Chromatography, High Pressure Liquid ; methods ; Drug Residues ; analysis ; Electrochemistry ; Ethidium ; analysis ; Luminescent Measurements ; methods ; Sensitivity and Specificity

Objective To investigate the effects of different afferent nerve injury on development of neuropathic pain and its relationship with brain-derived neurotrophic factor (BDNF) in spinal cord and dorsal root ganglion (DRG) in rats. Methods Twenty-four male SD rats aged 2 months weighing 200-250 g were randomly divided into 3 groups:group Ⅰ sham operation (group S); group Ⅱ sural nerve injury (group SUR) and group Ⅲ gastrocnemius-soleus nerve injury (group GS). Sural nerve and gastrocnemius-soleus nerve were transected in group SUR and GS respectively. Paw withdrawal threshold to von Frey filament stimulation was measured the day before and at day 3 and 7 after operation. The animals were killed at postoperative day 7 after the measurement of paw withdrawal threshold. The ipsllateral L5 DRG and L5 segment of the spinal cord were removed. BDNF expression in the spinal dorsal horn was determined. The percentage of BDNF positive neurons and ATF-3 positive neurons in the total DRG neurons and the percentage of BDNF positive neurons in the damaged neurons (ATF-3 positive) were calculated. Results Mechanical hyperalgesia developed after transection of gastrocnemius-soleus muscle in group GS. Mechanical pain threshold was sinificantly lower, while BDNF expression in the spinal dorsal horn and the percentage of BDNF positive neurons in total DRG neurons were significantly higher in group GS than in group S and SUR (P ＜ 0.01). There was no significant difference in all variables between group SUR and S (P＞0.05). There was no significant difference in the percentage of ATF-3 positive neurons in the total DRG neurons between group GS and SUR (P ＞ 0.05), but the percentage of BDNF positive neurons in the damaged neurons (ATF-3 positive) was significantly higher in group GS than in group SUR (P ＜ 0.05). Conclusion Transection of the afferent nerve innervating muscle can produce neuropathic pain through up-regulation of BDNF expression in spinal dorsal horn and DRG in rats, while transection of the afferent nerve innervating skin can not.

· AIM: To investigate the efficacy and safety of intravitreal injection of plasmin, hyaluronidase, or the combination of the two in inducing posterior vitreous detachment (PVD).· METHODS: 15 mini-type pigs were assigned to three groups (Group A, B and C), 5 in each group. One eye of each pig was intravitreally injected with the studying agent,and the fellow eye was used as control. Group A received a vitreous injection of hyaluronidase 50U (0.1mL); group B received plasmin 0.5U (0.1mL); group C received plasmin 0.5U (0.05mL) combined with hyaluronidase 50U (0.05mL). The fellow eyes in each group were injected with 0.1mL balanced salt solution (BSS). All the pigs were examined with slit-lamp biomicroscope, direct and indirect ophthalmoscope, B-scan and electroretinograph (ERG). After 7 days, the animals were killed and the eyes were enucleated and examined with light microscope, transmission electron microscope and scanning electron microscope.· RESULTS: B-scan examination showed that one eye of Group A and two eyes of Group B had partial PVD at 1st day after injection and one eye of Group C at 1 hour after injection. On the 7th day, B-scan, light microscopy and scanning electron microscopy demonstrated that all the eyes of Group A and Group B had partial PVD, while none of the control eyes had PVD. Rank sum test for scanning electron microscopy results of all the groups showed P ＜0.005.Furthermore, the comparisons between every two groups were made. The results of analyses were as follows: P＞0.05 between the drug injected eyes of Group A and Group B, P＜0.05 between Group B and Group C, Group A and Group C.The b-wave and a-wave amplitudes of ERG showed no significant difference either between preinjection and postinjection in all groups or between the drug injected eyes and the control eyes in each group. Light microscopy and transmission electron microscopy revealed no damage to the retinal structure.· CONCLUSION: Intravitreal injection of hyaluronidase 50U or plasmin 0.5U or their combination can produce PVD effectively and quickly without retinal functional or structural toxicity. The combination of the two proteases was proved to be synergetic.

Objective To evaluate the effect of surgical trauma on the cognitive function and expression of hepcidin and ferroportin 1 (FP1) in hippocampus in aged rats.Methods One hundred male Sprague-Dawley rats,aged 18 months,weighing 400-500 g,were randomly divided into 2 groups with 50 rats in each group:control group (group C) and surgical trauma group (group ST).The rats were anesthetized with chloral hydrate,but underwent no operation in group C.The rats Were anesthetized with chloral hydrate and underwent 30 min of modified exploratory laparotomy in group ST.Ten rats were chosen from each group at 24 h after operation and the cognitive function was assessed using Morris water-maze test for 6 consecutive days.Ten rats were sacrificed on 1st,3rd,5th and 7th days after beginning of Morris water-maze test and brains were removed for determination of hepcidin and FP1 expression in hippocampus by PCR and Western blot.Results Compared with group C,the escape latency was significantly prolonged,the time of staying at the original platform quadrant and frequency of crossing the original platform were decreased on 3rd,4th and 5th days after beginning of Morris water-maze test,and the expression of hepcidin was up-regulated and the expression of FP1 was down-regulated at each time point in group ST (P ＜ 0.05).Conclusion Surgical trauma can decrease the cognitive function in aged rats and the mechanism may be related to up-regulation of hepcidin expression and down-regulation of FP1 expression in hippocampus.

Animals ; Cytokines ; blood ; Endotoxemia ; blood ; therapy ; Endotoxins ; blood ; toxicity ; Enzyme-Linked Immunosorbent Assay ; Hemofiltration ; Interleukin-1 ; blood ; Interleukin-10 ; blood ; Models, Animal ; Swine ; Time Factors ; Tumor Necrosis Factor-alpha ; analysis

Objective To investigate the expression of Caspase-9 and heat-shock protein-90 (HSP 90) in rats after focal cerebral ischemia-reperfusion injury.Methods The male SD rats (200-250 g) were divided into three groups by the random number table: normal group, sham group and cerebral ischemia-reperfusion (CIR) group.Each group was sorted into four subgroups including group 6 h, group 24 h, group 48 h and group 72 h according to the reperfusion time.Suture-occluded method was adopted to prepare focal cerebral ischemia-reperfusion(CIR) injury in rat model.Enzymelinked immunosorbent assay (ELISA) method was used to detect the variations of Caspase-9 and HSP-90 expression in rats.Results The changes in Caspase-9 and HSP 90 expression in the brain cells were observed by ELISA method.The expression of Caspase-9 and HSP-90 was weakly expressed in sham group, and was at peak in CIR group within 24 h-48 h, then began to decline at 72 h after the reperfusion time.The differences in the expression of caspase-9 and HSP-70 between sham group and normal group were not statistically significant.Conclusions Apoptotic cells gradually increase along with reperfusion time and reach the peak at 48 h after cerebral ischemia-reperfusion.In ischemia half dark stripe, the expression of Caspase-9 and HSP 90 is increased in neuronal cells after cerebral ischemia-reperfusion, and the positive cells number is at peak at 48 h after cerebral ischemiareperfusion.Apoptosis of neuronal cells after cerebral ischemia and reperfusion is a dynamic evolutionary process.The expression of Caspase-9 and HSP 90 in nerve cells plays an important role in regulating cell apoptosis.

Objectives To investigate the changes of erythropoietin(EPO)expression in rats after focal cerebral ischemia/reperfusion injury.Methods Male Sprague-Dawley rats were randomly divided into normal,sham,cerebral ischemic/reperfusion(CIR)groups.Middle cerebral artery occlusion(MACO)model was established by Longa's method,and reperfusion was followed 2 hours after occlusion in CIR group.The rats' brain neurological deficit scores were evaluated at 24 h,48 h,72 h and 96 h after reperfusion.The protein expression of EPO was determined by immunohistochemistry staining and Western blotting in each time points.Results The rats' brain neurological deficit scores at 48 h,72 h and 96 h were significantly increased(3.40±0.32,3.60±0.17,3.70±0.21,all P<0.05)compared with those at 24 h(3.00±0.22)after reperfusion in CIR group.The results of immunohistochemistry staining and Western blotting showed that the positive expression of EPO proteins in rats started at 24 h(0.36±0.05,140.20±0.30)after cerebral ischemic/reperfusion injury,increased significantly at 48 h(1.09±0.10,145.40±0.16),reached the peak at 72 h(1.29±0.09,156.23±0.12),began to decline at 96 h(0.98±0.04,141.56±0.36).Conclusions Cerebral ischemia and reperfusion injury can induce increased expression of EPO protein,which suggests that EPO may have protective effect on nerve cells under the condition of ischemia and reperfusion.

Objective To investigate the safty and accuracy ot estimating the living donor's graft volume with IQQA liver imaging evaluation system.Methods Between June 2007 and July 2010,123living liver donors were enrolled to undergo 16-slice CT scanning,then graft volume was estimated by both IQQA and manu-traced multi-slice spiral computed tomography (MSCT) approach.The graft volume and time consuming between IQQA and manu-traced MSCT were compared. Pearson Correlation test was uesd to verify the correlation between the estimated graft volume estimated each method and actual graft weight detected in operation.Linear correlation analysis was done.Results The mean graft volume by IQQA and manu-traced MSCT was (856.76 ± 162.18) and (870.64 ±172.54) cm3 respectively preoperation.Paired t-test showed there was no statistically significant difference between IQQA and MSCT methods (P＞0.05).It took mean ( 16.9 ± 1.4) min to calculatethe graft volume by IQQA and (39.3 ± 2.1 ) min by manu-traced MSCT,respectively (P＜0.05).The real graft volume was (632.59 ± 13 1.73) cm3.Pearson correlation test showed the graft volume calculated by either IQQA or MSCT method had a significantly positive correlation with the real graft weight (MSCT r =0.921,IQQA r =0.896,P＜0.01 ).Graft weight could be expressed in the equation:Actual graft weight =- 150.303 + 1.025 × IQQA value or =- 94.397 + 0.955 × MSCT value.Conclusion IQQA system has same accuracy with MSCT method in predicting the graft volume but consuming less time.IQQA system promotes the recognition of clinician on liver three dimensional anatomic structure.

Tripterygium glycosides preparation which extracted from the traditional Chinese herb Tripterygium wilfordii (TWHY), was widely used to treat the autoimmune diseases. Previous works demonstrated that TWHF had potent anti-inflammatory and immunosuppressive properties. But the different quality and high incident rate of side effects of different manufactures inhibited its clinical application. Since TWHF had been generally known to play a therapeutical effect by synergism of multiple constituents, it was necessary to build the relationship between the HPLC fingerprint and bioactivity so as to ensure the quality safety and efficacy. The HPLC fingerprint showed that description and content of peaks from different manufactures were diverse. Only 11 common peaks were found. In this study, mice spleen cells stimulated by Con A were used to test the proliferation inhibition bioactivity of TWHF preparations, which were incubated with 30, 15, 7.5, 3.75, 1.88 and 0.94 mg x L(-1) TWHF preparations for 48 h. The results showed that mice spleen cells proliferation was inhibited by all TWHF preparations significantly compared with the control group, which suggested the TWHF preparations showed immune suppress activity. The TWHF preparations from 7 manufacture showed different IC50 value, which might belong to different contents which showed in the HPLC fingerprint. Moreover, a relationship between the HPLC fingerprint and the bioactivity were established to identify important constituents by grey relational analysis (GRA). The result showed that all the contents were relative with the IC50, especially No. 5 and 10 peaks, but No. 1 peak, which was proved to be triptolide, had few contribute to the inhibition of mice spleen cells proliferation. The study of relationship between the HPLC fingerprint and the IC50 by GRA could help to investigate mechanism of bioactive and provide an evidence for the quantification of multi-constituents.
Animals ; Anti-Inflammatory Agents ; analysis ; pharmacology ; Cell Proliferation ; drug effects ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Glycosides ; analysis ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; drug effects ; Tripterygium ; chemistry

Objective The presence of alloreactive memory T cells in recipient is a critical handicap to achieve transplantation tolerance.To make a mouse model which mimics the present transplant patient is important for research at this subject.Thus,we developed a novel re-transplant model and compared the alloresponse in this model with that in the conventional memory T cellstransfer model (transfer control).Methods The re-transplant model was established via microsurgery and vessel cannula techniques,and the experiment was composed of three groups:the re- transplant group,memory T cell-transfer group (transfer control) and the conventional blank group (blank control).The research indexes included survival time of donor heart,rejection score of allograft,and detection of proliferation and differentiation of the alloreactive memory/effector T cells by by flow cytometry (FCM) and in vitro mixed lymphocyte reaction (MLR).Results The median survival time of allograft in re-transplant recipients was significantly shortened compared to that of transfer control,but there was no significant difference in rejection score of graft between them (the score in retransplant group was the most intense of the three groups). Moreover, proliferation and differentiation of the alloreactive effector T cells were more intensive in re- transplant recipients than in the transfer control,which was confirmed by in vitro MLR and by FCM of the splenocytes for detecting CD44highCD62L-memory/effector phenotype cells.Conclusion The recall alloresponse in retransplantation is more intensive than that in memory-transfer setting and this re-transplant model is more close to the clinic situation than the memory-transfer model in rodents.