Neuroprotective effect of cycloheximide on hypoxic-ischemic brain injury in neonatal rats

This study was done to determine the neuroprotective effect of cycloheximide on neonatal hypoxic-ischemic brain injury. Seven day-old newborn rat pups were subjected to 90 min of 8% oxygen following a unilateral carotid artery ligation. The extent of cerebral infarction was evaluated at 1 and 4 week of recovery. Apoptosis was identified by performing terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and flow cytometry with a combination of fluoresceinated annexin V and propidium iodide. Brain infarction area was significantly increased at 4 week compared to 1 week after hypoxia-ischemia in the control group. With cycloheximide treatment, the number of TUNEL positive cells in the ipsilateral cerebral cortex at 48 hr and peri-infarct area at 1 and 4 week of recovery was significantly reduced, both apoptotic and necrotic cells by flow cytometry 48 hr after the injury were significantly reduced, and the extent of cerebral infarction at 1 and 4 week of recovery was also significantly attenuated compared to the hypoxia-ischemia control group. In summary, our data suggest that apoptosis plays an important role in the development of delayed infarction, and inhibition of apoptosis with cycloheximide significantly reduces the ensuing cerebral infarction in a newborn rat pup model of cerebral hypoxia-ischemia.     

Evidence of Nuclear DNA Fragmentation Following Hypoxia-Ischemia in the Infant Rat Brain, and Transient Forebrain Ischemia in the Adult Gerbil

Wistar rats, eight days old, were subjected to permanent bilateral forebrain ischemia, followed by hypoxia for 15 minutes. A cerebral infarct, mainly involving the cerebral neocortex, hippocampus, amygdala, striatum and subcortical white matter was produced. Neurons and glia showing punctate chromatin condensation and karyorrhectic cells were observed 12 hours after hypoxia-ischemia. Their number increased during the first two days and recruitment of cells with degenerating nuclei occurred until day five. In situ labeling of nuclear DNA fragmentation stained many normal-appearing nuclei, as well as punctate chromatin condensations and nuclear fragments in karyorrhectic cells. Delayed neuronal death in the CA1 area of the hippocampus was observed after 20 minutes of transient forebrain ischemia in the adult gerbil. In situ labeling of nuclear DNA fragmentation demonstrated stained punctate chromatin condensation in a few degenerating cells at 48 hours post-ischemia. Substantial labeling of CA1 neurons occurred in the fourth day.
Agarose gel electrophoresis of extracted brain DNA from ischemic infant rats and adult gerbils showed a ladder-type pattern which is typical of nuclear DNA fragmentation into oligonucleosomal fragments (internucleosomal cleavage). These findings suggest that endonuclease(s) activation may play a role in cell death induced by different forms of hypoxia-ischemia.     

Morphological characteristics of dying cells in axial structures of developing human embryos

Programmed cell death (PCD) is a widespread phenomenon in the development of vertebrates. In most cases, dying cells during development exhibit generalized morphological features typical of apoptosis. We analyzed the morphological features of dying cells in the developing axial structures of 5 human embryos between 5 and 8 weeks of postovulatory age. Cell death in the axial structures, i.e. spinal cord, notochord and surrounding mesenchyme and somites, was analyzed using light and electron microscopy. Tissue samples were taken from the cervicothoracic region of normal human conceptuses. Two morphological types of cell death were found: apoptosis which was characterized by round or semilunar nuclear chromatin condensations, condensation and shrinkage of the cytoplasm and formation of apoptotic bodies, and cell death without the morphological features of apoptosis which was characterized by pyknotic nuclear chromatin condensations, vacuolated cytoplasm and the formation of numerous intercellular spaces. Apoptotic death occurred during the 5th week of normal development in all the axial structures. Later, apoptotic death appeared in all the axial structures, with the exception of the notochord, where some dying cells displayed features of secondary necrosis. According to our findings, apoptosis seems to be the most frequently observed type of PCD, but it is not the exclusive type of morphological cell death during the development of axial structures in human embryos.     

Mild traumatic brain injury induces apoptotic cell death in the cortex that is preceded by decreases in cellular Bcl-2 immunoreactivity

Although mild traumatic brain injury is associated with behavioral dysfunction and histopathological alterations, few studies have assessed the temporal pattern of regional apoptosis following mild brain injury. Anesthetized rats were subjected to mild lateral fluid-percussion brain injury (1.1–1.3 atm), and brains were evaluated for the presence of in situ DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling, TUNEL) and morphologic characteristics of apoptotic cell death (nuclear and cytoplasmic condensation, presence of apoptotic bodies). Significant numbers of apoptotic TUNEL(+) cells were observed in the injured parietal cortex and underlying white matter up to 72 h post-injury (P<0.05 compared to sham-injured-injured), with maximal numbers present at 24 h. Apoptosis was confirmed by the presence of 180–200 bp nuclear DNA fragments in tissue homogenates. The appearance of apoptotic TUNEL(+) cells in the injured cortex was preceded by a marked decrease in immunoreactivity for the anti-cell death protein, Bcl-2, as early as 2 h post-injury. This decrease in cellular Bcl-2 staining was not accompanied by a concomitant loss of staining for the pro-cell death Bax protein, suggesting that post-traumatic neuronal death in the cortex may be dependent on altered cellular ratios of Bcl-2:Bax. In the hippocampus, no significant increase in apoptotic TUNEL(+) cells was observed compared to sham-injured-injured animals. However, selective neuronal loss was evident in the CA3 region at 24 h post-injury, that was preceded by an overt loss of neuronal Bcl-2 immunoreactivity at 6 h. No changes in either cellular Bcl-2 or Bax expression were observed in the thalamus or white matter at any time post-injury.

Taken together from these data, we suggest that apoptosis contributes to cell death in both gray and white matter, and that decreases in cellular Bcl-2 may, in part, be associated with both apoptotic and non-apoptotic cell death following mild brain trauma.     

新生大鼠低氧缺血脑损伤后c-fos在脑神经元中的表达

目的：探讨c-fos基因表达在新生大鼠脑低氧缺血损伤中所起的作用。方法：采用免疫组化及逆转录扩增方法,观察新生大鼠脑低氧缺血后皮质、海马组织中c-fos基因转录、翻译水平的表达现象。结果：脑低氧缺血后早期海马和皮质部位可同时诱导出c-fos mRNA和c-fos蛋白（与sham组比较P<0.05）;皮质表达的c-fos明显低于海马（P<0.05）,非结扎侧c-fos基因一过性表达增高,但与结扎侧比较有明显差异（P<0.05）。结论：新生大鼠脑组织在低氧缺血后具备能产生特殊基因表达改变的功能,其c-fos基因的表达可能对于脑损伤后细胞的恢复与生存有重要意义。     

Fas/CD95/APO-1 can function as a death receptor for neuronal cells in vitro and in vivo and is upregulated following cerebral hypoxic-ischemic injury to the developing rat brain

Fas/CD95/Apo-1 is a cell surface receptor that transduces apoptotic death signals following activation and has been implicated in triggering apoptosis in infected or damaged cells in disease states. Apoptosis is a major mechanism of neuronal loss following hypoxic-ischemic injury to the developing brain, although the role of Fas in this process has not been studied in detail. In the present study, we have investigated the expression and function of Fas in neuronal cells in vitro and in vivo. Fas was found to be expressed in the 14 day old rat brain, with strongest expression in the cortex, hippocampus and cerebellum. Cross-linking of Fas induced neuronal apoptosis both in neuronal PC12 cells in culture and following intracerebral injection in vivo, indicating that neuronal Fas was functional as a death receptor. This death was shown to be caspase dependent in primary neuronal cultures and was blocked by the selective caspase 8 inhibitor IETD. Finally, cerebral hypoxia-ischemia resulted in a strong lateralised upregulation of Fas in the hippocampus, that peaked six to twelve hours after the insult and was greater on the side of injury. These results suggest that Fas may be involved in neuronal apoptosis following hypoxic-ischemic injury to the developing brain.     

胸腺嘧啶激酶/丙氧鸟苷体外杀伤携带胸腺嘧啶激酶基因的C6胶质瘤细胞时细胞凋亡的检测

目的检测胸腺嘧啶激酶基因转染C6胶质瘤细胞后用丙氧鸟苷处理时细胞凋亡的发生。方法用脂质体介导的基因转染将单纯疱疹病毒胸腺嘧啶激酶基因转入C6胶质瘤细胞中,用G418筛选,建立稳定表达胸腺嘧啶激酶的C6细胞株;加入丙氧鸟苷作用不同时间,用原位末端标记、透射电镜和DNA凝胶电泳法检测。结果用丙氧鸟苷作用72h发现有大量原位末端标记阳性的细胞,阳性信号呈块状或半月形位于核膜内侧;透射电镜发现肿瘤细胞核内染色质呈块状或帽状位于核膜内侧,非染色质部分呈空泡状,细胞质有的固缩,细胞器基本保持完整;有的则高度肿胀,细胞内亚单位遭到破坏;DNA凝胶电泳发现用丙氧鸟苷作用72h细胞基因组DNA呈梯状带。结论实验结果提示胸腺嘧啶激酶/丙氧鸟苷系统作用于表达胸腺嘧啶激酶的C6细胞时该细胞主要是以凋亡的方式死亡的。     

Effect of the fungal mycotoxin patulin on the chromatin structure of fission yeast Schizosaccharomyces pombe

The fungal mycotoxin patulin is produced by several molds, especially by Aspergillus and Penicillium. The aim of this study was to clarify whether patulin causes alterations in plasma membrane permeability of Schizosaccharomyces pombe lead to cellular shrinkage charateristic to apoptosis or increases cell size indicating necrosis in cells. Transmission and scanning electronmicroscopy revealed that lower concentrations of patulin induced cellular shrinkage and blebbing, higher concentration caused expansion without cellular disruption. Large‐scale morphological changes of individual cells were followed by time lapse video microscopy. Patulin caused the elongation and stickiness of cells or rounded up their shapes. To visualize chromatin structures of S. pombe nuclei upon patulin treatment, protoplasts were isolated from S. pombe and subjected to fluorescent microscopy. Chromatin changes in the presence of 50 μM patulin concentration were characterized by elongated nuclei containing sticky fibrillary chromatin and enlarged round shaped nuclei trapped at the fibrillary stage of chromatin condensation. Short (60 min) incubation of S. pombe cells in the presence of high (500 μM) patulin concentration generated patches of condensed chromatin bodies inside the nucleus and caused nuclear expansion, with the rest of chromatin remaining in fibrillary form. Longer (90 min, 500 μM) incubation resulted in fewer highly condensed chromatin patches and in nuclear fragmentation. Although, high patulin concentration increased the size of S. pombe size, it did not lead to necrotic explosion of cells, neither did the fragmented nuclei resemble apoptotic bodies that would have indicated programmed cell death. All these morphological changes and the high rate of cell survival point to rapid adaptation and mixed type of fungistatic effects. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

脑内移植治疗帕金森病的移植神经元凋亡时程的研究

为了探讨将 E14腹侧中脑 ( VM)细胞悬液植入 Parkinson病大鼠纹状体后的移植神经元的凋亡时程及其数量的变化规律 ,将实验动物分为移植组和空白对照组 ,两组均包括移植后 1、4、7和 2 8d四个时间点。分别运用 TU NEL 法、TH免疫组化法、H-E染色及透射电镜观察了凋亡细胞的生化和形态特征 ,藉以估计其凋亡细胞的数量变化状况。结果表明 ,移植组中用 TU NEL法观察移植神经元具有 TU NEL 阳性反应胞核的细胞在移植后 1、4、7、2 8d所占的百分比分别为 0 .14 9%、0 .13 1%、0 .0 66%和0 .0 0 5 % ,移植后 1、4d与 7、2 8d之间的比较差异显著 ( P<0 .0 0 1)。 TH免疫组化反应显示 ,在移植后 1、4、7、2 8d TH免疫反应阳性细胞存活率分别为 15 .7%、6.3 %、5 .9%和 4.9% ,移植后 1d与 4、7、2 8d之间的比较差异显著 ( P<0 .0 0 1)。本研究结果提示 :( 1)移植的胚 VM细胞凋亡主要见于移植后的头 7d,这一期间内大约有 90 %移植神经元发生凋亡 ;( 2 )这种凋亡对移植的TH细胞存活率的影响仅见于移植后的 4d。     

Mechanisms underlying hypoxia-induced neuronal apoptosis

In vivo models of cerebral hypoxia-ischemia have shown that neuronal death may occur via necrosis or apoptosis. Necrosis is, in general, a rapidly occurring form of cell death that has been attributed, in part, to alterations in ionic homeostasis. In contrast, apoptosis is a delayed form of cell death that occurs as the result of activation of a genetic program. In the past decade, we have learned considerably about the mechanisms underlying apoptotic neuronal death following cerebral hypoxia-ischemia. With this growth in knowledge, we are coming to the realization that apoptosis and necrosis, although morphologically distinct, are likely part of a continuum of cell death with similar operative mechanisms. For example, following hypoxia-ischemia, excitatory amino acid release and alterations in ionic homeostasis contribute to both necrotic and apoptotic neuronal death. However, apoptosis is distinguished from necrosis in that gene activation is the predominant mechanism regulating cell survival. Following hypoxic-ischemic episodes in the brain, genes that promote as well as inhibit apoptosis are activated. It is the balance in the expression of pro- and anti-apoptotic genes that likely determines the fate of neurons exposed to hypoxia. The balance in expression of pro- and anti-apoptotic genes may also account for the regional differences in vulnerability to hypoxic insults. In this review, we will examine the known mechanisms underlying apoptosis in neurons exposed to hypoxia and hypoxia-ischemia.     

Nerve cell death types in the edematous human cerebral cortex

Cortical biopsies of 18 patients with clinical diagnosis of congenital hydrocephalus, brain trauma, and vascular anomaly were examined with the transmission electron microscope to study the distinct and overlapped morphological cell types of nerve cell death in the human edematous cerebral cortex. The nerve cells showed lobulated and shrunken nucleus, irregular enlargement and fragmentation of perinuclear cistern, with areas of apparently intact nuclear pore complexes alternating with regions of nuclear pore complex disassembly. The nucleolus appears unaltered in moderate edema and with distorted nucleolar subcompartments in severe edema. Most nonpyramidal nerve cells, astrocytes and oligodendrocytes underwent an oncotic-apoptotic-necrotic continuum featured by swollen nucleoplasm, cytoplasm, and cell organelles, chromatin condensation and marginalization, and formation of apoptotic bodies. In a lesser proportion other nonpyramidal nerve cells, astrocytes and oligodendrocytes only showed apoptosis or oncosis. Autophagic cell death characterized by presence of autophagic vacuoles of lysosomal origin was rarely seen. The above findings suggest that different mechanisms of nerve cell death occur in the human edematous cerebral cortex related with brain trauma, congenital hydrocephalus, vascular anomaly, and their anoxic-ischemic conditions. An oncotic-apoptotic continuum process leading to necrosis predominates in human cerebral cortex nerve cell populations. The nerve cell death is discussed in relation with the severity of brain edema, anoxic-ischemic conditions of brain parenchyma, oxidative stress, glutamate excitotoxicity, calcium overload, and caspase dependent and independent mechanisms.     

Failure to complete apoptosis following neonatal hypoxia-ischemia manifests as "continuum" phenotype of cell death and occurs with multiple manifestations of mitochondrial dysfunction in rodent forebrain

Controversy surrounds proper classification of neurodegeneration occurring acutely following neonatal hypoxia-ischemia (HI). By ultrastructural classification, in the first 24 h after neonatal hypoxia-ischemia in the 7-day-old (p7) rat, the majority of striatal cells die having both apoptotic and necrotic features. There is formation of a functional apoptosome, and activation of caspases-9 and -3 occurring simultaneously with loss of structurally intact mitochondria to 34.7+/-25% and loss of mitochondrial cytochrome c oxidase activity to 34.7+/-12.7% of control levels by 3 h after hypoxia-ischemia. There is also loss of the mitochondrial motor protein, kinesin. This combination of activation of apoptosis pathways simultaneous with significant mitochondrial dysfunction may cause incomplete packaging of nuclear and cytoplasmic contents and a hybrid of necrotic and apoptotic features. Evidence for an intermediate biochemistry of cell death including expression of the 17 kDa isoform of caspase-3 in dying neurons lacking a classic apoptotic morphology and degradation of the neuronal cytoskeletal protein spectrin by caspase-3 and calcium-activated calpains yielding 120 kDa and 145/150 kDa fragments, respectively, is also found. In summary, neonatal hypoxia-ischemia triggers apoptotic cascades, and simultaneously causes mitochondrial structural and functional failure. The presence of a "continuum" phenotype of cell death that varies on a cell-by-cell basis suggests that the phenotype of cell death is dependent on the energy available to drive the apoptotic pathways to completion.     

Age-dependence of heat-induced strand separation of DNA in situ in postmitotic cells of rat brain as revealed by acridine orange microfluorimetry.

Acridine orange microfluorimetry as well as Feulgen microfluorimetry were applied on smears of cerebral and cerebellar cortex of young (2-month-old) and old (24-27-month-old) female Wistar rats. The strand separation of DNA caused by thermal denaturation was measured between 50 degrees and 90 degrees C in the nuclei of cortical nerve cells and cerebellar granular cells. In the young nerve cell nuclei a considerable DNA-loss was observed after thermal denaturation, therefore, corresponding corrections should have been made on the heat denaturation curves. The old nerve cell nuclei of brain cortex showed a much higher heat stability than the young ones, whereas in the cerebellar granular cells the age-dependent difference was much less expressed. Our observations on chromatin in situ show the same tendency as do the findings obtained on isolated chromatin by others. The results are discussed first from a methodical point of view then the arguments are listed suggesting a possible role of interprotein cross-links in the age-dependent stabilization of the chromatin gel.     

A cysteine protease from<Emphasis Type="Italic"> Taenia solium</Emphasis> metacestodes induce apoptosis in human CD4+ T-cells

Here we investigated whether the depletion of CD4+ lymphocytes, observed in mononuclear cells incubated with Taenia solium metacestode E/S products or with living cysts was due to apoptosis. Using the deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), electron microscopy and DNA gel electrophoresis, we found signs of apoptosis in these cells. Results showed that cysteine protease activity was responsible for this effect, since E-64 prevented cell death in all cases. Electron microscopy studies showed that lymphocytes exhibited features of apoptosis such as cellular membrane integrity, strangling and fragmentation of nuclei, chromatin condensation, apoptotic bodies and loss of microvilli. In contrast, lymphocytes co-cultured with living metacestodes plus E-64 exhibited integrity of their structures. DNA fragmentation was detected by TUNEL assays and DNA gel electrophoresis. The results suggested that cell death induced by the cysteine protease from the T. solium metacestode may be involved in down-regulation of cell-mediated responses in infected hosts.     

Local and remote cellular responses following a surgical lesion in the Cebus apella cerebral cortex

Distribution of bromodeoxyuridine immunoreactive (BrdU-IR) cell nuclei was analyzed at proximal and remote cortical sites in adult Cebus apella monkeys after a programmed surgical lesion placed either in the prefrontal or in the striate cerebral cortex. Increased GFAP-IR and vimentin-IR astrocytes, as well as IsolectinB4 labeled microglial cells, were observed both at lesional and perilesional areas. After injury at either location, the BrdU nuclear incorporation spread to supragranular layers in remote cortical areas functionally related to the injured cortex, probably due to involvement of degenerated cortico-cortical association fibers. Double labeling with Ki-67 suggested that remote BrdU-IR nuclei belong to proliferating cells, but the cell type remains to be determined, since these nuclei did not correspond to NeuN, MAP2, GFAP, Vimentin and isolectinB4 labeled cells. This remote effect should be incorporated to current clinical and experimental appraisal of cortical lesions.     

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 72h but not 24h after status epilepticus. Internucleosomal DNA cleavage (DNA "laddering") occurred in the four most damaged brain regions examined by electron microscopy 24h after SE and the three most damaged regions 72h 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.     

Molecular mechanisms of neuronal death in the dorsal lateral geniculate nucleus following visual cortical lesions

We investigated the molecular mechanisms of cell death in the dorsal lateral geniculate nucleus of the rat, following suction lesion of the visual cortex at birth or in the third postnatal week, using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique and immunohistochemistry for caspase-3, -7, -8, and cleaved poly(ADP-ribose) polymerase.Following lesion at birth, TUNEL-positive neurons were found in the dorsal lateral geniculate nucleus between 24 h and 3 days after lesion, with a peak on the second day. Shorter survival times (12-18 h) resulted in labeling of very few neurons in dorsal lateral geniculate nucleus and of several neurons in the perilesional cortex. Activated caspase-3 was expressed from the first to the third days after lesion, whereas cleaved poly(ADP-ribose) polymerase and activated caspase-8 were expressed on the second and third day. Activated caspase-7 was expressed mainly in pretectal nuclei. Caspase-3 activation coincided with the appearance of TUNEL-positive profiles, but decreased earlier than TUNEL. In the ipsi- and contralateral cerebral cortex, all parameters were unchanged. In animals lesioned in the third week, rare apoptotic thalamic neurons were detected as TUNEL- and activated caspase-3-positive profiles 2 days after cortical ablation, and were still present 1 week after lesion.Thus, early target ablation has dramatic effects on neonatal thalamic neurons, which die following activation of caspases 3 and 8. In contrast, cortical neurons are relatively unaffected by target deprivation. Compared with early lesions, late lesions induce a limited thalamic cell death, that persists over time.     

Minocycline alleviates hypoxic-ischemic injury to developing oligodendrocytes in the neonatal rat brain

The role of minocycline in preventing white matter injury, in particular the injury to developing oligodendrocytes was examined in a neonatal rat model of hypoxia-ischemia. Hypoxia-ischemia was achieved through bilateral carotid artery occlusion followed by exposure to hypoxia (8% oxygen) for 15 min in postnatal day 4 Sprague-Dawley rats. A sham operation was performed in control rats. Minocycline (45 mg/kg) or normal phosphate-buffered saline was administered intraperitoneally 12 h before and immediately after bilateral carotid artery occlusion+hypoxia and then every 24 h for 3 days. Nissl staining revealed pyknotic cells in the white matter area of the rat brain 1 and 5 days after hypoxia-ischemia. Hypoxia-ischemia insult also resulted in apoptotic oligodendrocyte cell death, loss of O4+ and O1+ oligodendrocyte immunoreactivity, and hypomyelination as indicated by decreased myelin basic protein immunostaining and by loss of mature oligodendrocytes in the rat brain. Minocycline significantly attenuated hypoxia-ischemia-induced brain injury. The protective effect of minocycline was associated with suppression of hypoxia-ischemia-induced microglial activation as indicated by the decreased number of activated microglia, which were also interleukin-1beta and inducible nitric oxide synthase expressing cells. The protective effect of minocycline was also linked with reduction in hypoxia-ischemia-induced oxidative and nitrosative stress as indicated by 4-hydroxynonenal and nitrotyrosine positive oligodendrocytes, respectively. The reduction in hypoxia-ischemia-induced oxidative stress was also evidenced by the decreases in the content of 8-isoprostane in the minocycline-treated hypoxia-ischemia rat brain as compared with that in the vehicle-treated hypoxia-ischemia rat brain. The overall results suggest that reduction in microglial activation may protect developing oligodendrocytes in the neonatal brain from hypoxia-ischemia injury.     

Factors affecting the survival of cat retinal ganglion cells after optic nerve injury

Summary After partial transection of one optic nerve in adult cats the majority of retinal ganglion cells degenerate and die 1 week after axotomy, whilst other cell classes degenerate slowly and survive for a long period after the lesion. We have investigated the effects of intravitreal and intraperitoneal injections of MK-801, a NMDA-glutamate receptor antagonist, on the early degeneration of retinal ganglion cells after partial optic nerve section. Control animals received saline intravitreal injections. Retinal flat mounts were retrogradely labelled with horseradish peroxidase and counterstained with Cresyl Violet. We evaluated the ganglion cell loss in the three experimental groups 1 week after lesion and compared them with normal uninjured controls and injured untreated retinae. In untreated retinae 49% of ganglion cells die 1 week after the lesion. Systemic MK-801 treatment prolonged survival of 41% of retinal ganglion cells that would die without treatment. Intravitreal MK-801 or saline prolonged survival of 71% of retinal ganglion cells that would die without treatment, but the results of saline administration had a larger range of variability. In untreated retinae many pyknotic cells were observed. The decreased in number after systemic MK-801 treatment and in some retinae treated with intravitreal injections of saline solution. There were no pyknotic cells after local, intravitreal MK-801 treatment. These results support the hypothesis that NMDA-receptor mediated neurotoxicity plays an important role in the early retinal ganglion cell death after retrobulbar axotomy. They also support the existence of an endogenous source of neurotrophins whose release is triggered by eyeball injury. We conclude that the early death of retinal ganglion cells after axotomy occurs by a mechanism that can be controlled by neurotrophins and antagonists to NMDA-glutamate receptors.To whom correspondence should be addressed.