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Expression Changes of Nuclear Receptors-Glucocorticoid Receptor, Mineralocorticoid Receptor and Apoptosis in Locus Coeruleus Nerons of PTSD Rats

Author: LiMan
Tutor: ShiYuXiu
School: China Medical University
Course: Human Anatomy and Embryology
Keywords: posttraumatic stress disorder locus coeruleus nuclear receptors Glucocorticoid receptor Mineralocorticoid receptor Caspase-3 Caspase-9 cytochrome c oxidase western blotting neurons apoptosis
CLC: R749.5
Type: Master's thesis
Year: 2010
Downloads: 128
Quote: 0
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Abstract


ObjectivePosttraumatic stress disorder (PTSD) is a disorder that develops in response to a traumatically stressful life event. Despite the extensive studies of the neurobiological correlates of this disorder, the underlying mechanisms responsible for the development of PTSD remain poorly understood. Symptoms, resultant from experiencing a severe traumatic event, may include increased sensitivity to stress, increased startle responses, severe anxiety, hypervigilance, increased autonomic response, and increased recollection of past stress as flashbacks and nightmares. These signs and symptoms suggest that PTSD be associated with increased circulating cortisol levels. However, it is surprising that many reports show that cortisol levels are actually lower than normal levels. A decreased negative feedback for the cortisol secretion is found in major depression and, in contrast, an enhanced negative feedback has been found in PTSD.Several studies have reported increased urinary concentrations of norepinephrine and/or epinephrine in patients with PTSD, compared with patients with non-trauma-related anxiety. The majority of noradrenergic neurons are located in the locus coeruleus (LC). In addition, LC plays a major role in poor sleep, hypervigilance, and irritability/anger, which make up a part of the hyperarousal cluster in the diagnosis of PTSD.There are two types of adrenal steroid receptors, TypeⅠ(mineralocorticoid receptor, MR) and TypeⅡ(glucocorticoid receptor, GR). The former has a higher affinity for cortisol than does the latter, exerting an inhibitory influence on the activity of the HPA-axis. Upon stress, the cortisol levels increase sharply and the GR is occupied. The balance between the two corticosteroid receptors is critical to the stress response and behavioral adaptation thereafter. Both MR-and GR-mediated effects on the information processing facilitate behavioral adaptation and thereby stimulate the higher brain centers to exert the inhibitory control over the HPA-axis activity. In this process, one may consider that corticosteroid receptors function as transcription factors. Of note, individual differences in the number, affinity and efficiency of these signaling cascades activated by these receptors have direct effects on the cortisol levels and its biological activity.Clinical observations in patients with PTSD and in the relevant animal models point towards an involvement of GR and MR in PTSD.Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli. The intrinsic pathway of apoptosis is mediated by various stimuli that cause the release of cytochrome c oxidase (COX) from mitochondria into the cytoplasm, triggering caspases activation. The mitochondrial caspases cascade is initiated by the release of COX and caspase-9 from the intermembrane space into the cytosol. Caspase-9 then activates caspase-3, the execution caspase, leading to the manifestation of apoptotic processes including DNA fragmentation as well as mitochondrial inner membrane permeability transition. Several studies have reported a reduction in the volume of the hippocampus in PTSD patients and single-prolonged stress induces apoptosis in the amygdala in a rat model of PTSD. Bracha HS reported veterans with probable or possible WR-PTSD were found to have substantially lower locus coeruleus neuronal counts compared to four comparison subjects. Did locus coeruleus neurons have cell apoptosis, which may lead to the dysfunction of locus coeruleus of PTSD.Method1、Model building and groupingUsed the SPS model of PTSD which had been established by international science conference that convoked in Japan in 2005.It is carried out by the following consecutive steps:Immobilization,2h;Forced swimming,20min; Ether anesthesia;Undisturbed. The rats were randomly divided into six groups:control group and SPS afterld,4d,7d,14d and 28d. 2、Immunohistochemistry staining of locus coeruleus GR,MR and Caspase-3Took out the rats brain which had been fixed and dipped down.Made them into Paraffin sections, and then were stained by SABC method. And DAB coloration, last, neutral gummi mounted. The result was observed by light microscope.3、Imunofluorescent analysis of Caspase-9The sections were incubated with mouse monoclonal antibody against Caspase-9 over night at 4℃. After three times washing, the sections were incubated with FITC anti-mouse IgG for 30 min at room temperature. After being washed in PBS and mounted, fluorescence microscope was applied for observation.4、Western blotting to detect MR and GRThe obtained locus coeruleus tissues were then homogenized in a buffer containing 100 mM Tris-Cl and 4% sodium dodecyl sulfate (SDS) at 4℃. The membrane was incubated withⅠantibody andⅡantibody. Blots were subjected to autoradiography. The levels of GR and MR were normalized byβ-actin.5、Assessing the mitochondria change in locus coerleus using transmission electronmicroscopy (TEM)The sections at the above incubation in, then postfixed in 1% osmium tetroxide for 30-60min, general dehydration, embedding, and finally polymerized in pure Epon. Locus coerleus was localized on semi-thin sections, then observed with transmission electron microscopy.6、RT-PCR to detect GR,MR, Caspase-3,Caspase-9 and COXDissected the fresh locus coeruleus. Extracting the total RNA to reverse transcription and PCR amplification. Then, have gel electrophoresis and imaging. Last, analyzed integrated optical density.Results1、Results of immunohistochemical analysis of GR and MRGR was located in nucleus and MR was located in cytoplasm.We found a significant increase in GR expression in the SPS rats (1d,4d, and 7d), compared with the control rats. The MR in the SPS rats and significantly decreased on day 1, but increased on days 4,7,14 and 28.Caspase-3 was located in cytoplasm. Evaluation of Caspase-3 immunohistochemistry showed a significant increased in the SPS-1d, SPS-4d and SPS-7d compared with the normal control group, then gradually decreased in SPS-14d.2、Imunofluorescent microscopy observed of Caspase-9 expressionThe concentrations of FITC labelled Caspase-9 in the locus coeruleus were measured. The Caspase-9 increased more than control in locus coeruleus at SPS-1d and SPS-4d, the signal of green was stronger, then gradually decreased at SPS-7d and SPS-14d.3、GR and MR expression detected by Western blottingThe GR levels in the SPS rats were elevated on days 1,4, and 7, and then gradually decreased. We observed a significant down-regulation of MR protein in the SPS rats on day 1, but gradually increased afterwards. The ratio curve pattern over time was consistent with the one analyzed by immunohistochemistry.4、Morphological changes of the mitochondria by TEMLocus coeruleus neurons mitochondria morphology good at control group, positive products were mainly distributed in the mitochondrial membrane both inside and outside. SPS groups can see mitochondria swelling, vacuolization deformation, ridge fracture, membrane rupture, COX released into the cytoplasm.5、The mRNA levels of GR,MR,COX, Caspase-3 and Caspase-9Similar to the Western blotting, compared with the control group, the mRNA levels of GR in the SPS rats were significant increased on days 1,4, and 7, and then gradually decreased. The MR mRNA levels were sharply declined on day 1, but gradually increased on days 4,7,14, and 28.Compared with the control group, the mRNA levels of COX and Caspase-9 in the SPS rats were significant increased on days 1d and 4d then gradually decreased. The Caspase-3 mRNA levels peaked at SPS-7d, then decreased on SPS-14d.Conclusion1、After SPS, the GR locus ceruleus temporary increase, and then lowered, revealing PTSD rat locus coeruleus neurons in nuclear receptor-GR expression changes directly involved in the symptoms of PTSD.2、The changes of in the expression of MR in the locus ceruleus neurons of PTSD-like rats may play an important role in the long-term persistent neuropsychological sequelae of PTSD.3、The locus coeruleus neuronal apoptosis would play an important role in the dysfunction of locus coeruleus in post traumatic stress disorder patients.

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