-2表达的变化

采用ＴＵＮＥＬ染色及免疫组织化学技术对光化学法脑缺血后细胞凋亡及其相关基因ｂｃｌ－２表达的变化进行了研究。结果发现，缺血后１２ｈ，损伤侧皮层缺血区内凋亡细胞数及ｂｃｌ－２免疫反应阳性细胞数明显增加，一直持续至缺血后７２ｈ；并呈现下列时程变化：在缺血后３ｈ每张切片上几乎无凋亡细胞出现，以后逐渐增加，缺血后１２ｈ达到峰值，缺血后２４ｈ和缺血后７２ｈ逐渐减少，但仍高于假手术组水平。凋亡相关基因ｂｃｌ－２的表达在缺血后３ｈ以前不明显，缺血后１２ｈ逐渐增加，缺血后２４ｈ最多，以后逐渐下降。上述结果提示，缺血后凋亡细胞的时程变化可能与缺血后梗塞灶的发生和发展有关，而ｂｃｌ－２表达的变化可能与抑制细胞凋亡、发挥内源性细胞保护作用有关。     

Two Types of Apoptotic Cell Death of Rat Central Nervous System-Derived Neuroblastoma B50 and B104 Cells: Apoptosis Induced During Proliferation and After Differentiation

Abstract: We describe here two types of apoptotic cell death observed in the rat CNS-derived neuroblastoma B50 and B104 cells. One type was induced by dibutyryl cyclic AMP (DBcAMP) after differentiation, and the other was induced by treatment of proliferating cells with cycloheximide. When B50 and B104 cells were treated with 1 m M DBcAMP in the presence of 0.5% fetal calf serum, they began to extend neurites within 12 h and differentiated into neurons at 24 h, as reported previously. However, further cultivation with DBcAMP for up to 72 h led to flotation and, finally, death. Death was by apoptosis as shown by chromatin condensation and DNA fragmentation. Addition of a protein kinase A inhibitor or removal of DBcAMP after differentiation suppressed apoptosis, indicating the involvement of cyclic AMP and protein kinase A in apoptotic cell death. Cell death was also induced in proliferating cells without neurite outgrowth by treatment with cycloheximide. The death was also judged to be by apoptosis based on chromatin condensation and apoptotic body formation, although DNA fragmentation into small sizes was not detected. Both types of cell death showed similar responses to inhibitors for protein kinases and protein phosphatases.     

Apoptosis and appearance of Trp53-positive micronuclei in murine tumors with different radioresponses in vivo.

The purpose of this paper is to determine the relationship between the response to radiation and the appearance of apoptosis and micronuclei with Trp53 protein in murine tumors after irradiation. Two murine tumors, EL4, which was derived from a mouse lymphoma, and FM3A, which was derived from a mouse mammary carcinoma, were locally irradiated with 15 Gy and sections were stained with H&E and an anti-Trp53 antibody. The response to radiation was greater in EL4 tumors than in FM3A tumors. The frequency of apoptotic cells in EL4 tumors was 6.1 +/- 1.2% at time zero, reached a peak of 36.3 +/- 3. 8% at 6 h, and then decreased with time through 72 h to 2.5 +/- 1.5% after 15 Gy irradiation. In FM3A tumors, no apoptotic cells were detected at 0, 1, 3, 6 or 24 h after exposure. At 48 and 72 h, the frequency was only 3.0 +/- 0.6% and 1.3 +/- 0.3%. Apoptotic cells increased significantly at 3, 6 and 24 h after irradiation in EL4 tumors (P < 0.008) and at 48 and 72 h in FM3A tumors (P < 0.006). The frequency of Trp53-positive cells was 17.9 +/- 2.2 and 15.2 +/- 2.3% at time zero in EL4 and FM3A tumors, respectively, increased to 74.5 +/- 4.5% in EL4 cells (P = 0.001), and increased to 33.9 +/- 1. 1% in FM3A cells (P = 0.005) 1 h after irradiation. Trp53-positive micronuclei appeared in cells in both tumors from 24 to 72 h after irradiation. The frequency of Trp53-positive micronuclei was 3.8 +/- 0.5 and 13.5 +/- 1.3% at 24 h in EL4 and FM3A tumors, respectively, and gradually decreased by 72 h. After exposure to 15 Gy, Trp53-positive micronuclei increased significantly in FM3A tumors compared to EL4 tumors at both 24 and 48 h (P < 0.02). The frequency of these micronuclei increased with increasing dose in FM3A tumors, and the difference between these percentages after 3 Gy and after 5, 10 and 15 Gy was significant (P < 0.02). Many apoptotic cells were observed in the radiosensitive EL4 tumor after irradiation. Death by apoptosis may be related to an early response to radiation in these tumors. The appearance of micronuclei may be an important mechanism of cell death in FM3A tumors in which no apoptosis was induced.     

Sendai virus vector-mediated gene transfer of glial cell line-derived neurotrophic factor prevents delayed neuronal death after transient global ischemia in gerbils.

We have developed a cytoplasmic replicating virus vector of Sendai virus (SeV) that infects and replicates in most mammalian cells, including neurons, and directs high-level gene expression. To investigate the protective effect of SeV vector-mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF) on the delayed neuronal death caused by transient global ischemia in gerbils, SeV vectors carrying either GDNF (SeV/GDNF) or enhanced green fluorescent protein gene (SeV/GFP) were stereotaxically microinjected into the lateral ventricle. Four days after injection, occlusion of the bilateral common carotid arteries for 5 min produced transient global forebrain ischemia. Treatment with SeV/GDNF significantly decreased the delayed neuronal death of the hippocampal CA1 pyramidal neurons observed 6 days after the operation. TUNEL staining demonstrated that SeV/GDNF treatment markedly reduced the number of apoptotic cells in the hippocampal CA1 neurons, indicating that SeV/GDNF treatment prevented apoptosis. Furthermore, delayed neuronal death on the contralateral side of the hippocampal CA1 was also prevented to a similar extent as that on the ipsilateral side. These results suggest that SeV/GDNF prevents the delayed neuronal death induced by ischemia and is potentially useful for gene therapy for stroke.     

Transient contralateral rotation following unilateral substantia nigra lesion reflects susceptibility of the nigrostriatal system to exhaustion by amphetamine

Following unilateral 6-OHDA induced SN lesion, a transient period of contralateral rotation has been reported to precede the predominant ipsilateral circling. In order to clarify the nature of this initial contralateral rotation we examined the effect of the duration of recovery period after the lesion, on amphetamine-induced rotational behavior. Three days post lesion, most rats circled predominantly contralaterally to the lesion. Such contralateral rotation may result from either degeneration-induced breakdown of the DA pool, or lesion-induced increase of DA turnover in the spared neurons. A substantial degree of contralateral preference was still evident when amphetamine was administered for the first time 24 days after lesioning, indicating involvement of spared cells in the contralateral rotation. However, regardless of the duration of recovery (and irrespective of either lesion volume, amphetamine dose, or post-lesion motor exercise), amphetamine-induced rotation tended to become gradually more ipsilateral as the observation session progressed, and all rats circled ipsilaterally to the lesion in response to further amphetamine injections. These findings suggest that amphetamine has an irreversible effect on the post-lesion DA pool contributing to contralateral rotation.     

Melatonin is protective in necrotic but not in caspase-dependent, free radical-independent apoptotic neuronal cell death in primary neuronal cultures.

To assess the neuroprotective potential of melatonin in apoptotic neuronal cell death, we investigated the efficacy of melatonin in serum-free primary neuronal cultures of rat cortex by using three different models of caspase-dependent apoptotic, excitotoxin-independent neurodegeneration and compared it to that in necrotic neuronal damage. Neuronal apoptosis was induced by either staurosporine or the neurotoxin ethylcholine aziridinium (AF64A) with a delayed occurrence of apoptotic cell death (within 72 h). The apoptotic component of oxygen-glucose deprivation (OGD) unmasked by glutamate antagonists served as a third model. As a model for necrotic cell death, OGD was applied. Neuronal injury was quantified by LDH release and loss of metabolic activity. Although melatonin (0.5 mM) partly protected cortical neurons from OGD-induced necrosis, as measured by a significant reduction in LDH release, it was not effective in all three models of apoptotic cell death. In contrast, exaggeration of neuronal damage by melatonin was observed in native cultures as well as after induction of apoptosis. The present data suggest that the neuroprotectiveness of melatonin strongly depends on the model of neuronal cell death applied. As demonstrated in three different models of neuronal apoptosis, the progression of the apoptotic type of neuronal cell death cannot be withhold or is even exaggerated by melatonin, in contrast to its beneficial effect in the necrotic type of cell death.     

Lack of ceramide generation in TF-1 human myeloid leukemic cells resistant to ionizing radiation

The mechanism(s) by which ionizing radiation (IR) induces cell death is of fundamental importance in understanding cell sensitivity and resistance. Here we evaluated the response to IR of two subclones of the autonomous human erythromyeloblastic cell line TF-1: TF-1-34 (which expresses CD34) and TF-1-33 (which lacks CD34). In clonogenic assays, TF-1-34 cells were found to be relatively less sensitive to IR compared to TF-1-33 cells based on the D0 determination (3.01 vs 1.56 Gy). Furthermore, after IR at 12 Gy, TF-1-33 cell viability decreased by approximately 50% within 24 h, whereas TF-1-34 cell growth was unaffected during this time. Gradual loss of TF-1-34 cell viability was observed only after 48 h. Morphological and molecular analysis revealed that TF-1-33 cells died of apoptosis, and TF-1-34 cells of delayed reproductive cell death. While IR produced comparable amounts of DNA double strand breaks (DSB) in both cell lines, TF-1-34 retained DSB much longer than TF-1-33 suggesting that radioresistance and the defective apoptotic response of TF-1-34 cells was not related to a higher DNA repair capacity. However, the lack of an apoptotic response in TF-1-34 was correlated to the absence of a sphingomyelin (SM)-ceramide (CER) signaling pathway. Indeed, IR triggered in TF-1-33 cells but not in TF-1-34, SM hydrolysis (25% at 12 Gy) and CER generation (>50%) through the activation of neutral but not acid sphingomyelinase. Synthetic cell permeate CER induced apoptosis in both TF-1-33 and TF-1-34 cells. This study indicates that alterations of the SM-CER signaling pathway can significantly influence the cell death process as well as the survival of acute myeloid leukemia cells after IR exposure.     

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

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

大鼠局灶性脑缺血后不同时间点磷酸化Rb蛋白与凋亡神经元的共定位研究

目的探讨大鼠局灶性脑缺血后磷酸化Rb蛋白(p-Rb,ser 795)的表达定位与神经元凋亡的时空关系。方法制备大鼠大脑中动脉梗塞(MCAO)模型,分为假手术对照组、缺血1h再灌注12h,1d,3d,7d组。利用TUNEL法检测缺血周边区细胞凋亡情况;TUNEL与p-Rb荧光双标观察神经元凋亡与p-Rb表达、定位的关系。结果缺血半暗带内大部分TUNEL阳性细胞为神经元;大鼠MCAO再灌注12h和1d,TUNEL与p-Rb分别以重叠和镶嵌的方式共定位;再灌注3d,7d发生p-Rb核浆转移的神经元与TUNEL染色细胞仍然分别维持在高水平,但是两者却没有明显的共定位关系。结论 p-Rb可能参与短暂局灶脑缺血后神经元早期凋亡过程,间接或者不参与神经元晚期凋亡过程。     

ATP controls neuronal apoptosis triggered by microtubule breakdown or potassium deprivation.

BACKGROUND: Early loss of neurites followed by delayed damage of neuronal somata is a feature of several neurodegenerative diseases. Death by apoptosis would ensure the rapid removal of injured neurons, whereas conditions that prevent apoptosis may facilitate the persistence of damaged cells and favor inflammation and disease progression. MATERIALS AND METHODS: Cultures of cerebellar granule cells (CGC) were treated with microtubule disrupting agents. These compounds induced an early degeneration of neurites followed by apoptotic destruction of neuronal somata. The fate of injured neurons was followed after co-exposure to caspase inhibitors or agents that decrease intracellular ATP (deoxyglucose, S-nitrosoglutathione, 1-methyl-4-phenylpyridinium). We examined the implications of energy loss for caspase activation, exposure of phagocytosis markers, and long-term persistence of damaged cells. RESULTS: In CGC exposed to colchicine or nocodazole, axodendritic degeneration preceded caspase activation and apoptosis. ATP-depleting agents or protein synthesis inhibition prevented caspase activation, translocation of the phagocytosis marker, phosphatidylserine, and apoptotic death. However, they did not affect the primary neurite loss. Repletion of ATP by enhanced glycolysis restored all apoptotic features. Peptide inhibitors of caspases also prevented the apoptotic changes in the cell bodies, although the axodendritic net was lost. Under this condition cell demise still occurred 48 hr later in a caspase-independent manner and involved plasma membrane lysis at the latest stage. CONCLUSIONS: Inhibition of the apoptotic machinery by drugs, energy deprivation, or endogenous mediators may result in the persistence and subsequent lysis of injured neurons. In vivo, this may favor the onset of inflammatory processes and perpetuate neurodegeneration.     

Role of NAD(P)H:quinone oxidoreductase in the progression of neuronal cell death in vitro and following cerebral ischaemia in vivo

A direct involvement of the antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO1) in neuroprotection has not yet been shown. The aim of this study was to examine changes, localization and role of NQO1 after different neuronal injury paradigms. In primary cultures of rat cortex the activity of NQO1 was measured after treatment with ethylcholine aziridinium (AF64A; 40 micro m), inducing mainly apoptotic cell death, or oxygen-glucose deprivation (OGD; 120 min), which combines features of apoptotic and necrotic cell death. After treatment with AF64A a significant NQO1 activation started after 24 h. Sixty minutes after OGD a significant early induction of the enzyme was observed, followed by a second increase 24 h later. Enzyme activity was preferentially localized in glial cells in control and injured cultures, however, expression also occurred in injured neuronal cells. Inhibition of the NQO1 activity by dicoumarol, cibacron blue or chrysin (1-100 nM) protected the cells both after exposure to AF64A or OGD as assessed by the decreased release of lactate dehydrogenase. Comparable results were obtained in vivo using a mouse model of focal cerebral ischaemia. Dicoumarol treatment (30 nmol intracerebroventricular) reduced the infarct volume by 29% (p = 0.005) 48 h after the insult. After chemical induction of NQO1 activity by t-butylhydroquinone in vitro neuronal damage was exaggerated. Our data suggest that the activity of NQO1 is a deteriorating rather than a protective factor in neuronal cell death.     

Radiation-induced gastric epithelial apoptosis occurs in the proliferative zone and is regulated by p53, bak, bax, and bcl-2

Unlike the small intestine and colon where gamma-radiation-induced apoptosis has previously been well characterized, the response of murine gastric epithelium to gamma-radiation has not been investigated in detail. Apoptosis was therefore assessed on a cell positional basis in gastric antral and corpus glands from adult male mice following gamma-radiation. Maximum numbers of apoptotic cells were observed in both antrum and corpus at 48 h and at radiation doses greater than 12 Gy. However, the number of apoptotic cells observed in the gastric epithelium was much lower than observed in the small intestine or colon after similar doses of radiation. Hematoxylin and eosin, caspase 3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling detected similar numbers and cell positional distributions of apoptotic cells, hence hematoxylin and eosin was used for subsequent studies. The highest numbers of apoptotic cells were observed at cell positions 5-6 in the antrum and cell positions 15-18 in the corpus. These distributions coincided with the distributions of PCNA-labeled proliferating cells, but not with the distributions of H(+)-K(+)-ATPase-labeled parietal cells or TFF2-labeled mucous neck cells. Decreased numbers of apoptotic gastric epithelial cells were observed in p53-null, bak-null, and bax-null mice compared with wild-type counterparts 6 and 48 h after 12 Gy gamma-radiation. Significantly increased numbers of apoptotic gastric epithelial cells were observed in bcl-2-null mice compared with wild-type littermates 6 h after 12 Gy gamma-radiation. Radiation therefore induces apoptosis in the proliferative zone of mouse gastric epithelium. This response is regulated by the expression of p53, bak, bax, and bcl-2.     

Development of cerebral infarction, apoptotic cell death and expression of X-chromosome-linked inhibitor of apoptosis protein following focal cerebral ischemia in rats

The aim of this study was to investigate the role of apoptosis or necrosis in the development of delayed infarct, and the relationship between the level of XIAP gene, caspase-3 activation and ischemic cell death following transient focal cerebral ischemia. Adult male Sprague-Dawley rats underwent right middle cerebral artery occlusion (MCAo) for 50 min and reperfusion for 0.5, 4, 8, 24 h, 3, 7, 14 days. On TTC-stained coronal sections, delayed infarct was observed to develop in the whole MCA territory, especially in frontoparietal cortex after ischemia. Near total infarct was shown in striatum 24 h after MCAo, while delayed infarct was evident in the cortex. By day 3, the infarct had progressively expanded to the nearly whole area of the frontoparietal cortex. Flow cytometric analysis of Annexin-V (marks apoptosis) and PI (propidium iodide, marks necrosis) labeling cells showed that MCAo dominantly induced necrosis in ischemic core, striatum. Apoptosis contributed to delayed infarct and cell death in the border zone, dorsolateral cortex and hippocampus. The time-course of caspase-3 activation was consistent with the changes of apoptosis and infarct following MCAo. Further RT-PCR experiments indicated that there was a biphasic regulation of XIAP in time- and region-dependent manner after ischemia. In the infarct core (striatum), following a transient and slight increase during 0.5 h to 4 h post-MCAo, expression of XIAP mRNA markedly decreased. On the other hand, a longer and larger upregulation of XIAP was observed at early time points in border zone (0.5 to 8 h, in dorsolateral cortex; 0.5 to 24 h in hippocampus), then the level of XIAP reduced. A negative correlation was observed between apoptosis and regulation of XIAP gene in these regions. Our findings suggest a possible association between expression of XIAP gene, apoptosis and delayed infarct following ischemia.     

Optimal Percoll concentration facilitates flow cytometric analysis for annexin V/propidium iodine-stained ischemic brain tissues

We sought to determine the optimal Percoll concentration for ischemic rat brain prepared for flow cytometric (FC) measurements. Animals were subjected to the right middle cerebral artery (MCA) occlusion, and were euthanized at 3, 12, 24, and 72 h after reperfusion onset. The brains were processed by different concentrations (unisolated, 20, 25, 30, or 40%) of Percoll and stained with annexin V/propidium iodine (PI). Ischemic brain damage was evaluated by FC analysis and image analysis for histologic sections. The relative susceptibility of different phenotypes of cells to necrotic and apoptotic damage were evaluated by the FC analyses for the immunohistochemistry, PI, and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-processed brain tissues. Our results showed that FC analysis effectively detected the extent and maturation of apoptotic/necrotic brain damage, and the results were consistent with those determined from histologic brain sections. Neuron was more vulnerable to apoptosis than glia, whereas both cellular phenotypes were compatible in susceptibility for necrotic cell death. Percoll at a low concentration (20%) could effectively remove tissue debris without affecting membranous integrity of the injured neurons. Conversely, high percentages of Percoll (30-40%) substantially increased membranous damage for the injured cells. These results supported the application of FC to determine the extent and progression in time, as well as relative phenotypes of apoptotic/necrotic cell deaths following ischemic damage. We highlighted the use of Percoll at low percentages to facilitate the removal of tissue debris and to improve membrane integrity preservation for the injured neurons.