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.

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.

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.

Objective To investigate the expression of CyelinD1 protein in nephroblastoma.Methods The tissue microarray were made in 28 cases of nephroblastoma and 19 cases of renal tissue adjacent to tumor.The expression of CyclinD1 and p53 proteins were detected by immunohistochemistry.Results The positive rate of the expression of CyclinD1 and p53 protein was significantly different between nephroblastoma and renal tissue adjacent to tumor (P < 0.05).The positive rate of the expression of CyclinD1 and p53 protein was significantly different in different degree of differentiation of nephroblastoma (P < 0.05).The expression of CyclinD1 and p53 protein was positively correlated (P < 0.05).Conclusions The CyclinD1 and p53 might be used as the marks for estimating the degree of differentiation of nephroblastoma.The over-expression of CyclinD1 and the mutation of p53 might play the vital role in nephroblastoma.

OBJECTIVE:To study the feasibility of overflow dissolution method for evaluating the drug in vitro sustained re-lease performance. METHODS:Overflow dissolution method was established by simulating the drugs elimination in vivo. Using Nifedipine sustained-elease tablets(Ⅰ)from 2 different manufacturers as model drug A,B,concentration-time curve,cumulative release rate- time curve,release velocity-time curve of model drugs in release pool at 3 different overflow speed (0,1.50,3.00 mL/min)were investigated. RESULTS:When overflow speed was 0,the cumulative dissolution was consistent with that of the con-ventional dissolution method. As the overflow speed increased,cmax of drug A,B was decreased [A:(8.89±0.20),(5.21±0.04), (3.51±0.03)μg/mL;B:(7.62±0.05),(4.80±0.09),(2.89±0.04)μg/mL];cumulative release rate was increased [A:(85.47± 2.45)%,(94.29 ± 2.44)%,(96.04 ± 2.56)%;B:(73.28 ± 1.13)%,(78.46 ± 1.94)%,(82.50 ± 1.69)%] ;tmax was ahead (A:1.5,1.0,0.5 h;B:2.0,1.0,0.5 h). CONCLUSIONS:Overflow dissolution method has avoided the inhibition of too large drug concentration on drug release,making complete drug release and more accurate evaluation of in vivo sustained release performance of the preparation.

[ 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.

Objective: To study the influences of Tiaozhixin(TZX) on NO/ET, 6 keto PGF 1a /TXB 2 and hemorrheology rats with hyperlipoidemia. Methods: The rat hyperlipoidemia and early atheroscleorsis models were established by feeding high lipid diet for 40 days. Meanwhile TZX was taken by oral administration at the dosages of 40、80g/kg. The levels of NO、ET 1、6 keto PGF 1a and TXB 2 in serum were determind, and the hemorrheology markers were observed. Results: TZX could raise the levels of NO in serumobviourly ET 1 level of the normal and model rats. The large dose of TZX could increase 6 keto PGF 1a content remarkably which benefits maintenance of the balances of 6 keto PGF 1a /TXB 2. It could lower the whole blood specific viscosity, whole blood reduction specific viscosity, plasma specific viscosity; aggregation index of RBC; shorten RBC electrophoresis time; also decrease fibrinogen content; inhibit the platelet aggregation of normal rats induced by ADP. Conclusion: TZX can improve the abnormal hemorrheology and recover the balance of TXB 2/6 keto PGF 1a and NO/ET of rats with hyperlipoidemia, which might be one of mechanisms of antiatherosclerosis action.

Objective To identify the changes of DNA methylation profile in the process of malignant transformation of BEP2D cell induced by α particles.Methods The genomic DNAs were isolated from the malignant transformation BERP35T4 cells and immortalized human bronchial epithelial cell line BEP2D.Genomic DNAs were digested by MseI and ligated of PCR linkers.Methylated DNAs were digested by BstUI and amplified by PCR.The methylated DNA probes were prepared by labeling with Cy3 and Cy5 fluorescence dyes individually and hybridized to the methylation CpG-Island microarray.The hybridization results were scanned and analyzed.Intensity values were quality controlled and normalized.The normalized data were used to identify the differentially expressed genes based on a 1.5 fold difference of the expression level.Results There were 16 genes which showed changes of methylation level in malignant transformation BERP35T4 cells, 9 of them were hypermethylation and 7 were hypomethylation.These genes were including the SKIP gene, PPP3CC gene, MAP2K6 gene, KIR2DL1 gene, KIR2DL4 gene, KIR3DP1 gene, ZNF493 gene, ZNF100 gene, NKX2-5 gene, TFAP2D gene, DR1 gene, KCNJ16 gene, CCDC18 gene, FNBP1L gene, IRX4 gene, EPB41L3 gene, TCP10 gene and so on.Conclusions The DNA methylation might have effects on ionizing radiation drived tumorigenesis.

BACKGROUND: Quantitative pharmaco-electroencephalography (QPEEG) can reflect cerebral cortical function, which can be certainly affected by general anesthetics. Anesthesia depth has good correlation with the anesthetic dosage, so if we can find out the areas of brain and band of QPEEG which is relative to the anesthetic dosage, the band may be taken as the index to reflect the depth of anesthesia. OBJECTIVE: To observe the effects of propofol on the alpha2-band (α2- band) of QPEEG in rabbits. DESIGN: A randomized control animal experiment. SETTING: Jiangsu Provincial Key Laboratory of Anesthesiology, Xuzhou Medical College. MATERIALS: The experiment was carried out in the animal laboratory of Xuzhou Medical College from October 2004 to August 2005. Thirtysix healthy adult rabbits were randomly divided into propofol 2.5, 5 and 10 mg/kg groups with 12 rabbits in each, including 6 were used to observe the change of percentage of each band power of QPEEG, and the other 6 were used to observe the latency and duration for the disappearance of righting reflex in the rabbits. METHODS: The experiment was performed between 14:00-17:00 every day. Rabbits in the three groups were treated with intravenous injection of propofol of 2.5, 5 and 10 mg/kg respectively within 30 seconds. ① The conscious rabbits were fixed onto the platform in a prone osition, and the QPEEG was recorded with the method of power spectrum analysis before administration and at 20, 30, 40, 50, 60, 70, 80, 90, 100 and 110 s and 2, 5, 10, 15, 20 and 30 minutes after administration respectively. The sampling time for each time point was 5 s. ② The latency and duration for the disappearance of righting reflex in the rabbits were recorded. RESULTS: ll the 36 rabbits were involved in the analysis of esults. ① After the intravenous injection of propofol, the righting reflexes all disappeared within 1 minute. The greater the dosage, the shorter the latency and the longer the duration r=0.79, P ＜ 0.01). ② Compared with before administration, propofol of 2.5 mg/kg had no obvious influence on the percentage of α2-band power (P ＞ 0.05); The percentages of α2-band power in the brain areas were increased after administration in the propofol 5 mg/kg group (P ＜ 0.05); Except that there were no significant differences in the left and right parietal regions between the propofol 10 mg/kg group and the propofol 5 mg/kg group, the percentages of α2-band power in the other brain areas in the propofol 10 mg/kg group were decreased as compared with those before administration and those in the other two groups (P ＜ 0.05), and the changes above were more obvious in the frontal and temporal regions.CONCLUSION: The influence of propofol on the percentage of α2-band power of QPEEG is biphasic, it is suggested that α2-band would be an index to reflect the anesthesia depth of propofol.

Objective To investigate the role of RhoA /Rho-kinase pathway in rat models of left heart disease-as-sociated pulmonary hypertension ( PH-LHD) .Methods Twenty male SD rats (3-4 week-old, 90-100 g) were randomly divided into two groups (10 rats in each group):the group C ( control group) with sham operation, and group H ( pulmo-nary arterial hypertension) .The rat model of left heart disease-associated pulmonary hypertension was established by supra-coronary aortic banding in the group H, and the sham surgery was applied for the rats in the group C ( The titanium clip was fixed at the mediastinal tissue adjacent to the artery rather than the ascending aorta).On day 60 after the operation, the cardiac functions, including right ventricular systolic pressure and pulmonary artery pressure were evaluated.After that, all rats were sacrificed and treated with cardiopulmonary lavage in vivo until the lung became white.Then the left lung tissues were fixed in 4%paraformaldehyde for pathological observation while the right lung tissues were frozen for mRNA detec-tion.Results Compared with the group C, both ventricular systolic pressure and pulmonary artery pressure in the group H were increased significantly (P<0.01).Pathological data demonstrated that the pulmonary artery walls in H group were much thicker than that in the group C.Moreover, vascular wall hypertrophy index in the group H was increased greatly compared with that in the group C (P<0.01).QPCR data showed that mRNA levels of Rho kinase, RhoA and ET-A R in the group H were up-regulated compared with the group C ( P<0.01) .Conclusions Rat model of left heart disease-asso-ciated pulmonary arterial hypertension can be successfully established by supracoronary aortic banding.Rho-kinase-media-ted pathway may contribute to the pathogenesis and progress of left heart disease-associated pulmonary arterial hypertension.