Objective To develop a technique that can directly demonstrate collateral sprouting of intact nerve fibers at endtoside neurorrhaphy site. Methods Five Wistar adult rats were used in this study. The common peroneal nerves at one side were sectioned at the level of knee joint, and their distal ends were sutured to the tibial nerves after removal of a 1 mm-diameter window in the epineurium. Three months after the operation, the nerve segments at neurorrhaphy site and the normal tibial nerves at the contralateral site were harvested. The specimens were fixed in 10% formaldehyde and postfixed in 1% osmium tetroxide, thenmacerated in glycerol. Single fiber was teased out in pure glycerol under an operative microscope, then transferred to a slide and observed under light microscope. The nerve segments at neurorrhaphy site and distal peroneal nerves were alsoharvested for histological evaluation. Results At the neurorrhaphy site, small nerve fibers sprouted from a donor nerve fiber near node’s of Ranvier. While such phenomena were not found in normal tibial nerve. From the longitudinal sectionof neurorrhaphy site, bundles of nerve fibers ranged from tibial nerve to peroneal nerve were observed. Lots of regenerative nerve fibers emerged in distal peroneal nerve. Conclusion The phenomena of collateral sprouting at end-to-side neurorrhaphy site can be demonstrated directly by nerve fiber micro-tease technique.
【摘要】 目的 探討辣椒素對不同年齡SD大鼠內臟感覺神經元上辣椒素受體（TRPV1）介導的離子通道的影響。 方法 急性分離7~9 d和21~23 d大鼠迷走神經結狀神經節神經元，利用全細胞膜片鉗技術在分離的神經元上記錄辣椒素激活TRPV1受體后通道電流的變化。 結果 ①7~9 d和21~23 d大鼠內臟感覺神經元的膜電容分別為（18.57±8.60）和（19.85±9.47） pF，（Pgt;0.05）；②辣椒素能夠激活7~9 d和21~23 d大鼠內臟感覺神經元上TRPV1并產生相似的內向電流，兩組產生的峰電流密度分別為（48.59±18.87）、（55.91±20.52） pA/pF（Pgt;0.05）；③反復應用辣椒素使TRPV1受體發生失敏現象。 結論 大鼠內臟感覺神經元的TRPV1受體通道在出生后已經發育成熟，且對辣椒素激活的通道電流有相似的變化。【Abstract】 Objective To investigate the effects of capsaicin on transient receptor potential vanilloid 1 (TRPV1) receptor-mediated ion channel currents of visceral sensory neurons in different-aged Sprague-Dawley rats. Methods We isolated the vagal nodose ganglion neurons of rats at an age of 7-9 days or 21-23 days acutely. With the whole cell patch clamp technique, we recorded the current changes of TRPV1 channels activated by capsaicin. Results ① Membrane capacitances of the visceral sensory neurons were (18.57±8.60) and (19.85±9.47) pF in rats of 7-9 and 21-23 days, respectively (Pgt;0.05). ② Capsaicin activated the TRPV1 channels and generated inward currents in all the rats; and the peak current densities of the rats of 7-9 days and 21-23 days were respectively (48.59±18.87) and (55.91±20.52) pA/pF (Pgt;0.05). ③ Repeated applications of capsaicin produced a phenomenon of desensitization in TRPV1 channels. Conclusion TRPV1 receptor channels of visceral sensory neurons in rats have matured after birth, and the current changes of TRPV1 channels activated by capsaicin are similar.
Objective To explore the human stromal cell-derived factor 1α (hSDF-1α) and human vascular endothel ial growth factor 165 (hVEGF165) mRNA expressions of the transfected cells after hSDF-1α gene and hVEGF165 gene were transfected into rat myoblasts in vitro so as to lay a foundation for further study on the synergistic effects of 2 genes on tissue engineered skeletal muscle vascularization. Methods The myoblasts of 1-day-old Sprague Dawley rats were cultured and purified by trypsin digestion assay in vitro and were identified by immunohistochemistry staining of Desmin. pproximately 70%-80% of confluent myoblasts were transfected with enhanced green fluorescent protein (EGFP)-hSDF-1α and EGFP-hVEGF165 genes in vitro (transfected group) and were not transfected (control group). The expressions of hSDF-1αand hVEGF165 mRNA and protein in the transfected cells were detected by RT-PCR, ELISA, and Western blot espectively.Results The cultured cells were identified as myoblasts by immunohistochemistry staining of Desmin. The expression ofgreen fluorescent protein was observed in transfected cells, indicating that hSDF-1α and hVEGF165 genes were transfected into myoblasts successfully. The mRNA and protein expressions of the 2 genes were positive in the transfected group by RT-PCR and Western bolt assay at 2, 4, 6, and 8 days after transfection, and were negative in the control group. The expressions of hSDF- 1α and hVEGF165 showed a stable low level in the control group, but the expressions of the proteins increased at 2 days and then showed gradual downtrend with time in the transfected group by ELISA assay. There were significant differences in the expressions of hSDF-1α and hVEGF165 proteins between different time points in the transfected group, and between 2 groups (P lt; 0.05). Conclusion hSDF-1α and hVEGF165 genes are successfully transfected into myoblasts in vitro, and mRNA and proteins of hSDF-1α and hVEGF165 can be expressed in the transfected myoblasts, which may provide the experimental evidence for the expressions of hSDF-1α and hVEGF165 mRNA and proteins in vivo successfully.
目的 觀察局灶性腦缺血梗死區周圍層粘連蛋白（Laminin）及基質金屬蛋白酶（MMP）-9表達，探討其在腦缺血再灌注損傷發病機制中的作用。 方法 將45只體重250～300 g、4個月齡的Sprague Dawley（SD）雄性大鼠隨機分為假手術（Sham）組與局灶性缺血再灌注（MCAO）組。MCAO組又分6、24、72 h及7 d組，每組各9只。光學顯微鏡下觀察腦組織病理改變，免疫組織化學染色檢測各組Laminin及MMP-9的表達情況。 結果 再灌注后6 h，即有MMP-9表達明顯增高，表達高峰出現在再灌注后24 h，3 d時有所下降，至7 d時仍明顯高于基礎水平（P＜0.01）。Laminin的表達于再灌注6 h開始降低，24 h降至最低，3 d后開始緩慢回升。 結論 腦缺血再灌注后，病變區MMP-9與Laminin表達變化可反映腦血管壁受損狀況，并與腦缺血損傷及修復過程有關。
ObjectiveTo investigate the effect of pharmacologic delay with pioglitazone, a peroxisome proliferator-activated receptor γ (PPAR-γ) agonist, on extended perforator flap survival in a rat model. MethodsSeventy male Sprague Dawley rats, weighing 250-300 g, were randomly divided into control group (n=35) and experimental group (n=35). A three-territory flap was made, including two choke zones. Pioglitazone was dissolved in 1.5 mL saline. Oral doses of pioglitazone[10 mg/(kg·d)] was given by gavaged for 5 days in the experimental group, while the same volume of saline was given in the control group at same time point. After 7 days, the flap survival area was measured and angiographic diagnosis was made. The tissue samples were harvested from choke zone Ⅱ for histological study and vascular endothelial growth factor (VEGF) expression detection by immunohistochemical staining. The content of nitric oxide (NO) in choke zones I and Ⅱ was measured at immediate, 1, 3, 5, and 7 days after operation. ResultsThe flap general change of 2 groups was similar. Varying degrees of necrosis occurred with the extension of time in 2 groups. At 7 days after operation, the flap survival rate was 87.73%±3.25% in the experimental group and 76.07%±2.92% in the control group, showing a significant difference (t=-10.338, P=0.000). The number of true anastomosis in choke zones I and Ⅱ was 5.40±1.14 and 3.00±0.71 in the experimental group, and was 3.20±0.84 and 0.80±0.84 in the control group respectively, showing significant differences between the 2 groups (t=-3.479, P=0.008;t=-4.491, P=0.002). The microvessel density and the expression of VEGF in choke zone Ⅱ of experimental group were (33.16±7.73)/mm2 and 4 368.80±458.23, respectively, which were significantly higher than those of control group[(23.29±5.91)/mm2 and 2 241.24±554.43] (t=5.073, P=0.000;t=-14.789, P=0.000). The content of NO in the experimental group were significantly higher than those in the control group at other time points (P<0.05) except for at immediate after operation. ConclusionPharmacologic delay with pioglitazone can improve extended perforator flap viability through increasing ischemia-induced angiogenesis and choke vessels vasodilation in rat models.
ObjectiveTo study the feasibility and advantages of preparing an animal model of defecation reconstruction after spinal cord injury in rats by mechanical polishing method. MethodsForty adult female Sprague Dawley rats (weighing, 250-300 g) were randomly divided into 2 groups (n=20). The lamina was opened by mechanical polishing method to expose the cauda equina in experimental group, then bilateral L5 and S1 nerve roots end-to-end anastomosis was done under 10 times microscope, and finally cauda equina between the L5 and L6 (except S1) was cut. The lamina was opened by traditional bites method in control group, and the other treatment methods were in agreement with the experimental group. The operative time, intra-operative blood loss, and situation of rats at postoperative 3 days were recorded. ResultsThe operative time of experimental group[(93.05±7.60) minutes] was significantly shorter than that in control group[(131.30±11.68) minutes] (t=12.279, P=0.000); intra-operative blood loss in experimental group[(4.33±0.46) mL] was significantly lower than that in control group[(7.36±0.58) mL] (t=18.293, P=0.000). At 3 days after operation, 18 rats (90%) survived in experimental group, and 12 rats (60%) survived in control group; difference was significant in the survival rate between 2 groups (χ2=4.800, P=0.028). ConclusionTo establish an animal model of defecation reconstruction after spinal cord injury in rats by mechanical polishing method is feasible, and it has shorter operative time, less blood loss, and lower postoperative mortality than the traditional bites method. But there is a certain learning curve and requirement to master microsurgical techniques.
Objective To investigate whether the peri pheral administration of amitri ptyl ine and bupivacaine produces anti-hyperalgesic effect and to screen the neurotoxicological effect on sciatic nerve blockade in a rat model of neuropathic pain. Methods Twenty-four adult male SD rats [weighing (200 ± 20) g] were made the models of chronic constriction injury (CCI) and randomly divided into 3 groups (n=8) 5 days after operation: group A (amitriptyl ine), group B (bupivacaine) and group C (normal sal ine). 0.5 mL 0.5% amitriptyl ine, 0.5% bupivacaine or normal sal ine were given in group A, group B, and group C, respectively through implanted cannulas after 5, 7 and 9 days of CCI once a day for successive 3 days. The motor function was measured before administration and 1, 2, 4, 8, 12 and 24 hours after every administration. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured before administration and 1, 3, 5 and 7 days after the third administration. The operated sciatic nerve samples were obtained for neuropathological examination under l ight microscope. Results Twenty-four CCI rats were all survival without infection, palsy and catheter fall ing off. Compared with group C, the rats of group A and group B both produced significant ambulation deficits after every administration (P lt; 0.05). The ambulation deficits lasted 2 hours (group B) and 8 hours (group A) respectively. But the ambulation deficits of CCI rats were all reversible. The MWT and TWL of group A 1 and 3 days after the third administration increased when compared with those before administration and 5 and 7 days after the third administration, and when compared with group B and group C (P lt; 0.05). There was no significant difference (P gt; 0.05) in l ight microscopic neuropathological examination among three groups. Epineurial tissue and endoneurium tissue integrity, tidy arrangement of fibers, less inflammatory cell and no marked degeneration of myel inated fibers were observed. Conclusion Repeated sciatic nerve blockade with 0.5% amitriptyl ine has peripheral anti-hyperalgesic effects on neuropathic pain of rats. No morphological evidence of neurotoxicity in the sciatic nerve of rats is observed in 0.5% amitriptyl ine.
Objective To establish model of thymectomy in adult rats. Methods The animal models were built by resection of the thymus and simultaneously emptying the air under xiphoid in the rats underwent thoracotomy. Results Of 30 rats, 1 died of postoperative atelectasis, 1 died of excessive bleeding because of puncturing the pulmonary vein by mistake during the operation. Twenty-eight rats survived more than 30 days. A successful rate of 93.3% was achieved in the making of thymectomy model. Conclusion The results show that the model is easy to operate and the success rate is very high, and can be used in the experiment of thymectomy in the rats.
Objective Bone marrow mesenchymal stem cells (BMSCs) play an important role in repairing nerve injury, meanwhile external temperature has significant effect on BMSCs transplantation, prol iferation, and differentiation. To investigate the effect of BMSCs transplantation and mild hypothermia on repair of rat spinal cord injury (SCI). Methods Forty-five female adult SD rats (weighing 200-250 g) were made the models of hemitransection SCI and divided randomly into 3 groups according to different treatments: group A (SCI group), group B (BMSCs transplantation group), and group C [BMSCs transplantation combined with mild hypothermia (33-35 ) group]. At 1, 2, 4, 6, and 8 weeks after injury, the fuction of hind l imb was evaluated with Basso Beattie and Bresnahan (BBB) score and incl ined plane test. At 4 weeks after injury, histopathology and BrdU immunohistochemistry staining were performed. At 8 weeks after injury, horseradishperoxidase (HRP) retrograde nerve trace and transmission electron microscope (TEM) testing were performed to observe the regeneration of axon. Results After 4 weeks, the function of hind l imb obviously recovered in groups B and C, there were significant differences in BBB score between groups B, C and group A (P lt; 0.05), between group B and group C (P lt; 0.05). There was no significant difference (P gt; 0.05) in tilt angle among 3 groups after 1 and 2 weeks, and there were significant differences (P lt; 0.05) among 3 groups after 4 weeks. HE staining showed that significant cavity could be seen in group A, l ittle in group B, and no cavity in group C. BrdU immunohistochemistry staining showed that the number of positive cells was 0, 90.54 ± 6.23, and 121.22 ± 7.54 in groups A, B, and C, respectively; showing significant differences (P lt; 0.01) among 3 groups. HRP retrograde neural tracing observation showed that the number of HRP positive nerve fibers was 10.35 ± 1.72, 43.25 ± 2.65, and 84.37 ± 4.59 in groups A, B, and C, respectively, showing significant differences (P lt; 0.01) among 3 groups. TEM observation showed that a great amount of unmyel inated nerve fibers and myel inated nerve fibers were found in central transverse plane in group C. Conclusion The BMSCs transplantation play an impontant role in promotion of recovering the function of hind l imb after SCI, and mild hypothermia has synergism effects.
Objective To study the allograft effect of two kinds of tissue engineered oral mucosa lamina proprias on skin fullthickness wounds. Methods The cultured Wistar rat oral mucosa fibroblasts (OMF) were incorporated into collag en or chitosancollagen to construct the tissue engineered oral mucosa laminaproprias, and then the OMFs were labeled with BrdU. The fullthickness round skin defects were made with a round knife (diameter, 0.8 cm) on the backs of 36 Wistar rats (2125 weeks old), which were divided into 2 experimental groups: the fibroblastpopulated collagen lattices (FPCL) group (grafted by FPCLs) and the fibroblastpopulated chitosan collagen lattices (FPCCL) group (grafted by FPCCLs), and the control group (only covered with gauges). All the wounds were observed by the naked eyes or the light microscope, and were measured 4, 7, 14, and 21 days postoperatively. Results There were no infection during the wound healing period. At 7 days after the grafting, the wounds in the 3 groups were covered by scab and/or gauze; at 14 days, the gauze and scab on the wounds in the three groups were all replaced by the new epidermis naturally except one scab each in the FPCCL group and the control groups,which was replaced at 17 days.All the centers of the new epidermis were measurable as the pink red points. At 21 days, all the new skins were smooth without hairs, and their color was similar to the normal one. At 4, 7, and 14 days,there was an indication that the wound diameters became significantly smaller in the three groups; but after the 14th day, there was no significant indication of this kind. At 7 days, the wound diameter in the FPCL group was significantly smaller than that in the FPCCL group and the control group (Plt;0.01). Under the lightmicroscope, at 4 days postoperatively, the decayed tissue on the surfaces of the recipient wounds in the FPCL group and the FPCCL group was separated from the lower granular tissue in which there were many inflammatory cells, fibroblasts, and new vessels. There was a similar-phenomenon in the control group. Each skin wound in the three groups was only partly keratinocyted at 7 days postoperativel y. The recipient wounds were wholly keratinocyted with when rete ridges observed at 14 and 21 days, but in the control group the wounds were keratinocyted with no rete ridges. Fibers in the new dermis were thin. The OMFs with Brdu appeared in the granular tissue and new dermis at 4, 7, 14, and 21 days postoperatively, which could be illustr ated by the immunohistochemical staining. The positive OMFs and the granular tissue joined in the repair of the skin defe cts without any allergic reaction during the period of the wound healing. Conclusion The oral mucosa fibroblasts as the new seed cells can join i n the repair of the skin defects effectively and feasibly. The fibroblastpopul ated collagen lattices and the fibroblastpopulated chitosan collagen lat tices can repair skin defects effectively and feasibly, too. And the quality of the new skins was better in the two experimental groups than in the control group.