Effect of hyperthermia and anoxia on glucocorticoid and mineralocorticoid receptor expression in neonatal rat hippocampus

Brief periods of neonatal asphyxia are frequently observed. Within the CNS, the hippocampus is known to be particularly vulnerable to the damaging effects of hypoxia/ischaemia. The hippocampus contains the highest concentration of both mineralocorticoid (MR) and glucocorticoid (GR) receptors and the balance between MR/GR activation influences cell birth and death. MR occupation appears to promote prosurvival actions, while GR overactivation favours neurodegeneration. It has been widely recognized that core body temperature is a critical determinant of the severity of hypoxic–ischemic brain injury; indeed, hyperthermia exacerbates the degree of damage. Therefore, the aim of the present investigation was to study the effect of elevated body temperature in newborn rats under control conditions or during neonatal exposure to a critical anoxia, on changes of MR and GR mRNA expression in the rat hippocampus. 2-day-old rats were exposed to anoxia in 100% nitrogen atmosphere. Rectal temperature was kept at 33 °C (typical for the rat neonates), or elevated to a level typical for febrile (39 °C) adults. Control rats were exposed to atmospheric air under the respective thermal conditions. The changes in MR and GR mRNA expression in hippocampus were examined 24 h after exposure. Our data show that hyperthermia with or without added anoxia, causes induction of MR mRNA expression in neonatal rat hippocampus without any effect on GR mRNA expression. We suggest this elevation of MR plays an important role in modulating the survival of neurons in the injured hippocampus.     

Expressions of hippocampal mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the single-prolonged stress-rats

Post-traumatic stress disorder (PTSD) is a stress-related mental disorder caused by traumatic experience. Single-prolonged stress (SPS) is one of the animal models proposed for PTSD. Rats exposed to SPS showed enhanced inhibition of the hypothalamo-pituitary-adrenal (HPA) axis, which has been reliably reproduced in patients with PTSD. Mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the hippocampus regulate HPA axis by glucocorticoid negative feedback. Abnormalities in negative feedback are found in PTSD, suggesting that GR and MR might be involved in the pathophysiology of these disorders.In the present study, we performed immunohistochemistry and western blotting to examine the changes in hippocampal MR- and GR-expression after SPS. Immunohistochemistry revealed decreased MR- and GR-immunoreactivity (ir) in the CA1 of hippocampus in SPS animals. Change in GR sub-distribution was also observed, where GR-ir was shifted from nucleus to cytoplasm in SPS rats. Western blotting showed that SPS induced significantly decreased MR- and GR-protein in the whole hippocampus, although the degree of decreased expression of both receptors was different. Meanwhile, we also found the MR/GR ratio decreased in SPS rats. In general, SPS induced down-regulation of MR- and GR-expression. These findings suggest that MR and GR play critical roles in affecting hippocampal function. Changes in MR/GR ratio may be relevant for behavioral syndrome in PTSD.     

Colocalization of mineralocorticoid receptor and glucocorticoid receptor in the hippocampus and hypothalamus

We investigated the distribution and colocalization pattern of the two corticosteroid receptors, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), in the hippocampus and hypothalamus, the main target regions of corticosterone in the rat brain, using double immunofluorescence histochemistry in conjunction with specific polyclonal antibodies against MR and GR. In the hippocampus, MR- and GR-immunoreactivity (ir) were colocalized in CA1 and CA2 pyramidal neurons and granule cells of the dentate gyrus, while only MR-ir was seen in the CA3 pyramidal neurons. Colocalization of MR- and GR-ir was also observed in the parvocellular region, but not in the magnocellular region of the paraventricular nucleus (PVN). Subcellular distribution of MR-ir was more cytoplasmic in comparison with that of GR-ir, while the ratio of cytoplasmic to nuclear distribution of these receptors was different among the regions. After adrenalectomy (ADX), the distribution pattern of both receptors was changed to cytoplasmic, although the degree of the change of distribution was different among each region. Replacement of corticosterone after ADX recovered the distribution pattern to that of the sham-operated animals. These results suggest that the balance of MR and GR in the cell underlies the potential regulation of corticosteroid through the hippocampus and hypothalamus.     

Corticosteroids stimulate the amphibious behavior in mudskipper: potential role of mineralocorticoid receptors in teleost fish

It has long been held that cortisol, a glucocorticoid in many vertebrates, carries out both glucocorticoid and mineralocorticoid actions in teleost fish. However, 11-deoxycorticosterone (DOC) has been identified as a specific endogenous ligand for the teleostean mineralocorticoid receptor (MR). Furthermore, the expressions of MR mRNA are modest in the osmoregulatory organs, but considerably higher in the brain of most teleosts. These recent findings suggest that the mineralocorticoid system (DOC/MR) may carry out some behavioral functions in fish. To test this possibility, we examined the effects of cortisol and DOC administration in the amphibious behavior in mudskipper (Periophthalmus modestus) in vivo. It was found that mudskippers remained in the water for an increased period of time when they were immersed into 5 μM DOC or cortisol for 8 h. Additionally, an exposure to 25 μM DOC for 4 to 8 h caused a decreased migratory frequency of mudskippers to the water, reflected a tendency to remain in the water. It was further observed that after 8 h of intracerebroventricular (ICV) injection with 0.3 pmol DOC or cortisol the staying period in the water increased in fish. The migratory frequency was decreased after ICV DOC injection which indicated that fishes stayed in the water. Concurrent ICV injections of cortisol with RU486 [a specific glucocorticoid-receptor (GR) antagonist] inhibited only the partial effects of cortisol. Together with no changes in the plasma DOC concentrations under terrestrial conditions, these results indicate the involvement of brain MRs as cortisol receptors in the preference for an aquatic habitat of mudskippers. Although the role of GR signaling cannot be excluded in the aquatic preference, our data further suggest that the MR may play an important role in the brain dependent behaviors of teleost fish.     

Cytosolic glucocorticoid receptor expression in the rat vestibular nucleus and hippocampus following unilateral vestibular deafferentation

It has been suggested that vestibular compensation, the process of behavioural recovery that occurs following peripheral vestibular damage, might be partially dependent on the release of glucocorticoids (GC) during the early stages of recovery from the lesion. One possibility is that glucocorticoid receptors (GRs) in the vestibular nucleus complex (VNC) might change following the lesion, altering their response to GCs. We sought to test this hypothesis by quantifying the expression of cytosolic GRs in the bilateral VNCs at 10 h, 58 h and 2 weeks following unilateral vestibular deafferentation (UVD) in rat, using western blotting. We also examined GR expression in the CA1, CA2/3 and dentate gyrus (DG) subregions of the hippocampus and measured serum corticosterone levels. Compared with sham surgery and anaesthetic controls, we found no significant changes in GR expression in the ipsilateral or contralateral VNCs at any time post-UVD. However, we did find a significant decrease in GR expression in the ipsilateral CA1 at 2 weeks post-UVD. Serum corticosterone levels were significantly lower in all groups at 58 h post-op. compared to 10 h and 2 weeks; however, there were no significant differences between the UVD and control groups at any time point. These results suggest that changes in GR expression in the VNC are unlikely to contribute to the development of vestibular compensation. However, long-term changes in GR expression in CA1 might be related to chronic deficits in hippocampal function and spatial cognition following vestibular damage.     

The effects of the glucocorticoid receptor antagonist RU486 and phospholipase A2 inhibitor quinacrine on acoustic injury of the mouse cochlea

Glucocorticoids are used clinically for the treatment of acoustic injury. However, the protective mechanism of glucocorticoid in acoustic injury has not been completely clarified. Also, the effects of phospholipase A2 (PLA2) on acoustic injury have not been examined to the best of our knowledge. The purpose of the present study was to examine the effects of methylprednisolone, a glucocorticoid receptor inhibitor (RU486) and a phospholipase A2 inhibitor (quinacrine) on cochlear injury induced by acoustic overexposure. Seventy-eight mice were exposed to a 4kHz pure tone at 128dB SPL for 4h. The auditory brainstem response (ABR) was used to examine the hearing thresholds. Cochlear morphology was examined to estimate the outer hair cell loss induced by acoustic overexposure. Methylprednisolone and quinacrine significantly alleviated the hearing threshold shift and hair cell loss induced by acoustic overexposure. RU486 antagonized the protective effect of methylprednisolone. The present findings suggest firstly that glucocorticoids exert protective effects against acoustic injury; secondly, that the protective effect of methylprednisolone was exerted by binding glucocorticoid receptors, and finally that activation of PLA2 may be involved in acoustic injury.     

The relationship between the expression of the glucocorticoid receptor in biopsied colonic mucosa and the glucocorticoid responsiveness of ulcerative colitis patients

The objective of this study was to clarify the relationship between the frequency of infiltrating cells expressing the glucocorticoid receptors (GR) α and β in biopsied colonic mucosa and the glucocorticoid (GC) responsiveness of ulcerative colitis (UC) patients. Active UC patients (n = 38) were divided into GC-sensitive and GC-resistant groups. GRβ⁺ cells were significantly higher in the GC-resistant group than in the GC-sensitive and control groups. GRα mRNA was expressed in all UC patients, while GRβ mRNA was expressed in only 1 patient in the GC-sensitive group (n = 6) and 7 patients in the GC-resistant group (n = 8). Double-positive cells for GRβ and CD4 or CD19 were frequently observed. The Foxp3⁺ cell count was significantly higher in the GC-sensitive group than in the GC-resistant group, but double Foxp3⁺GRβ⁺ cells were not observed. These results indicated that the sensitivity of GC therapy could probably be predicted by immunostaining biopsy specimens for GRβ and Foxp3.     

Effect of neonatal respiratory infection on adult BALB/c hippocampal glucocorticoid and mineralocorticoid receptors

The current study investigated the effects of neonatal infection with Chlamydia muridarum bacteria on glucocorticoid (GR) and mineralocorticoid (MR) receptors in the adult mouse hippocampus. In male adults infected at birth, circulating corticosterone was significantly increased when compared to same sex controls; while neonatal infection resulted in female adults with significantly increased GR mRNA compared to same sex controls. When comparing males and females after neonatal infection, males had significantly less GR protein than females. Interestingly, after control treatment, males had significantly more GR mRNA, MR mRNA, and GR protein with significantly lower corticosterone than females. Neonatal respiratory infection significantly impacts adult hippocampal GR and MR, and circulating corticosterone in a sex-specific manner potentially altering stress responsivity.     

多发性肌炎/皮肌炎患者外周血单个核细胞中糖皮质激素受体α、βmRNA表达与糖皮质激素疗效相关性的研究

目的探讨多发性肌炎/皮肌炎（PM/DM）患者外周血单个核细胞（PBMC）中糖皮质激素受体（GR）正常表位α和突变体表位β mRNA表达与PM/DM患者对糖皮质激素（GC）疗效的相关性。方法采用逆转录-聚合酶链反应（RT-PCR）方法对18例GC治疗敏感、10例GC治疗抵抗的PM/DM患者PBMC中GRα、β mRNA的表达水平进行了检测,同时采用放射免疫分析法测定血清中的皮质醇含量,并与20例正常对照进行比较。结果血清皮质醇含量在GC治疗敏感和抵抗的PM/DM患者及正常对照间差异均无统计学意义（均P〉0.05）。GC治疗敏感和抵抗的PM/DM患者PBMC中GRα mRNA的表达水平均显著低于正常对照（均P〈0.01）,且GC治疗抵抗的PM/DM患者显著低于GC治疗敏感的PM/DM患者（P〈0.05）。GC治疗敏感和抵抗的PM/DM患者PBMC中GRβ mRNA的表达均显著高于正常对照（均P〈0.05）,但在GC治疗敏感和抵抗的PM/DM患者间则差异无统计学意义（P〉0.05）。结论GRα mRNA表达水平降低可能与PM/DM患者对GC治疗抵抗相关,GRβ mRNA表达水平在PM/DM患者的GC治疗抵抗中无显著作用。     

Immunocytochemical application of monoclonal antibodies to rat liver glucocorticoid receptor

Seven per cent (10/145) of hybridomas raised against partially purified activated glucocorticoid receptor from rat liver produced monoclonal antibody to receptor. Six IgM secreting clones selected for further investigation bound equally well to activated and non-activated receptor from fresh rat liver, but significantly less well (11-25 per cent) to receptor from frozen rat liver. No interaction was found with oestrogen receptor from rat uterus but extensive cross reaction occurred with progesterone receptor. Although none of the antibodies bound to glucocorticoid receptor from human or porcine liver or lymphoid cells, several cross-reacted with mouse liver glucocorticoid receptor. Immunoelectroblotting of proteins from fresh and frozen rat liver cytosol showed the antibodies bound to 90,000 and 40,000 MW forms of receptor respectively. Immunostaining of both frozen and paraffin embedded sections of rat tissue showed that receptor is preserved during fixation and processing of tissues. Using both indirect immunoperoxidase and immunogold silver staining methods, the pattern of receptor staining observed correlates with the known glucocorticoid responsiveness of the tissues studied.     

大鼠烫伤后肝胞浆糖皮质激素受体和酪氨酸转氨酶的变化

成年大鼠,雌,背部用沸水烫伤,轻烫组的烫伤面积为5.3cm直径的圆面积。烫10秒。重烫组为6.5cm直径,15秒。于烫伤后1、3、6和9小时断头处死,测定血浆皮质酮(B)、肝胞浆糖皮质激素受体(GCR)和酪氨酸转氨酶活性(TAT)。结果烫后3和6小时,尽管重烫组血浆B很高,但TAT却明显低于轻烫组(P<0.001),反映了严重烫伤组靶细胞对内源性糖皮质激素(GC)的反应性降低。烫后1、3小时重烫组GCR的结合容量(R_0)也明显低于轻烫组,提示重烫组靶细胞对GC的反应性降低至少部分地是由于GCR减少所致。对严重烫伤时靶细胞对GC的反应性降低的可能的病理和临床意义作了讨论。     

Acute stress increases neuropsin mRNA expression in the mouse hippocampus through the glucocorticoid pathway

Stress affects synaptic plasticity and may alter various types of behaviour, including anxiety or memory formation. In the present study, we examined the effects of acute stress (1 h restraint with or without tail-shock) on mRNA levels of a plasticity-related serine protease neuropsin (NP) in the hippocampus using semiquantitative RT-PCR and in situ hybridization. We found that NP mRNA expression was dramatically increased shortly after exposure to the acute restraint tail-shock stress and remained at high level for at least 24 h. The level of NP mRNA would be correlated to the elevated plasma concentration of the glucocorticoid corticosterone (CORT) and to the stress intensity. Application of CORT either onto primary cultured hippocampal neurons (5 nM) or in vivo to adrenalectomized (ADX) mice (10 mg/kg B.W., s.c.) mimicked the effect of stress and significantly elevated NP mRNA. These results suggest that the upregulation of NP mRNA after stress is CORT-dependent and point to a role for neuropsin in stress-induced neuronal plasticity.     

乙型肝炎患者糖皮质激素受体亚型mRNA表达水平的研究

目的研究乙型肝炎患者外周血单个核细胞（PBMC）的糖皮质激素受体（glucocorticoid receptor,GR）亚型含量变化与乙型肝炎临床分型的相关性。方法应用实时荧光定量RT-PCR方法分别检测正常对照组（20例）、慢性肝炎组（31例）、肝硬化组（25例）、重型肝炎组（29例）PBMC内GR-α与GR-β亚型mRNA的表达水平。结果慢性肝炎患者GR-αmRNA相对含量为（22.9±2.8）×10-2、GR-βmRNA相对含量为（2.8±0.8）×10-4;重症肝炎患者GR-αmR-NA相对含量为（36.6±3.9）×10-2、GR-βmRNA相对含量为（2.6±0.5）×10-4;肝硬化炎患者GR-αmRNA相对含量为（7.2±1.5）×10-2、GR-βmRNA相对含量为（3.6±1.3）×10-4;健康对照组GR-αmRNA相对含量为（5.6±0.5）×10-2、GR-βmRNA相对含量为（3.0±0.9）×10-4。慢性肝炎组和重症肝炎组患者GR-αmRNA表达水平与正常对照组比较均明显增高,差异有显著性（t=19.71,P〈0.001;t=23.73,P〈0.001）,各组患者GR-β表达水平与正常对照组比较无显著性差异（P〉0.05）。结论乙型肝炎患者PBMC的GR-αmRNA含量变化与乙型肝炎临床分型相关。     

音乐对大鼠海马NMDA受体表达的作用

目的：研究音乐刺激对大鼠海马组织NMDA受体及其受体蛋白mRNA表达的影响。方法：用免疫组织化学和PCR技术，定量检测了NMDA受体蛋白和编码该受体的mRNA表达。结果：音乐刺激后，NMDA受体蛋白及其mRNA表达，在持续音乐刺激组分别高出对照组40％和58％，生后音乐组高出33％和42％，而胎期音乐组与对照组相比不显著(4％和6％)。结论：音乐刺激能显著增强大鼠海马组织NMDA受体和mRNA表达，而且其增强效应具有音乐刺激时间依赖性，且生后的作用较为明显。     

大鼠海马γ-氨基丁酸-A受体在生后发育中的表达

目的:研究大鼠生后发育过程中海马组织γ-氨基丁酸-A(GABA-A)受体的表达规律。方法:用免疫组织化学和PCR技术,检测不同年龄大鼠海马组织GABA-A受体及编码该受体的mRNA表达,并用图像分析方法进行定量研究。结果:大鼠海马组织在生后3 d已经出现GABA-A受体免疫反应,以后逐渐增强,到生后30 d达到最高值,海马各区GABA-A受体免疫反应强度没有显著的差别;编码GABA-A受体的mRNA表达也有类似的增龄性增多,但其表达高峰值提前到14 d。结论:生后大鼠海马GABA-A受体表达在一定时期内呈增龄性表达增强的趋势。     

C-Jun蛋白对糖皮质激素受体表达及转录激活能力的影响

目的观察促进与抑制c-Jun蛋白表达对糖皮质激素受体(GR)表达和转录激活能力的影响,探索c-Jun与GR功能的相互关系.方法采用基因重组技术,构建PAVU6-c Jun siRNA干涉质粒,电穿孔法分别转染U937细胞c-Jun蛋白表达真核载体与PAVU6-c Jun siRNA干涉质粒,观察c-Jun表达对GR表达和转录激活能力的影响.结果成功构建PAVU6-c JunsiRNA干涉质粒,转化U937细胞后GR的表达和转录激活活性有增强,而转染c-Jun表达载体pCMV-c JunGR的表达和转录激活活性有所减弱.结论c-Jun蛋白表达水平对GR的表达和转录激活活性有影响,抑制c-Jun蛋白表达,一定程度上可促进GR表达并增强其转录激活功能.     

Glucocorticoid receptor gene polymorphisms and glucocorticoid sensitivity of subdermal blood vessels and leukocytes

A considerable variability in the sensitivity to glucocorticoids (GCs) exists between individuals and these differences have been implicated in the etiology of psychiatric diseases such as depression. Glucocorticoid receptor (GR) gene polymorphisms might account in part for variability in GC responsiveness. We assessed the association between four common GR gene (NR3C1) polymorphisms (ER22/23EK, N363S, BclI, 9beta) and markers of glucocorticoid sensitivity in two target tissues (subdermal blood vessels, peripheral leukocytes) in 206 healthy individuals. The BclI GG genotype group showed the least degree of skin blanching, reflecting a lower GC sensitivity of subdermal blood vessels (p=.01). No association between GR genotype and GC sensitivity of peripheral leukocytes was observed. In the same subjects we previously observed an association between GR genotype and GC sensitivity of the pituitary. Polymorphism of the GR gene might constitute a vulnerability or protection factor for stress related disorders and altered GC sensitivity.     

连续单一应激后大鼠海马5-HT_(1A)受体活性与糖皮质激素受体表达的关系

目的:观察连续单一应激(SPS)大鼠海马糖皮质激素受体(GR)变化与5-HT1A受体的关系,探讨创伤后应激障碍的发病机制。方法:选用雄性成年Wistar大鼠45只,将大鼠随机分为对照组、模型组和阻断组,每组15只。模型组和阻断组给予SPS应激,其中阻断组大鼠在接受SPS前用55-HT1A受体阻断剂WAY100635预处理。采用免疫组织化学和免疫印迹技术测定海马GR水平,采用逆转录-聚合酶链式反应检测GRmRNA表达变化。结果:(1)免疫组织化学结果显示,模型组大鼠海马GR表达高于对照组(P0.01),阻断组则低于模型组(P0.05);(2)WesternBlot结果显示,模型组大鼠海马GR相对表达高于对照组(P0.01),阻断组低于模型组(P0.01);(3)RT-PCR结果表明,与对照组比较,模型组大鼠海马GRmRNA表达增强(P0.01);与模型组比较,阻断组表达量降低(P0.05)。结论:SPS海马GR表达变化与5-HT1A受体有关。     

Immunohistochemical analysis of the colocalization of corticotropin-releasing hormone receptor and glucocorticoid receptor in kisspeptin neurons in the hypothalamus of female rats

Kisspeptin, a neuropeptide encoded by Kiss1 gene, plays pivotal roles in the regulation of reproductive function. Recently various stressors and stress-induced molecules such as corticotropin-releasing hormone (CRH) and corticosterone have been shown to inhibit Kiss1 expression in rat hypothalamus. To determine whether CRH and glucocorticoids directly act on kisspeptin neurons, we examined the colocalization of CRH receptor (CRH-R) and glucocorticoid receptor (GR) in kisspeptin neurons in the female rat hypothalamus. Double-labeling immunohistochemistry revealed that most kisspeptin neurons in the anteroventral periventricular nucleus and periventricular nucleus continuum (AVPV/PeN), and arcuate nucleus (ARC) expressed CRH-R. We also observed a few close appositions of CRH immunoreactive fibers on some of kisspeptin neurons in AVPV/PeN and ARC. On the other hand, most kisspeptin neurons in AVPV/PeN expressed GR, whereas only a few of kisspeptin neurons in ARC expressed GR.