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.     

Clonidine diminishes c-jun gene expression in the cardiovascular sensitive areas of the rat brainstem

The present study investigated the effect of clonidine on the basal and inducible c-jun and c-fos mRNA expression in the nucleus tractus solitarius (middle, mNTS, and rostral, rNTS) and the rostral ventrolateral medulla (caudal, cRVLM, and rostral, rRVLM). Conscious rats received saline, clonidine (30 microg/kg, i.v.), saline plus sodium nitroprusside (NP), or clonidine plus NP. Under basal conditions (saline-infused rats), c-jun mRNA was expressed in the mNTS and rRVLM but not in the rNTS or cRVLM whereas c-fos mRNA was not detectable. Clonidine attenuated the increases in c-fos in the mNTS and cRVLM and c-jun gene expression in the mNTS and rRVLM caused by NP-evoked hypotension and also reduced the basal expression of c-jun mRNA in the mNTS and rRVLM. These findings establish a causal link between clonidine inhibition of c-fos expression in brainstem and its hypotensive action, and provide the first evidence that clonidine attenuates the expression of the closely linked c-jun gene in neurons implicated in centrally mediated hypotension.     

Basic FGF-like immunoreactivity in the developing and adult rat brainstem

Although a variety of in vitro and in vivo actions of basic fibroblast growth factor (bFGF) on neuronal cells have been documented, the physiological role of this protein in the nervous system is still contested. Since the distribution of a molecule in the nervous system may provide cues for an understanding of its possible roles, we have begun to study its cellular localization in the central and peripheral nervous system using immunocytochemistry with an anti-bFGF-specific antibody. Here we provide an account on the distribution of bFGF-like immunoreactivity (bFGF-IR) in the brainstem of the developing and adult rat. Basic FGF-IR was found to be widely distributed in motor and sensory nuclei. In all nuclei examined, only subpopulations of neurons were stained. Different staining patterns were found. For example, in the red nucleus weakly or unstained perikarya were surrounded by numerous immunoreactive fibers, often in close contact with the neuronal surface. In the reticular formation and facial nerve, many neuronal cell bodies showed a strong IR that extended into the processes. Glial cells were consistently unstained. During early postnatal development changes of the distribution of bFGF IR were found. From this wide distribution pattern of bFGF-IR, we conclude that bFGF may have more general and, possibly, diverse functions rather than a restricted role for a particular subset of neurons. Variations in the staining pattern of nerve cell bodies in a single nucleus may suggest a function related to neuronal activity.     

Clonidine diminishes c-jun gene expression in the cardiovascular sensitive areas of the rat brainstem

The present study investigated the effect of clonidine on the basal and inducible c-jun and c-fos mRNA expression in the nucleus tractus solitarius (middle, mNTS, and rostral, rNTS) and the rostral ventrolateral medulla (caudal, cRVLM, and rostral, rRVLM). Conscious rats received saline, clonidine (30 μg/kg, i.v.), saline plus sodium nitroprusside (NP), or clonidine plus NP. Under basal conditions (saline-infused rats), c-jun mRNA was expressed in the mNTS and rRVLM but not in the rNTS or cRVLM whereas c-fos mRNA was not detectable. Clonidine attenuated the increases in c-fos in the mNTS and cRVLM and c-jun gene expression in the mNTS and rRVLM caused by NP-evoked hypotension and also reduced the basal expression of c-jun mRNA in the mNTS and rRVLM. These findings establish a causal link between clonidine inhibition of c-fos expression in brainstem and its hypotensive action, and provide the first evidence that clonidine attenuates the expression of the closely linked c-jun gene in neurons implicated in centrally mediated hypotension.     

Expression of bcl-2, bax, and caspase-3 in the brain of the developing rat

Naturally occurring neuronal death (NOND) is generally considered to be apoptotic. Apoptosis is an active form of cell death in which the regulation of specific proteins produces anti- or pro-apoptotic signals. Two of the protein families involved in this regulation are the bcl proteins and caspases. A quantitative immunoblotting technique was used to examine the temporal expression of bcl-2, bax, and two isoforms of caspase 3 (an active 20 kDa isoform and the inactive 32 kDa precursor) throughout the developing neuraxis. Long-Evans rat fetuses were collected on gestational day (G) 16 and G19, and pups were harvested on postnatal day (P) 0, P3, P6, P12, P21, and P30. Brains were divided into five segments: cortex, thalamus, midbrain, medulla/pons, and cerebellum. In general, the expression of bax increased and the ratio of bcl-2 expression to bax expression decreased concurrent with published data on the onset of NOND in a given area. The timing of these events was paralleled by an increase in the expression of active caspase 3. Unlike the bcl proteins, caspase 3 expression returned toward fetal levels as the brain matured. The timing of the changes in bcl protein and caspase expression show that both protein families are involved in promoting neuronal death. Reductions in caspase expression (and not bcl-2 and bax expression) are key to ending the period of NOND.     

Glutamate receptor phenotypes in the auditory brainstem and mid-brain of the developing rat

Glutamate receptors mediate most excitatory synaptic transmission in the adult vertebrate brain, but their activation in developing neurons also influences developmental processes. However, little is known about the developmental regulation of the subunits composing these receptors. Here we have studied age-dependent changes in the expression of α-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) and N-methyl-d -aspartate (NMDA) receptor subunits in the cochlear nucleus complex (CN), the superior olivary complex (SOC), the nuclei of the lateral lemniscus, and the inferior colliculus of the developing rat. In the lateral superior olive, the medial nucleus of the trapezoid body, and the ventral nucleus of the lateral lemniscus, the distribution of AMPA receptor subunits changed drastically with age. While GluR1 and GluR2 subunits were highly expressed in the first 2 postnatal weeks, GluR4 staining was detectable only thereafter. GluR1 and GluR2 immunoreactivities rapidly decreased during the third postnatal week, with the GluR1 subunits disappearing from most neurons. In contrast, the adult pattern of the distribution of AMPA receptor subunits emerged gradually in most of the other auditory nuclei. Thus, progressive as well as regressive events characterized AMPA receptor development in some nuclei, while a monotonically maturation was seen in other regions. In contrast, the staining patterns of NMDA receptor subunits remained stable or only decreased during the same period. Although our data are not consistent with a generalized pattern of AMPA receptor development, the abundance of GluR1 subunits is a distinctive feature of early AMPA receptors. As similar AMPA receptors are present during plasticity periods throughout the brain, neurons undergoing synaptic and structural remodelling might have a particular need for these receptors.     

Active caspase-3 in the sudden infant death syndrome (SIDS) brainstem

In a retrospective postmortem study, we examined the neuronal expression of active caspase-3, a specific apoptotic marker, in the brainstem of 67 infants dying from sudden infant death syndrome (SIDS), and 25 age-matched control infants (non-SIDS). Neuronal immunostaining for active caspase-3 was semi-quantitatively scored in nuclei from five brainstem levels: rostral, mid and caudal pons, and rostral and caudal medulla. Regardless of the cause of death (SIDS vs. non-SIDS), age-related differences in active caspase-3 expression were identified, predominantly in the medulla. No gender-related differences were identified. Comparing SIDS to non-SIDS cases, increased active caspase-3 expression was restricted to four nuclei in the caudal pons (abducens, facial, superior olivary, and pontine nuclei) and two nuclei in the rostral medulla (hypoglossal and dorsal motor nucleus of the vagus). We conclude that neuronal apoptosis is increased in the brainstem of SIDS compared to non-SIDS infants.     

Ethanol-induced caspase-3 activation in the in vivo developing mouse brain

Recently several methods have been described for triggering extensive apoptotic neurodegeneration in the developing in vivo mammalian brain. These methods include treatment with drugs that block NMDA glutamate receptors, drugs that promote GABA(A) neurotransmission, or treatment with ethanol, which has both NMDA antagonist and GABAmimetic properties. A single intoxication episode induced by any of these agents is sufficient to cause widespread neurodegeneration throughout many brain regions. The cell death process transpires rapidly from early to late stages within several hours. As the neurons die, they become TUNEL positive and show, by both light and electron microscopy, all of the classical morphological characteristics of apoptosis. In the present study, using immunocytochemical methods, we document that ethanol intoxication of 7-day-old infant mice causes a widespread pattern of caspase-3 activation corresponding to the pattern of apoptotic neurodegeneration that is occurring simultaneously.     

Postnatal development of preproenkephalin mRNA containing neurons in the rat lower brainstem

Postnatal developmental changes of preproenkephalin (PPE) gene expression in rat brainstem neurons were studied by in situ hybridization histochemistry. On the basis of PPE mRNA expression, brainstem neurons were categorized into three types: 1) type I neurons were characterized by constant or increasing expression of PPE mRNA during postnatal development; 2) type II neurons started to express PPE mRNA several days after birth and continued to do so thereafter; and 3) type III neurons showed transient expression of PPE mRNA or stopped expressing the mRNA during early postnatal development. Type I PPE neurons were observed in diverse brainstem structures including the mesencephalic and pontine central gray matter, various reticular and raphe nuclei, the ventral tegmental area of Tsai, the interpeduncular nucleus, the nucleus of the brachium of the inferior colliculus, the ventral and dorsal tegmental nuclei of Gudden, the sphenoid nucleus, the laterodorsal tegmental nucleus, Barrington's nucleus, the parabrachial region, the lateral lemniscus and its related nuclei, the trapezoid nucleus, the rostral and ventromedial periolivary nuclei, the mesencephalic trigeminal and principal sensory trigeminal nuclei, the locus coeruleus, the subcoeruleus nucleus, the medial and spinal vestibular nuclei, the dorsal and ventral cochlear nuclei, the medial and lateral cerebellar nuclei, the Roller nucleus, and the intermedius nucleus of the medulla. Type II PPE neurons were found in the superior colliculus, the inferior colliculus, the central part of the dorsal tegmental nucleus, and as Golgi neurons in the granular layer of the cerebellum. Type III PPE neurons were located in the substantia nigra, the red nucleus, the superior olive, the motor trigeminal nucleus, the facial nucleus, the inferior olive, the dorsal motor nucleus of the vagus, and the hypoglossal nucleus. Such region-specific expression of the PPE gene during postnatal ontogeny suggests that rat brainstem PPE neurons may be involved in a variety of developmental events, such as cell proliferation, differentiation, and migration.     

Activation of intracellular pathways with forskolin and phorbol ester increases LHRH mRNA level in the rat hypothalamus superfused in vitro

Two intracellular signal transduction mechanisms such as cAMP-protein kinase a and phosphatidylinositol (PI) turnover-protein kinase c are known to be dually involved in the regulation of luteinizing hormone-releasing hormone (LHRH) release. However, it is not yet evident that the activation of two intracellular pathways affects the LHRH gene expression. The present study aims, therefore, to determine whether the activation of two intracellular pathways affects changes in LHRH mRNA. To this end, we took advantage of an in vitro superfusion system, where rat hypothalamic tissues were superfused with media containing forskolin (FKN) and/or phorbol-12-myristate-13-acetate (PMA). Superfusates were collected at 10-min intervals and LHRH release was determined by radioimmunoassay. After a 2-h superfusion period, the post-superfusion hypothalami were recovered and poly (A) RNA fractions were isolated. Alterations in LHRH mRNA in response to FKN and/or PMA were determined by an RNA-blot hybridization assay using a 32P-end-labeled LHRH oligonucleotide (29-mer) probe. In vitro perfusion of hypothalamic fragments with PMA and/or FKN stimulated LHRH release as well as LHRH mRNA. The combined infusion of FKN and PMA did not produce an additive effect on the LHRH mRNA levels, but it was effective in synergistically increasing LHRH secretion in vitro. These data clearly demonstrate that the biosynthetic machinery of LHRH is influenced by activation of two intracellular pathways, both cAMP-protein kinase a and phosphatidyl-inositol turnover-protein kinase c, indicating the transsynaptic regulation of hypothalamic LHRH gene expression.     

Increased activity and involvement of caspase-3 in radiation-induced apoptosis in neural cells precursors from developing rat brain

Using primary cultures of neural precursor cells of cortex from developing rat brain, we demonstrated the involvement of caspase-3 in the apoptotic process induced by gamma irradiation. The precursor nature of cells was confirmed by nestin and GFAP immunoreactivity and by the capacity of differentiation in neuronal and glial cells after 5 days in culture. Neural precursors were irradiated with single doses ranging from 0.1 to 4Gy. Cellular death, determined 24 h post-irradiation (pi) was dose-dependent and the induction of apoptosis was confirmed by nuclear condensation, DNA fragmentation and hypodiploid DNA peak represented by the "sub G1" region. For the higher doses, apoptosis was evident after 4-6 h pi and increased during 24 h. Caspase-3 activity increased with doses and was maximal at 4-6 h pi with 3Gy and remained similar with 4Gy. The protection from radiation-induced apoptosis by caspase-3 inhibitor, zDEVD-fmk, confirmed that this enzyme is involved in the apoptotic mechanism in this system. The possibility of using this tissue culture system for studying the effects of ionizing radiation on morphological and molecular differentiation was considered.     

Pro-opiomelanocortin mRNA and peptide co-expression in the developing rat pituitary.

Pro-opiomelanocortin (POMC) is synthesized in both the pituitary gland and the brain. Various peptide products of this precursor, namely beta-endorphin, ACTH and alpha-MSH are co-localized in the anterior lobe corticotrophs, all intermediate lobe cells and in hypothalamic neurons. Messenger RNA (mRNA) for POMC has further been shown to exist in these tissues. In this study, we have shown that POMC mRNA, and peptide accumulation as detected by in situ hybridization and immunocytochemistry, respectively, occur simultaneously within the rat pituitary gland during ontogeny and that their maturation occurs in parallel during prenatal and early postnatal development.     

Distribution of preprocholecystokinin mRNA in motoneurons of the rat brainstem and spinal cord.

The distribution of cholecystokinin (CCK)-immunoreactive neurons in the central nervous system has been questioned because many antisera raised against CCK have been found to cross-react with calcitonin gene-related peptide. The use of in situ hybridization histochemistry has allowed investigators to determine which CCK-immunoreactive cells actually contain the messenger RNA (mRNA) coding for preprocholecystokinin (preproCCK), thus indicating that the peptide is synthesized in these cells. In this study, we report the distribution of preproCCK mRNA in the brainstem and spinal cord of the rat. The main findings of this study are the localization of preproCCK mRNA in motoneurons of cranial nerves IV, V, VI, VII and XII, as well as in motoneurons of the cervical and lumbrosacral levels of the spinal cord. Additionally, cells in lamina III at the cervical and lumbar enlargements contain preproCCK mRNA, suggesting that cells expressing CCK may be important in the processing of sensory information from the appendages.     

Testosterone stimulates LH-RH-like mRNA level in the rat hypothalamus

To elucidate whether testosterone (T) replacement to castrated male rats may promote luteinizing hormone-releasing hormone (LH-RH) biosynthesis in the hypothalamus, an RNA-blot hybridization assay was employed. Poly(A) mRNA fractions from hypothalami (40-50) of intact, castrated plus vehicle, and castrated plus T group were isolated, blotted on nitrocellulose paper and hybridized with 32P-end labelled LH-RH oligonucleotides (29 mer) which is complementary to the rat LH-RH mRNA. LH-RH-like mRNA level markedly attenuated 2 weeks following castration and T replacement significantly increased LH-RH-like mRNA level, comparable to about 80% of that observed in intact male rats. In addition, LH-RH contents and release from hypothalami were studied, to examine the correlation with LH-RH gene expression and secretory activity of LH-RH. Castration significantly reduced LH-RH contents and LH-RH release in vitro. T replacement restored LH-RH contents and release comparable to those shown in the intact group. This study demonstrates for the first time that T replacement increases LH-RH-like mRNA level in the hypothalami of castrated rats suggesting that T may act at the pretranslational level. This change in LH-RH gene expression is parallel with alteration of LH-RH content and release.     

caspase-3/8 mRNA在增殖期病理性瘢痕组织中的表达

背景：细胞凋亡参与了肉芽组织向瘢痕组织转化过程中多种细胞成分大量减少的动态过程,而Caspase家族介导的细胞凋亡是细胞凋亡的主要途径。 目的：从基因水平探讨caspase-3 mRNA及caspase-8 mRNA在增生性瘢痕组织中的作用。 方法：实验所用病理性瘢痕组织及正常皮肤组织均取自本院烧伤整形外科住院、伤口愈合6个月以后的增殖期病理性瘢痕组织18例。以正常皮肤组织18例做对照。利用RT-PCT技术分别检测半年以上增生性瘢痕组织中成纤维细胞caspase-3 mRNA及caspase-8 mRNA的表达情况。 结果与结论：在增生性瘢痕组织中,caspase-3 mRNA及caspase-8 mRNA表达的相对值分别为8.31±1.47和7.12±1.24,均明显低于正常皮肤组织(P < 0.05)。提示caspase-3/8在瘢痕形成过程中起着得要的作用,增生性瘢痕的形成可能与组织中caspase-3/8表达低下有关。     

Differential changes of bax, caspase-3 and p21 mRNA expression after transient focal brain ischemia in the rat

Recent studies of transient focal ischemia have focused interest on apoptotic mechanisms of neuronal cell death involving constitutive pro-apoptotic proteins. The finding of specific patterns of novel gene expression might indicate the activation of pro-apoptotic genes in previously ischemic areas. Thus, we investigated gene expression for the pro-apoptotic regulators, Bax and caspase-3, after transient focal brain ischemia, together with the p53-regulated cell cycle inhibitor, p21/WAF1/CIP1. Reversible occlusion of the middle cerebral artery for 2 h was carried out in halothane-anesthetized rats using the poly-L-lysine coated filament method. In situ hybridization was performed at 0, 1, 3, 6 h and 1, 3 and 7 d of recirculation and in sham controls. Radioactive antisense probes served for detection of bax, p21 and caspase-3 mRNAs on brain sections, and quantitative film autoradiography was combined with image-averaging techniques. Bax mRNA tended to decline after focal brain ischemia within 1 d. p21 mRNA was upregulated with a perifocal pattern at 3 h and 1 d after ischemia whereas the ischemic regions themselves failed to show significant upregulation. Caspase-3 mRNA was elevated in the resistant dorsomedial cortex at 1 d. A pro-apoptotic pattern of novel gene expression, involving Bax and caspase-3, was not observed after transient focal brain ischemia. Rather, the perifocal expression of p21 and caspase-3 mRNAs observed at 1 d after ischemia points to reactive changes in resistant brain areas.