Effect of ginsenoside Rg1 on behavioral changes and autophagy of hip-pocampal neurons of rats with post-traumatic stress disorder / 中国病理生理杂志

AIM:To investigate the effects of ginsenoside Rg1 on the behavioral changes and the autophagy of hippocampal neurons of the rats with post-traumatic stress disorder (PTSD).METHODS:The Sprague-Dawley rats were randomly divided into 5 groups:control group, model group, fluoxetine group, low-dose ginsenoside Rg1 group and high-dose of ginsenoside Rg1 group.The combination of single prolonged stress and foot stock was performed to induce PTSD-like animal model.The rats in fluoxetine group was administered with fluoxetine by gavage at dose of 10 mg/kg for 21 d, while the rats in low and high doses of ginsenoside Rg1 groups were administered with ginsenoside Rg1 by gavage at doses of 20 mg/kg and 40 mg/kg for 21 d, respectively.The rats in control group and model group were both given saline by gavage for 21 d.The open-field test and stiff behavior test were used to examine the behavioral changes of the rats.The morphological structure and numerical changes of the hippocampal neurons were observed by Nissl-staining method.We adopted immunofluorescence labeling to observe the beclin 1 and LC3 positive hippocampal neurons and the levels of beclin 1 and LC3-Ⅱ/LC3-Ⅰratio in rat hippocampus.RESULTS:Compared with control group, decreased vertical movement time and horizontal movement time in open-field test and increased rate of stiff behavior in the stiff behavior test were observed in model group.Hippocampal neurons in model group were loosely arranged with vacuole-like structures and different degrees of cell shrinkage in contrast with control group.More beclin 1 and LC3 positive cells were identified, and higher protein levels of beclin 1 and ratio of LC3-Ⅱ/LC3-Ⅰ in model group were found as compared with control group.However, increase in movement in open-field test and decrease in stiff behavior were detected in the rats treated with low-and high-dose ginsenoside Rg1 as compared with the model rats.Meanwhile, vacuole structures, the numbers of beclin 1 and LC3 positive neurons, the protein expression of beclin 1 and LC3, and the total cell numbers were increased.Higher dose of ginsenoside Rg1 had more profound effects on these observed results.CONCLUSION:Ginsenoside Rg1 alleviates the abnormal behaviors in the PTSD rats, which might be related to the inhibition of abnormal autophagy of hippocampal neurons.

Tripotolide ameliorates inflammation and apoptosis induced by focal cerebral ischemia/reperfusion in rats / 浙江大学学报·医学版

To investigate the effects of triptolide on inflammation and apoptosis induced by focal cerebral ischemia/reperfusion in rats.The rat model of focal cerebral ischemia/reperfusion injury was established according to Longa's method. A total of 80 SD rats were randomly divided into 5 groups:normal control, sham group, DMSO group, middle cerebral artery occlusion (MCAO) group, and MCAO with tripolide treatment group. TTC staining was used to examine the site and volume of cerebral infarction, and Longa score was employed for neurological disorders measurement. Number of astrocytes was measured by fluorescence staining, and neuronal apoptosis was determined by TUNEL staining. The expressions of inducible nitric oxide synthase(iNOS), cyclooxygenase 2(COX-2) and NF-κB proteins were detected by immunohistochemistry, and the expression of iNOS, COX-2 mRNA was detected by real-time PCR.Compared with DMSO group and MCAO group, brain edema was improved (80.03±0.46)% (<0.05), infarct volume was reduced (8.3±1.4)% (<0.01), Longa score was decreased (1.38±0.20,<0.05) in triptolide treatment group. Meanwhile triptolide also dramatically reduced the number of GFAP-positive astrocytes (<0.05), alleviated protein expression of COX-2 (91.67±1.31), iNOS (95.24±5.07) and NF-κB (75.03±2.06) triggered by MCAO (all<0.05), and induced a down-regulation of cell apoptosis as showed by TUNEL assay (64.15±3.52,<0.05).Triptolide can reduce the cerebral infarction volume, attenuate brain edema and ameliorate the neurological deficits induced by cerebral ischemia-reperfusion injury rats, indicating that it might be used as a potential anti-inflammatory agent.

Effect of JNK expression in dorsal root ganglia on histological change of foot skin in diabetic rats / 中国病理生理杂志

[ ABSTRACT] AIM:To observe the change of skin histology in diabetic rats and to investigate the possible me-chanism of c-Jun N-terminal kinase (JNK) protein in the dorsal root ganglion (DRG) during the process.METHODS:Diabetic animal model was established in the male SD rats by intraperitoneal injection of streptozotocin.Plantar skin speci-mens of the rats were collected from control group, DM 2-week group (DM2), DM 4-week group (DM4), and DM 8-week group ( DM8) .Immunohistochemical staining and HE staining were used to observe the change of PGP 9.5 immunoreactive nerve terminals and the structures of the skin tissues.The protein expression of PGP 9.5 in the plantar skin tissues, and JNK and p-JNK protein in the DRG within lumbar 5, 6 (L5, 6), and sacral 1 (S1) spinal cord segments were detected by Western blotting.RESULTS:PGP 9.5 immunoreactive nerve terminals of the plantar skin of the rats mainly distributed in the basal layer of the epidermis and papillary dermis.Compared with control group, PGP 9.5 positive nerve terminals in DM4 group showed reduced density and sparse distribution.PGP 9.5 positive nerve terminals in DM8 group showed signifi-cantly reduced distribution, thinner nerve diameter, shorter length and distorted shape.Histological changes of the thinner epidermal tissue, reduced epidermal cell layers, uneven cell distribution and arrangement in DM4 group, and significantly reduced epidermal cell layers, swollen and blurred cells, increasing cell gap, lack of stratified epidermis arrangement for part of epidermis, atropal and degenerated dermal collagen fiber, significantly decreased subcutaneous fat in DM8 group were observed.The results of Western blotting showed that the protein expression of PGP 9.5 in the plantar skin tissue of DM rats was progressively decreased along with the disease, while the protein level of p-JNK in L5, 6-DRG or S1-DRG showed a gradual increasing trend.PGP 9.5 immunoreactive positive nerve terminal density of plantar skin in DM rats had a negative correlation with the protein level of p-JNK in L5, 6-DRG and S1-DRG (P<0.01), but showed a significant positive correlation with the plantar skin thickness (P<0.01).CONCLUSION:The protein level of p-JNK within L5, 6-DRG or S1-DRG in DM rats shows a progressive enhancement.At the same time, there is a significant change in the skin tissue density and structure.The changes of skin tissue and nerve morphology in DM rat may be related to the activation of JNK/SAPK pathway in L5, 6-DRG or S1-DRG cells.Blocking or inhibiting JNK/SAPK pathway may delay the diabetic pe-ripheral neuropathy and reduce the risk of skin lesions.

Distribution and source of calcitonin gene-related peptide immunoreactive nerve terminal in prepuce of penis and frenulum of prepuce of rats / 中国组织工程研究

BACKGROUND: Frenulum of prepuce of penis contained many nerve terminals is an extremely sensitive region. If the frenulum is injured in circumcision or other operations, the complication, such as postoperative spontaneous pain of penis, sexual disturbance and so on, will occur. But there still is no define explanation for this up to now.OBJECTIVE: To observe the distribution of immunoreactive nerve terminal of calcitonin gene-related peptide (CGRP) in prepuce of penis and frenulum of prepuce of adult SD rats, and look for the source of CGRP immunoreactive nerve terminal in frenulum of prepuce.DESIGN: A single sample trial.SETTING:Department of Anatomy, School of Medicine, Zhejiang University.MATERIALS: The experiment was performed at Department of Anatomy,School of Medicine. Zhejiang University from September 2004 to May 2005. A total of 20 adult male SD rats were selected, and were raised in warm, quiet, photophygous environment for 1 week before the trial so as to make the rats fit for the environment and maintain their basal state.METHODS: The rats were assigned randomly into 2 groups. Ten rats in the first group were treated with the immunohistochemical method to observe the distribution of CGRP immunoreactive nerve terminal in prepuce of penis and frenulum of prepuce of adult rats. Ten rats in the second group were treated with fluorogold (FG) retrograde labeled combined with CGRP immunofluorescence labeled method to look for the source of CGRP immunoreactive nerve terminal in frenulum of prepuce of penis. MAIN OUTCOME MEASURES: ①The morphology and distribution of CGRP immunoreactive nerve terminal in prepuee of penis and frenulum of prepuce of adult SD rats were observed under light microscope. ②The distributive density and difference of CGRP immunoreactive nerve terminal in prepuce of penis and frenulum of prepuce were detected and compared (represented by A). ③Morphology and distribution of FG retrograde labeled -positive, CGRP single-labeled positive and FG/CGRP double-labeled positive neurons in dorsal root ganglion were observe under fluorescence microscope. ④Mean quantity of FG retrograde labeled positive, CGRP single abeled positive and FG/CGRP double-labeled positive neurons in dorsal root ganglion was counted.RESULTS: Totally 20 rats were involved in the analysis of results. ① Amber-coloured CGRP immunoreactive nerve terminal appeared in prepuce of penis and frenulum of prepuce of adult rats. These nerve terminal mainly occurred in basal layer of epidermis and papillary layer of dermis, distributed as twig shape or intestiniform; mostly of them were bundled, different in length, and some of them showed enlarged nodosity. ②The distributive density of CGRP immunoreactive nerve terminal in frenulum of prepuce of penis was significantly larger than that in prepuce of penis (2.15±0.32, 1.02±0.22,t =-2.03,P＜0.01). ③Combined with the FG retrograde labeled method it was found that these nerve terminal was derived from neurons of dorsal root ganglion opposed to the sixth lumbar spinal cord and the neurons of dorsal root ganglion opposed to the first acral spinal cord. FG retrograde labeled positive neurons differed in length. The cell body showed round or orbicular-ovate, without obvious prominence. Bright inaurate fine particle appeared in cytoplasm, no label in nucleus. Most cells arranged in line along nerve tract or diffusedly distributed. Most CGRP single-labeled positive neurons were middle or small cells found by CGRP immunofluorescence labeling. Dyeing was too dark.Reaction product distributed evenly in cytoplasm, which showed bright dark green (FITC labeled color). The same positive section was observed comparatively under different excitation light. It was found that FG/CGRP double-labeled positive cells were middle or small, and its amount accounted for a half of the total number of FG retrograde positive cells.CONCLUSION: CGRP may participate the transmission of sensory information in prepuce of penis and frenulum of prepuce of rats. The CGRP immunoreactive nerve terminal in frenulum of prepuce of penis of rats is sourced from neurons of dorsal root ganglion opposed to the sixth lumbar spinal cord and the neurons of dorsal root ganglion opposed to the first sacral spinal cord.

THE DISTRIBUTION AND ORIGIN OF CGRP IMMUNOREACTIVE NERVE TERMINALS IN THE PREPUCE AND FRENULUM OF PENIS / 解剖学报

Objective To study the distribution and origin of calcitonin gene-related peptide(CGRP) immunoreactive nerve terminals in the prepuce and frenulum of penis of human and mice. Methods The CGRP-immunoreactive nerve terminals in the prepuce and frenulum of penis were detected by the method of immunohistochemistry.The fluoro-gold(FG) retrogradely tracing and CGRP immunofluorescence were used to tracing the origin of CGRP-immunoreactive terminals in the penile frenulum of rats. Results In the prepuce and frenulum of adult penis,CGRP-immunoreactive nerve terminals were mainly observed in the basal stratum and spinal stratum of epidermis distributing in a cluster.These nerve terminals were arborescent and beady in appearance.The density of these nerve terminals was significantly higher in frenulum than that in prepuce.The distribution of CGRP-immunoreactive nerve terminals in rats was similar to that of in adult.FG retrogradely tracing method found that the FG retrolabelled neurons were localized in L-6DRG and S-1-DRG.Combined CGRP immunofluorescence method indicated that a number of DRG neurons were showed CGRP-immunoreactive.CGRP-positive neurons were mainly moderate or small-sized,intense immunostained,bright green granules appeared as FITC fluorescence in the cytoplasm.These positive-neurons were arranged in rows or spots among nerve bundles.A half of FG-labelled neurons also showed CGRP-immunoreactive expression in L-6-DRG and S-1-DRG respectively.All of FG/CGRP double-labelled neurons were moderate or small-sized.Conclusion Large numbers of CGRP-immunoreactive nerve terminals were observed in the penile frenulum of human and rats.These terminals were originated from the peripheral branches of CGRP-immunoreactive neurons in L-6-DRG and S-1-DRG of rats.It is suggested that CGRP may take part in the transmission of nociception in the prepuce and frenulum of penis.