应用型本科院校《动物病理学》课程教学改革探索与实践

以应用型人才培养的实施为切入点,构建产教融合、注重实践的动物病理学教学课程改革方案,利用"产学研"基地、新媒体辅助技术等手段,采取"以赛促学"、"双师同课"等方式,从课程目标、教学模式、教学方法、实践教学、考核评价方面进行探索和实践,为应用型地方本科院校动物医学专业课程改革提供参考。

《动物病理学》教学方法的改革与探索

《动物病理学》是动物医学各专业及相关专业的一门非常重要的专业基础课程,其理论性和实践性极强。针对该特点,在教学过程中,教师应该运用讲授与自学有机结合、启发式教学、应用多种教学手段、合理布置作业、理论联系实际和改革考试方法等措施,激发学生的学习兴趣,达到提高教学质量的目的。

Resveratrol prolongs allograft survival after liver transplantation in rats

AIM： To study the immuno-modulatory effect of resveratrol （RES） on allograft rejection after liver transplantation in rats. METHODS： Male Sprague-Dawley （SD） rats were selected as donors and male Wistar rats as recipients for a rejection model. The recipients were divided into four groups after orthotopic liver transplantation （OLTx）. In the RES A, B, and C groups, RES was given intra-peritoneally once a day （25, 50, and 100 mg/kg, respectively） after OLTx, whereas in the control group, vehicle buffer was given intra-peritoneally once a day. The survival time, serum chemistry, production of cytokines, activation of transcription factor NF-kB, and histopathologic findings were then compared among these groups. RESULTS： The mean survival time after OLTx in the RES C group was significantly longer than that in the control group （16.7＋-1.2 d ,vs9.3＋-0.6 d, P〈0.01）. On the 7th posttransplant day the serum albumin level significantly improved in the RES C group, the serum total bile acid and alanine aminotransferase （ALT） levels were significantly lower in the RES C group, the serum IL-2 and INF-y levels were significantly lower in the RES C group, and the activation of transcription factor NF-kB in peripheral blood T lymphocytes was significantly suppressed in the RES A, B, and C groups in comparison to those in the control group. On the 7^th post-transplant day, a histological examination revealed apparent difference in the severity of rejection between the RES C group and control group. CONCLUSION： RES has an immuno-suppressive property as well as protective effect on hepatocytes under allograft rejection. It might serve as a novel agent for reducing the severity of hepatic allograft rejection in rats.

Histopathological comparison of topical therapy modalities for acute radiation proctitis in an experimental rat model

AIM： To evaluate the prevalent topical therapeutic modalities available for the treatment of acute radiation proctitis compared to formalin. METHODS： A total of 120 rats were used. Four groups （n = 30） were analyzed with one group for each of the following applied therapy modalities： control, mesalazine, formalin, betamethasone, and misoprostol. A single fraction of 17.5 Gy was delivered to each rat. The rats in control group rats were given saline, and the rats in the other three groups received appropriate enemas twice a day beginning on the first day after the irradiation until the day of euthanasia. On d 5, 10, and 15, ten rats from each group were euthanized and a pathologist who was unaware of treatment assignment examined the rectums using a scoring system. RESULTS： The histopathologic scores for surface epithelium, glands （crypts） and lamina propria stroma of the rectums reached their maximum level on d 10. The control and formalin groups had the highest and mesalazine had the lowest, respectively on d 10. On the 15^th d, mesalazine, betamethasone, and misoprostol had the lowest scores of betamethasone. CONCLUSION： Mesalazine, betamethasone, and misoprostol are the best topical agents for radiation proctitis and formalin has an inflammatory effect and should not be used.

Experimental study on operative methods of pancreaticojejunostomy with reference to anastomotic patency and postoperative pancreatic exocrine function

AIM:To assess the patency of pancreaticoenterostomy and pancreatic exocrine function after three surgical methods. METHODS: A pig model of pancreatic ductal dilation was made by ligating the main pancreatic duct. After 4 wk ligation, a total of 36 piglets were divided randomly into four groups. The piglets in the control group underwent laparotomy only; the others were treated by three anastomoses: (1) end-to-end pancreaticojejunostomy invagination (EEPJ); (2) end-to-side duct-to- mucosa sutured anastomosis (ESPJ); or (3) binding pancreaticojejunostomy (BPJ). Anastomotic patency was assessed after 8 wk by body weight gain, intrapancreatic ductal pressure, pancreatic exocrine function secretin test, pancreatography, and macroscopic and histologic features of the anastomotic site. RESULTS: The EEPJ group had significantly slower weight gain than the ESPJ and BPJ groups on postoperative weeks 6 and 8 (P < 0.05). The animals in both the ESPJ and BPJ groups had a similar body weight gain.Intrapancreatic ductal pressure was similar in ESPJ and BPJ. However, pressure in EEPJ was significantly higher than that in ESPJ and BPJ (P < 0.05). All three functional parameters, the secretory volume, the flow rate of pancreatic juice, and bicarbonate concentration, were significantly higher in ESPJ and BPJ as compared to EEPJ (P < 0.05). However, the three parameters were similar in ESPJ and BPJ. Pancreatography performed after EEPJ revealed dilation and meandering of the main pancreatic duct, and the anastomotic site exhibited a variable degree of occlusion, and even blockage. Pancreatography of ESPJ and BPJ, however, showed normal ductal patency. Histopathology showed that the intestinal mucosa had fused with that of the pancreatic duct, with a gradual and continuous change from one to the other. For EEPJ, the portion of the pancreatic stump protruding into the jejunal lumen was largely replaced by cicatricial fibrous tissue. CONCLUSION: A mucosa-to-mucosa pancreatico- jejunostomy is the best choice for anastomotic patency when compared with EEPJ. BPJ can effectively maintain anastomotic patency and preserve pancreatic exocrine function as well as ESPJ.

A simple and sensitive method to assess ischemia occurrence in the setting of focal cerebral ischemia in rat: A comparative study

Objective: Neurological evaluation is commonly applied to identify ischemia in focal cerebral ischemia model though it might not be sensitive. In present study, we hired sleeping time to assess ischemia occurrence. Methods: Permanent middle cerebral artery occlusion was induced in Sprague-Dawley rats under pentobarbital and ketamine anesthesia respectively. Sleeping time was recorded. Neurological evaluation was conducted by modified Bederson’s scoring system at 4 h and histopathological evaluation was performed at 3 d after middle cerebral artery occlusion. Results: Slices of brain stained by TTC, H&E and hoechst 33258 revealed extensive lesion in the two ischemic groups. The sensitivity to identify ischemia by neurological evaluation was 62.5%, but it was 81.3% and 80% respectively when evaluating by sleeping time (pentobarbital: ≥90.7 min, ketamine: ≥36.1 min). The sensitivity to identify ischemia by sleeping time was significantly higher than that by neurological evaluation (P<0.05). Conclusion: Our results suggested that to identify ischemia by sleeping time is a simple and sensitive method in the setting of focal cerebral ischemia in rat.

Changes of CD8+CD28-T regulatory cells in rat model of colitis induced by 2,4-dinitrofluorobenzene

AIM:To determine the changes of CD8+ T subsets especially CD8+CD28-T regulatory cells in rat model of experimental colitis induced by 2,4-dinitrofluorobenzene (DNFB). METHODS:The rat model of experimental colitis was induced by enema with DNFB.Ten days later,colonic intraepithelial and splenic lymphooltes were isolated from colitis animals (n=16) and controls (n=8).The proportion of CD8+ T cells,CD8+CD28+ T cells and CD8+CD28-T regulatory cells were determined by flow cytometry. RESULTS:The model of experimental colitis was successfully established by DNFB that was demonstrated by bloody diarrhea,weight loss and colonic histopathology.The proportion of CD8+ T cells in either splenic or colonic intraepithelial lymphocytes was not significantly different between colitis animals and controls (spleen:34.6±7.24 % vs 33.5±9.41%, colon:14.0±8.93 % vs 18.0±4.06 %,P>0.05).But CD8+CD28- T regulatory cells from colitis animals were significantly more than those from controls (spleen:11.3±2.26 % vs 5.64±1.01%, colon:6.50±5.37 % vs 1.07±0.65 %,P<0.05).In contrast, CD8+CD28+ T cells from colitis animals were less than those from controls (spleen:23.3±6.14 % vs 27.8±9.70 %,P=0.06; colon:7.52±4.18 % vs 16.9±4.07 %,P<0.05).The proportion of CD8+CD28-T regulatory cells in splenic and colon intraepithelial CD8+ T cells from colitis animals was higher than that from controls (spleen:33.3±5.49 % vs 18.4±7.26 %, colon:46.0±14.3 % vs6.10±3.72 %,P<0.005). CONCLUSION:Experimental colitis of rats can be induced by DNFB with simplicity and good reproducibility.The proportion of CD8+CD28-T regulatory cells in rats with experimental colitis is increased,which may be associated with the pathogenesis of colitis.

A Macaca mulatta model of fulminant hepatic failure

AIM:To establish an appropriate primate model of fulminant hepatic failure (FHF).METHODS:We have,for the first time,established a large animal model of FHF in Macaca mulatta by intraperitoneal infusion of amatoxin and endotoxin.Clinical features,biochemical indexes,histopathology and iconography were examined to dynamically investigate the progress and outcome of the animal model.RESULTS:Our results showed that the enzymes and serum bilirubin were markedly increased and the enzyme-bilirubin segregation emerged 36 h after toxin administration.Coagulation activity was significantly decreased.Gradually deteriorated parenchymal abnormality was detected by magnetic resonance imaging (MRI) and ultrasonography at 48 h.The liver biopsy showed marked hepatocyte steatosis and massive parenchymal necrosis at 36 h and 49 h,respectively.The autopsy showed typical yellow atrophy of the liver.Hepatic encephalopathy of the models was also confirmed by hepatic coma,MRI and pathological changes of cerebral edema.The lethal effects of the extrahepatic organ dysfunction were ruled out by their biochemical indices,imaging and histopathology.CONCLUSION:We have established an appropriate large primate model of FHF,which is closely similar to clinic cases,and can be used for investigation of the mechanism of FHF and for evaluation of potential medical therapies.

Study of a Novel Small Caliber Vascular Graft in a Canine Model with Optical and Scanning Electron Microscopy

A novel biological small-diameter vascular graft was evaluated in a canine model. 3 cm long segments with 4 mm I.D. were implanted end-to-end in the carotid position of 12 dogs for 6 months. Color Doppler sonography was performed at the first week post-operation, and angiography was then administered to 9 grafts at 4th week, 12th week and 24th week respectively to monitor the graft pantency and blood flow characteristics. Vascular samples containing the grafts were collected at 1st week, 8th week, 12th week and 24th week after implantation. Morphological changes of the grafts were observed by optical and scanning electron microscopic (SEM) studies and compared with that of the original prosthesis and the normal host vessel. All grafts were patent throughout the experiment except one graft. Histopathology and SEM demonstrated both a nearly complete inner capsule of varied thickness lining the graft luminal surface and connective tissue adventitia formation at one-week post-operation. The neointima became confluent at 8 weeks and then compact but had no signs of hyperplasia up to 12 weeks; meanwhile on the neointimal surface newly grown endothelial-like cells were migrating from the stoma to the middle portion. The grafts also illustrated endothelialization in many “islands” in the mid-segment luminal surface of the grafts. In addition, the closer distance the cells towards the stoma were, the more morphological similarity the cells with the normal endothelial were. Taken together, the biological vascular graft remained patent for 24 weeks as a carotid prosthesis, characterized by the early and complete neointima formation plus endothelialization starting before 12 weeks post grafting. Therefore, the graft seems suitable for reconstruction of vascular lesions in dogs. Further studies may be carried out to extend the graft application for the clinical use.

Expression of nitric oxide synthase in T-cell-dependent liver injury initiated by ConA in Kunming mice

Objective: To investigate whether nitric oxide synthase (NOS) is expressed in T-cell-dependent liver injury initiated by concanavalin A (ConA) in Kunming mice and study the possible effect of nitric oxide(NO) on liver injury models. Methods: Liver injury in Kunming mice was induced by administration of ConA through tail vein. Expression of NOS in the liver was detected by NADPH diaphorase staining method. The possible effect of NO on liver injury models was obtained by L-NAME injection to suppress synthesis of NO. Results: NOS has a strong expression in hepatocytes after ConA injection, especially in those close to the central vein, while only a weak expression was found in the epithelial cells in control group. Liver injury became more serious when NO synthesis was inhibited by L-NAME, accompanied by great malondialdehyde(MDA) increase in serum and severe intrahepatic vascular thrombosis. Conclusion: NOS markedly expressed in ConA-induced liver injury, which may subsequently promote nitric oxide synthesis. Increasement of nitric oxide has a protective effect on ConA-induced liver injury.

A Correlative Study of CT, MR and Pathology in Rabbit Liver after Embolization by a China-Formulated Lipiodol Emulsion

OBJECTIVE To explore the MR characteristics following lipiodol retention in rabbit liver and to evaluate the sensitivity of CT （CT value 〉400 HU） and MR in displaying the hepatic degeneration and necrosis following embolization. METHODS Thirty-two rabbits were randomly divided into 3 groups. In the control group （n=8）, 2 ml of normal saline was injected into the right branch of the portal vein. In the first experimental group（n= 12）, 4 ml of lipiodol emulsion was injected into the main portal vein. In the second experimental group （n= 12）, 2 ml of lipiodol emulsion was injected into the right branch of the portal vein. CT and MR images were obtained before and after surgery in each group. The histopathologic condition was determined for all liver tissue specimens. RESULTS In the control group, CT and MR did not show any significant changes in the livers after surgery. After the operations in the experimental groups, the regional CT attenuation was 601±101 HU in the largest slice, which had no abnormal signals on T1Wl and T2Wl. In the first group, histologic examinations showed there were concentrated lipiodol droplets around the portal areas. In the second group, serious degeneration and necrosis in the right hepatic lobe occurred in 9 rabbits. T1Wl displayed homogenous or non-homogenous low signals and T2Wl mainly displayed a high signal. However, these pathologic changes did not appear on CT scanning due to high attenuation of the lipiodol. CONCLUSION There were no remarkable hepatic changes on MR in rabbits following good retention of the formulated lipiodol emulsion mixture of lipiodol and urografin（CT value 〉 400 HU）. MR displayed serious degeneration and necrosis of the liver following embolization.

MECHANISM OF PATHOLOGICAL CHANGES OF INTRAVENTRICULAR HEMORRHAGE IN DOGS

Objective To probe the mechanism of pathological changes of intraventricular hemorrhage(IVH). Methods The evaluation of neurological status,serial CT scans and pathological examination were applied on the canine model of IVH. The ventricular volume and blood clot volume were measured based on the CT images. Results The normal adult canine ventricle tend to be slitlike. After injection, the ventricle was obviously dilated by the blood clot. The linear regression of ventricular volume against blood clot volume was significant in the first week. From then on, however, while the clots continued to shrink, the ventricular volume showed progressive enlargement. The clots were lysed completely within 3 to 4 weeks. The linear regression of the degree of ventricular dilatation against the first clot volume was also significant. In the pathological examination, we found the ependymal lining of ventricular system was destroyed and neurons in the subependymal areas developed acidophil necrosis, which was prominent around Sylvian aqueduct. Conclusion Hemorrhagic ventricular dilatation(HVD) is a prominent feature of IVH and also is a strong indicator for poor prognosis. Ischemic changes of periventricular neurons in some important structures may be the most direct cause for poor outcome of IVH. It may be induced by periventricular vascular structures compressed by HVD, increased intracranial pressure, cerebral vasospasm and others.

PATHOLOGICAL AND MORPHOLOGICAL RESEARCH OF EXPERIMENTAL ACETABULAR DYSPLASIA

Objective To investigate the pathological mechanism of hip dysplasia. Methods The leftknee joints of eighteen rabbits were fixed in extending position with plaster cylinder for four weeks, but their hip joints were flexed. The right side served as control. Roentgenogram was made in all animals. The changes of the x-ray films and the pathological findings between left and right hips were compared. Results Appearance of hip dysplasia was obvious at four weeks after plaster fixation. There were pathological changes, including shallow ace-tabulum and flat femoral head, increased acetabular index and decreased acetabular head index on the x-ray films. Conclusion The hip dysplasia is the result of prolonged extending position of the knee joint. Abnormal knee posture seems to be one of the important factors of hip dysplasia. This kind of deformation may be worsened with time.

The biomechanics of point contact-dynamic compression plate and its effects on bone perfusion

Objective： To compare the mechanical properties of point contact-dynamic compression plate （PC-DCP） and its effects on cortical bone perfusion with that of dynamic compression plates （DCP） in goat tibiae. Methods： Twenty pairs of matched flesh goat tibiae were used. A transverse fracture model was established. The fractures with a 3ram interspace between the fracture ends were subject to fixations with the DCPs and the PC- DCPs respectively, then the four-points bending tests and the torsion tests were conducted to compare the mechanical properties of the PC-DCP with that of DCP. Another 13 sexually mature goats underwent fixations with the DCPs and the PC-DCPs, respectively, at the mid-shafts of the intact bilateral tibiae. Ischemic zones were observed at four time points （1 day, 2, 6, and 12 weeks after operation） using disuiphine blue staining technique. Remits： There were no significant differences in mechanical properties, such as bend- and torsionresistance, between the DCPs and the PC-DCPs. One day, 2, and 6 weeks after operation, on the side of DCP fixation, outer cortical bone ischemia under the plate persisted, and this condition did not reverse until 12 weeks after operation. However, on the side of PC-DCP fixation, cortical bone iscbemia occurred only in the periphery of the screw holes and at the contact sites of the PC NUTs 1 day after operation, and it disappeared at 2 weeks after operation. Conclusions： The PC-DCP has similar biomechanical properties of the DCP, but is less detrimental to local bone blood circulation than the conventional plates.

Effect of puerarin on neural function and histopathological damages after transient spinal cord ischemia in rabbits

Objective: To investigate the effect of puerarin on the neural function and the histopathological changes after ischemic spinal cord injury in rabbits. Methods: Thirty male New Zealand white rabbits were randomly divided into three groups as follows: puerarin group (n=10) receiving intravenous infusion of 30 mg/kg puerarin for 10 minutes, control group (n=10) receiving intravenous infusion of the same volume of normal saline as puerarin for 10 minutes, and sham operation group (n=10) undergoing only the surgical exposure of the abdominal aorta. Temporary spinal cord ischemia was induced by infrarenal aortic occlusion for 20 minutes and followed by reperfusion. The neural status was scored with the Tarlov criteria at 8, 12, 24 and 48 hours after reperfusion. All the animals were killed at 48 hours after reperfusion and the spinal cords (L 5) were removed immediately for histopathological study. Results: The neural function scores at 8, 12, 24 and 48 hours after reperfusion were higher in the puerarin group and sham operation group than those in the control group (P< 0.05). More normal motor neurons in the anterior horn of spinal cord were present in the puerarin group and sham operation group than those in the control group (P< 0.01). There was a strong correlation between the final neural function scores and the number of normal motor neurons in the anterior horn of spinal cord (r= 0.839, P< 0.01). Conclusions: Puerarin can significantly ameliorate the neural function and the histopathological damages after transient spinal cord ischemia in rabbits.

Pathological study of rabbits' femoral arteries subjected to gunshot wounds combining with seawater immersion

Objective: To investigate pathological characteristics of gunshot wounds concomitant seawater immersion in rabbits’ femoral arteries. Methods: Thirty rabbits were divided randomly into 3 groups: simple gunshot-wound group (Group I, n=10), gunshot wound with seawater immersion for 30 mins (Group II, n=10), and 60 mins group (Group III, n=10). Femoral arteries were impacted by (0.38 g) steel spheres fired with a (7.62 mm) rifle. After being wounded, rabbits in Groups II and III were immersed in seawater for 30 or 60 mins, but those in Group I were not. At 2, 4, 6, 8, 12 hours following injury, a 40 mm segment of the artery on each side of the gunshot point were excised and observed by light and electron microscopy. Results: The patterns of arterial injuries were mainly contusion and transection. Completely transected artery was classified as primary-wound-tract area, contused area and shocked area. Compared with those in Group I, the primary-wound-tract and contused areas in Group II manifested obvious swelling in the arterial wall especially at the outside 2/3 of the media. Vacuolar structures were often seen in smooth muscle cells of the media. Intercellular space among the smooth muscle cells were filled with homogeneous acidophilic substances. Deep rugae among endothelial cells flattened or rugal folds lost their longitudinal orientation, and marked fibrin and platelet deposition were noticed. No significant difference was detected between Group II and III. The pathological changes in the shocked area were similar in 3 groups. Conclusions: For gunshot wounds concomitant seawater immersion in rabbits’ femoral arteries, there was a marked swelling of cells and intercellular space in primary-wound-tract area and contusion area. The influence of these pathological changes on surgical reparation deservers further study.