Increased expression of mRNA encoding interleukin-1beta and caspase-1, and the secreted isoform of interleukin-1 receptor antagonist in the rat brain following systemic kainic acid administration

Kainic acid, an analogue of glutamate, injected systemically to rats evokes seizures that are accompanied by nerve cell damage primarily in the limbic system. In the present study, we have analyzed the temporal profile of the expression of the cytokines interleukin-1beta (IL-1beta) and IL-1 receptor antagonist (IL-1ra), and the related IL-1beta-converting enzyme (ICE/caspase-1), in different regions of the rat brain in response to peripheral kainic acid administration (10 mg/kg, i.p.). In situ hybridization histochemistry experiments revealed that IL-1beta mRNA-expressing cells, morphologically identified as microglial cells, were mainly localized to regions showing pronounced neuronal degeneration; hippocampus, thalamus, amygdala, and certain cortical regions. The strongest expression of IL-1beta mRNA was observed after 12 hr in these regions. A weak induction of the IL-1beta mRNA expression was observed already at 2 hr. Similar results were obtained by RT-PCR analysis, showing a significantly increased expression of IL-1beta mRNA in the hippocampus and amygdala after 12 hr. In addition, RT-PCR analysis revealed that IL-1ra mRNA, and specifically mRNA encoding the secreted isoform of IL-1ra (sIL-1ra), was strongly induced in the hippocampus and amygdala at 12 and 24 hr post-injection. RT-PCR analysis of mRNA encoding caspase-1 showed a significantly increased expression in the amygdala after 12 hr. In conclusion, in response to systemic kainic acid injection IL-1beta mRNA is rapidly induced and followed by induction of IL-1ra mRNA and caspase-1 mRNA, supporting a role of the IL-1 system in the inflammatory response during excitotoxic damage.     

Soman-induced interleukin-1 beta mRNA and protein in rat brain.

Exposure to high doses of the toxic organophosphate compound soman, also known as a chemical warfare agent, causes a progression of toxic symptoms including hyper-secretions, convulsions, respiratory depression, and finally death. In previous studies, we have demonstrated pronounced effects following soman intoxication in dopaminergic, GABAergic, and cholinergic systems in rat brain. The aim of this study was to investigate the effects on the pro-inflammatory cytokine interleukin-1beta (IL-1beta), indicated as mRNA and protein production, at different time intervals following soman intoxication. The peak levels of mRNA was observed 30 min following soman exposure, while a significant increase in the protein was observed at 6 h. Immunohistochemistry analysis revealed the presence of IL-1beta protein in astrocytes and endothelial cells. In addition to the previously observed effects of soman, there is an induction of IL-1beta in the brain. This effect, which is highly correlated to convulsions, implicates IL-1beta as a possible mediator for long-term brain damage observed after soman intoxication.     

Potentiation of kainic acid epileptogenicity and sparing from neuronal damage by an NMDA receptor antagonist

The time course and severity of the excitotoxic syndrome induced in rats by s.c. injection of 10 mg/kg kainic acid (KA) was modified by pretreatment with MK801, a non-competitive inhibitor of the NMDA receptor, at doses of 0.1, 1 and 10 mg/kg. A dose-dependent increase in the severity of the KA-induced electrographic (EEG) manifestations of epilepsy was seen after MK801. This consisted of an earlier appearance and higher number of EEG seizures, longer time spent in seizures, and an earlier onset of status epilepticus. In contrast, behavioral seizures were increased only in the 0.1 mg/kg MK801 group, but abolished by higher doses. On the contrary, wet dog shakes were progressively reduced with increasing doses of MK801. Four of the 9 animals receiving KA-only group and 3 of the 10 animals in the 1 and 10 mg MK801 groups were sacrificed 5 days after KA. The brain of the KA-only rats presented diffuse gross and microscopic evidence of hemorrhagic necrosis and neuronal damage; the MK801 rats showed only minimal neuronal loss in the CA3 hippocampal sector. This study demonstrates that neuronal damage and epileptiform activity can be dissociated. Furthermore, it confirms the protective effect of MK801 against neuronal damage caused by multiple factors. Lastly, it emphasizes the need for EEG monitoring in order to accurately assess any epileptic/antiepileptic effect.     

Inhibition of morphine withdrawal by the NMDA receptor antagonist MK-801 in rat is age-dependent

This study investigated the effects of the NMDA receptor antagonist MK-801 on the development of morphine dependence in 7-, 14-, and 21-day-old rat pups. For 6.5 days, starting at 1, 8, or 15 days of age, rats were pretreated with MK-801 (0.03 or 0.1 mg/kg, bid) or saline; 15 min later, morphine sulfate (10 mg/kg) or saline was injected to induce opiate dependence. On the afternoon of the seventh day, pups were injected with MK-801 (0.1 mg/kg) or saline and 15 min later with naltrexone (1 mg/kg) to precipitate withdrawal. Pups were then placed in a warm chamber with the litter and their behavior scan-sampled every 15 sec for a total of 15 min. MK-801 failed to inhibit morphine withdrawal in the 7-day-old rat, but did attenuate the development of morphine dependence in both the 14- and 21-day-old rats. These results suggest that the NMDA receptor is not functionally active in opiate withdrawal until around the second to third week of postnatal life in the rat and that there exists a transition period for the NMDA receptor to play a role in the development of opiate dependence and withdrawal.     

Region-specific changes in NMDA receptor mRNA induced by chronic morphine treatment are prevented by the co-administration of the competitive NMDA receptor antagonist LY274614

The steady-state mRNA levels of the NMDA receptor NR1 subunit were determined by a quantitative solution hybridization assay in selected CNS regions associated with antinociception in the rat. Tissues were obtained by microdissection from rats treated chronically with morphine alone or in combination with LY274614, a competitive NMDA receptor antagonist. Morphine treatment for 7 days resulted in the development of tolerance to morphine's analgesic effect and produced a significant decrease in the steady-state NR1 mRNA levels in the spinal cord dorsal horn (by 16%), and an elevation in nucleus raphe magnus and medial thalamus (by 26 and 38%, respectively). The NR1 mRNA levels were unchanged in the lateral paragigantocellular nucleus, locus coeruleus, periaqueductal grey, and sensorimotor cortex. NMDA receptor binding in the spinal cord measured with [3H]MK-801 was reduced approximately 50% by chronic morphine treatment. Co-administration of LY274614 (s.c. at 24 mg/kg/24 h via an osmotic pump) not only attenuated the development of morphine tolerance but also prevented the changes in the NR1 mRNA levels induced by chronic morphine administration. Neither a 7-day infusion of LY274614 nor an acute injection of morphine (10 mg/kg, s.c.) changed the NR1 mRNA levels. These results suggest that changes in the expression of the NR1 mRNA induced by chronic morphine in three CNS regions involved in antinociception are associated with the development of morphine tolerance and in the spinal cord, morphine tolerance is associated with the downregulation of NMDA receptors.     

红藻氨酸诱导大鼠癫痫发作脑组织HSP70表达的免疫组化及免疫印迹分析

目的探讨热休克蛋白（ＨＳＰ）７０在癫痫发作后的表达及作用。方法用免疫组化及免疫印迹分析法观察红藻氨酸诱导大鼠癫痫发作后ＨＳＰ７０的表达及其动态演变过程。结果正常海马结构含有一定量的组成性ＨＳＰ７０（ＨＳＣ７０），发作后３ｈ即有ＨＳＰ７０表达，２４ｈ达高峰（Ｐ＜０．０１），４８ｈ开始下降，持续至少７ｄ。在２４ｈ，ＨＳＰ７０免疫反应（ＨＳＰ７０－ＩＲ）阳性细胞主要分布在边缘结构。结论ＨＳＰ７０表达是癫痫发作所致细胞损伤的标志，并可能预示细胞死亡或存活，对于观察持续性神经细胞的活动特别是细胞损伤有较大意义     

Provocation of kainic acid receptor mRNA changes in the rat paraventricular nucleus by insulin-induced hypoglycaemia

Hypoglycaemia induced by insulin injection is a powerful stimulus to the hypothalamic-pituitary-adrenal (HPA) axis and drives the secretion of corticotropin-releasing hormone and vasopressin from the neurones in the paraventricular nucleus (PVN), as well as the downstream hormones, adrenocorticotropic hormone and corticosterone. In some brain regions, hypoglycaemia also provokes increases in extracellular fluid concentrations of glutamate. Regulation of glutamatergic mechanisms could be involved in the control of the HPA axis during hypoglycaemic stress and one potential site of regulation might be at the receptors for glutamate, which are expressed in the PVN. Insulin (2.0 IU/kg, i.p.) or saline was administered to adult male Sprague-Dawley rats and the animals were sacrificed 30 min, 180 min and 24 h after injection. The amount of several kainic acid-preferring glutamate receptor mRNAs (i.e. KA2, GluR5 and GluR6) were assessed in the PVN by in situ hybridisation histochemistry. Injection of insulin induced a rapid fall in plasma glucose concentrations, which was mirrored by an increase in plasma corticosterone concentrations. KA2 and GluR5 mRNAs are highly expressed within the rat PVN, and responded to hypoglycaemia with robust increases in expression that endured beyond the period of hypoglycaemia itself. However, GluR6 mRNA is expressed in the areas adjacent to the PVN and hypoglycaemic stress failed to alter expression of this mRNA. These experiments suggest that kainic acid-preferring glutamate receptors are responsive to changes in plasma glucose concentrations and may participate in the activation of the PVN neurones during hypoglycaemic stress.     

Regulation of progestin receptor expression in the developing rat brain by a dopamine d1 receptor antagonist

Steroid receptors within the developing brain influence a variety of cellular processes that endure into adulthood, altering both behaviour and physiology. Therefore, it is important to understand how steroid receptor expression is regulated during early brain development. Most studies indicate that oestradiol, by acting upon oestrogen receptors, increases the expression of progestin receptors in the developing brain. We have recently observed an additional mechanism by which dopamine can increase the expression of progestin receptors in developing female rat brain. That is, we found that a dopamine D1 receptor agonist can further increase progestin receptor expression by activating oestrogen receptors in a ligand-independent manner within restricted areas of female brain; however, it is unclear whether dopamine D1 receptors are involved in the normally occurring expression of progestin receptors in developing male and female brain. To investigate this, we examined whether a dopamine D1 receptor antagonist can disrupt the normal developmental expression of progestin receptors in both male and female rat brain. We report that treatment with a dopamine D1 receptor antagonist reduces progestin receptor expression within some, but not all, regions of the developing rat brain in a sex-specific manner. Some of the current findings also suggest that dopamine might be acting to prevent sex differences in progestin receptor expression in some areas while contributing to a sex difference in other areas.     

Application of real-time polymerase chain reaction to quantitate induced expression of interleukin-1beta mRNA in ischemic brain tolerance

A short duration of ischemia (i.e., ischemic preconditioning) was shown to result in significant tolerance to subsequent ischemic injury. Since previous reports suggest that interleukin-1beta (IL-1beta) may be involved in both ischemic damage and neuroprotection, the present work examined the expression of IL-1beta mRNA in cortical brain tissue after an established preconditioning (PC) stimulus known to produce significant brain tolerance to focal stroke after 1-7 days. Significant induction of IL-1beta mRNA was observed in the ipsilateral cortex at 6 hr (87+/-9 copies of the mRNA per microgram of brain tissue compared to 16+/-5 copies in sham-operated samples, P < 0.001, n = 4) and 8 hr (46+/-4 copies, P < 0.01, n = 4) after PC by means of real-time Taqman polymerase chain reaction (PCR). The peak expression of IL-1beta mRNA after PC was significantly (P < 0.01) lower than that after permanent occlusion of the middle cerebral artery (MCAO), i.e., 87+/-9 and 546+/-92 copies of RNA per microgram tissue at peak levels for PC and focal stroke, respectively. Immunohistochemistry studies revealed a parallel induction of IL-1beta in the ipsilateral cortex after PC. The maximal expression of IL-1beta was observed during the first week post-PC, showing marked parallelism with the duration of ischemic tolerance. These data suggest that the significant but low levels of IL-1beta induction after PC may contribute to ischemic brain tolerance.     

Interleukin-6, interleukin-1 beta and interleukin-1 receptor antagonist levels in epileptic seizures

PurposeData are accumulating to support the involvement of inflammatory mechanisms in the pathogenesis and course of epilepsy.MethodsThe aim of this study was to examine seizure-induced changes in plasma concentrations of interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1Ra), and interleukin-1 beta (IL-1β) in 23 patients with epilepsy undergoing a video-electroencephalography (EEG) study. Patients were divided into groups based on epilepsy type as follows: temporal lobe epilepsy (TLE) (n = 6), extra-temporal lobe epilepsy (XLE) (n = 8) and idiopathic generalised epilepsy (IGE) (n = 9). Serum levels of IL-1β, IL-1Ra and IL-6 were measured at baseline, immediately after the epileptic seizure, and at 3 h, 6 h, 12 h and 24 h after the seizure.ResultsWe demonstrated a significant increase in plasma levels of IL-6 and IL-1Ra that peaked at 12 h into the post-ictal period (p < 0.05). IL-1β levels did not differ from the baseline levels. We did not observe any differences in post-ictal cytokine release patterns between the TLE, XLE and IGE groups.ConclusionThe present study confirms the findings that epileptic seizures induce the production of IL-6 and IL-1Ra.     

Ornithine decarboxylase is differentially induced by kainic acid during brain development in the rat

The induction of brain ornithine decarboxylase (ODC) as a consequence of systemic kainic acid administration was studied in the hippocampus and the olfactory cortex-amygdala area of 10-day-old rat pups and 30-day-old young rats. In pups, ODC levels were moderately increased (plus 50–80%) 4 h after kainic acid administration, coming back quickly to control levels afterwards. In young rats, instead, ODC levels were dramatically increased by 17–25-fold, 16 h after kainic acid administration and decreased towards basal levels 48–72 h after injection. The present results suggest that the process of excitotoxic ODC induction can be split in two phases: a first phase characterized by moderate induction and essentially linked to the overstimulation of brain circuits and a second phase, during which a dramatic enzyme stimulation is accompanied by the appearance of neurodegenerative pathology.     

海人酸致癫痫大鼠海马GABAB受体亚单位GBR1a mRNA表达的研究

目的　通过研究海人酸致癫痫大鼠海马 GABAB受体亚单位 GBR1a m RNA表达的变化 ,为进一步探明癫痫发病的受体分子生物学机制奠定基础。方法　在立体定位仪下 ,将海人酸注射至大鼠杏仁核制备癫痫模型。将大鼠随机分为正常组和海马致癫痫组 ,分别于不同时间取材 ,进行脑组织 GABAB受体亚单位 GBR1a m RNA原位杂交检测。结果　正常组海马各区均有极少量的 GABAB受体亚单位 GBR1a m RNA的分布 ;海人酸致癫痫组( 6 h,12 h,2 4h) GABAB受体亚单位 GBR1a m RNA水平显著高于对照组 ( P<0 .0 1)。结论　正常大鼠海马各区存在着少量的 GBR1a m RNA表达 ,海人酸致痫后 6 h,12 h,2 4h,GBR1a m RNA的表达水平上调。     

Modulation of IL-1 beta gene expression by lipid peroxidation inhibition after kainic acid-induced rat brain injury

Brain injury was induced by intraperitoneal administration of kainic acid (KA, 10 mg/kg). Animals were randomized to receive either IRFI 042 (20 mg/kg i.p.), a lipid peroxidation inhibitor, or its vehicle (NaCl 0.9% DMSO 10% 1 ml/kg i.p.) 30 min before KA administration. A first set of animals was sacrificed 6 h after KA injection to measure malondialdehyde (MDA) content, glutathione-reduced (GSH) levels and the mRNA for interleukin-1beta (IL-1beta) in the cortex and in the hippocampus. A second set of animals was sacrificed 48 h after KA administration for histological analysis. All animals were observed for monitoring the behavioral sequelae and for evaluating latency of convulsions. Sham brain injury rats were used as controls. Intraperitoneal administration of IRFI 042 significantly decreased brain MDA (cortex: KA + vehicle = 0.285 +/- 0.04 nmol/mg protein; KA + IRFI 042 = 0.156 +/- 0.02 nmol/mg protein, P < 0.005; hippocampus: KA + vehicle = 0.350 +/- 0.03 nmol/mg protein; KA + IRFI 042 = 0.17 +/- 0.04 nmol/mg protein, P < 0.005), prevented the brain loss of GSH in both cortex (KA + vehicle = 7.81 +/- 1 micromol/g protein; KA + IRFI 042 = 12.1 +/- 1 micromol/g protein; P < 0.005) and hippocampus (KA + vehicle = 5 +/- 0.8 micromol/g protein; KA + IRFI 042 = 9.4 +/- 1.8 micromol/g protein; P < 0.005), reduced both brain IL-1beta mRNA expression and oedema, and increased latency of convulsions. Histological analysis showed a reduction of cell damage in IRFI 042-treated samples. The present data indicate that lipid peroxidation inhibition reduces IL-1beta gene expression and protects against kainic acid-induced brain damage.     

Association of interleukin-1beta and interleukin-1 receptor antagonist genes with disease severity in MS

OBJECTIVE: To investigate whether polymorphisms in the interleukin (IL)-1beta and IL-1 receptor antagonist (IL-1RA) genes are associated with both susceptibility to and clinical characteristics of MS. BACKGROUND: Genetic susceptibility to MS is determined by many partially identified genes. The genes encoding various cytokines are logical candidates for MS susceptibility and phenotype. METHODS: Genotypes were determined from 148 patients with clinically definite MS and 98 healthy controls. All the patients were unrelated, Dutch, and white. Patient files were reviewed for disease type, initial symptoms, age at onset of disease, and rate of disease progression. RESULTS: No significant differences in genotypes, allele frequencies, or carrier frequencies were found between MS patients and healthy controls. Stratification for disease type (relapsing-remitting, primary progressive, or secondary progressive) did not provide significant differences between patients and controls. However, a specific IL-1RA/IL-1beta combination was associated with disease severity. MS patients with the IL-1RA allele 2+/IL-1beta allele 2- combination had a higher rate of progression on the Expanded Disability Status Scale when compared with the other possible combinations (p = 0.007). CONCLUSIONS: IL-1RA and IL-1beta are disease severity genes rather than disease susceptibility genes. Furthermore, these gene polymorphisms may define subgroups of patients with a worse prognosis.     

Differential regulation of metallothionein-I,II, and III mRNA expression in the rat brain following kainic acid treatment

Although metallothioneins (MTs) are believed to be involved in the protection against neural stresses, spatio-temporal regulation of MT isoforms following neural insults has not been thoroughly examined. In this study, we found that systemic application of kainic acid (KA) rapidly induced MT-I and II expression in neurons localized in hippocampal formation, piriform cortex, and amygdala of the adult rat, whereas the level of MT-III mRNA was decreased in KA-vulnerable areas. At 96 h after KA treatment, while the neuronal expression of MT-I and II returned to basal level, the glial expression of MT-I, II and III was increased in the reactive astrocytes. Differential regulation of MT isoforms in neuron and gila suggests that each isoform might have distinct role in the cell-type dependent cellular responses against KA-evoked neural injuries.     

Serotonergic activation stimulates the pituitary-adrenal axis and alters interleukin-1 mRNA expression in rat brain

Interactions between neurotransmitters and immunomodulators within the central nervous system may be functionally relevant for communication between the immune system and the brain. Previous studies indicate that cytokines such as interleukin-1 (IL-1) alter activity of the serotonergic system at multiple levels. This study tested the hypothesis that serotonergic activation modulates cytokine mRNA expression in brain. Serotonergic activation was induced by injecting rats intraperitoneally (i.p.) prior to dark onset with the serotonin precursor L-5-hydroxytryptophan (5-HTP; 100 mg/kg). Cytokine mRNA expression in discrete brain regions at selected time points was determined by means of ribonuclease protection assay. Plasma corticosterone concentrations were also measured to determine if the hypothalamic-pituitary-adrenal axis is activated in response to this treatment, which potentially could exert feedback regulating cytokine message expression in brain. Plasma corticosterone was elevated for 4 h after 5-HTP administration. At this time IL-1alpha mRNA expression was reduced in the hippocampus, hypothalamus, and brainstem, and IL-1beta mRNA was reduced in the hippocampus. Six hours after 5-HTP injection, IL-1beta mRNA increased in the hypothalamus. These results show that activation of the serotonergic system affects cytokine message expression in rat brain, possibly by actions of corticosterone.     

Inhibition of leptin-induced IL-1beta expression by glucocorticoids in the brain

Leptin is an important circulating signal for the regulation of food intake and body weight. Glucocorticoids were suggested to play a physiological role in the feedback inhibition of immune/inflammatory responses. In the present study, we examined whether these neuroendocrine effects of glucocorticoids are linked to changes in the leptin-induced expression of IL-1beta and STAT3 activation in the brain. Intravenous injection of leptin induced IL-1beta expression in the hypothalamus. Pretreatment with dexamethasone dose dependently inhibited leptin-induced IL-1beta expression in the hypothalamus. Moreover, dexamethasone inhibited leptin-induced IL-1beta expression in the primary cultured glial cells. In contrast, pretreatment with dexamethasone did not inhibit leptin-induced STAT3 phosphorylation in the hypothalamus. These effects of dexamethasone may not be due to the change in the expression level of the leptin receptor Ob-Ra and Ob-Rb isoforms. Therefore, it is suggested that glucocorticoid negatively regulates leptin-induced IL-1beta expression in the brain.