目的 建立犬开放性气胸海水浸泡的实验模型 ,探讨实验动物早期死亡原因。 方法 2 0条健康成年杂种犬随机分为两组。对照组 :实验动物受伤后直接观察 ;实验组 :动物受伤后置入人工配制的海水中。监测血流动力学、呼吸、血液渗透压、血液电解质、动脉血气变化以及肺部病理改变。 结果 实验组死亡率明显高于对照组 ,平均生存时间为 45分钟。实验组经海水浸泡后有急性呼吸和循环功能衰竭、严重电解质平衡紊乱、高渗血症、重度肺损伤以及严重代谢性和呼吸性酸中毒。 结论 开放性气胸后海水浸泡可引起一系列严重的病理生理变化 ,其结果是导致实验动物早期死亡的重要原因。
Objective To explore an effective method to culture and purify canine bladder transitional epithelial cells.Methods Bladder tissue was obtained from healthy puppy under sterile conditions. Bladder mucosa was removed from the remaining tissue with fine scissor and minced into small pieces, and then were dissociated into single cell suspensions with 0.125% trypsin. The bladder epithelial cells were cultured in defined keratinocyte serum free medium. The cells were passaged and purified by 0.05% trypsin and 0.02% EDTA. Morphological characterization were studied under inverted phase contrast microscope and transmission electron microscope. Expression of cell specific marker protein was assessed by immunohistochemistry. Results Canine bladder transitional epithelial cells could be efficiently cultivated and expanded in serum-free medium without fibroblast contamination. The cells could be passaged 4-6 times without a distinguished decrease in cell proliferation. The cells were characterized by well-developed micro filament and desmosome junction under transmission electron microscope. Immunohistochemical staining with broadly reacting anticytokeratin antibodies (AE1/AE3) confirmed the epithelial phenotype of the cells.Different generations of cells showed diploid cells. Conclusion A large number of bladder transitional epithelial cells can be obtained from small bladder tissue with our digestion method. The cultured bladder epithelial cells can be proliferated to sufficient quantities for further reconstructive purposes.
Objective To investigate the effect of imbedding chemotherapy of sustained release of 5-fluorouracil on the healing of colonic stoma in dog. Methods Twenty-eight adult hybrid dogs were randomly divided into chemotherapy group （n=22) and control group (n=6). The canine sigmoid colon were firstly detached and then anastomosed via median abdominal incision, 200 mg sustained release of 5-fluorouracil was imbedded in the mesentery 1.0-1.5 cm away from colonic stoma in chemotherapy group, whereas the control substance was injected into the dogs in control group. Tissue samples were collected from mesentery and stomas on 3, 5, 7, 10 and 15 days after operation, respectively, in order to observe the healing of stoma. The drug concentrations in the stoma and in the tissues that were 0, 1, 3, 5, 7, 10 and 15 cm away from the imbedding point were also measured by high performance liquid chromatographymethod at different phases. Results The tissues from colonic stoma only showed inflammatory reaction at early stage, with no necrosis and cellular degeneration. It was observed that the stoma healed basically on the tenth day after operation. The drug concentrations in the tissues gradually decreased at the range of 0-15 cm over time, but all of which were higher than the anti-tumor effective concentration （0.10 μg/g）. Conclusion The imbedding chemotherapy of sustained release of 5-fluorouracil in mesentery has little effect on the healing of stoma, and it could remain an effective anti-tumor concentration in a period of time.
Objective To make a comparison for the change of maximum tensile intensity and stiffness of a whole implant that is placed into bone tunnel with various lengths tendon, by using beagle dog’s autogenous flexor tendons to reconstruct anterior cruciate l igament (ACL). Methods Sixty male beagle dogs were included in the experiment (weighting 13-16 kg). Three dogs were used for intact flexor tendon of both knees (normal control group), 3 dogs for the intact ACL andfemur-graft-tibia complex (auto control group) and 54 dogs (108 knees) for models of reconstructed ACL (6 experimentalgroups according to different lengths of tendon: 5, 9, 13, 17, 21 and 25 mm in the bone tunnel). The tensile intensity and stiffness were measured after 45, 90 and 180 days separately after operation. Results In the normal control group, the maximum tensile intensity of the intact flexor tendon was (564.15 ± 36.18) N, the stiffness was (59.89 ± 4.28) N/ mm. In the auto control group, the maximum tensile intensity of the intact ACL was (684.75 ± 48.10) N, the stiffness was (74.34 ± 6.99) N/ mm, all ruptured through the intra-articular portion of the graft. The maximum tensile intensity of femur-graft-tibia complex in the auto control group was (301.92 ± 15.04) N, the stiffness was (31.35 ± 1.97) N/mm. After 45 days of operation, all failure occurred at the tibial or femoral insertion site. After 90 days of operation, 24 of the breakpoints were scattered in tendon-bone junction, 12 (3 in 17 mm group, 5 in 21 mm group, 4 in 25 mm group) ruptured through the intra-articular portion. After 180 days of the operation, all breakpoints were distributed inside joint of the implant. The maximum tensile intensity and the stiffness were ber in 17, 21 and 25 mm groups than in 5, 9 and 13 mm groups after operation (P lt; 0.05). Conclusion Tendon with 17 mm length, which will be implanted into bone tunnel, is an appl icable index, in reconstruction of ACL by autogenous tendons.
Objective To observe the complication after embolizing the bilateral internal il iac arteries and the median sacral artery of dogs by different combinations and embolization levels with gelfoam particle, and to provide a reference for safety appl ication of gelfoam in cl inic. Methods Sixteen common grade adult healthy dogs (weighing 10-13 kg, 14 males and 2females) were randomly divided into 5 groups. Under the monitoring of digital subtraction angiography (DSA), the embolization was performed with gelfoam particle (diameter, 50-150 μm) in bilateral internal il iac arteries and the main branch of the median sacral artery (group A, n=3), in bilateral internal il iac arteries and the first branch of the median sacral artery (group B, n=3), in the main branch of bilateral internal il iac arteries (group C, n=3), in the unilateral internal il iac artery and the main branch of the median sacral artery (group D, n=4), and in the main branch of unilateral internal il iac artery (group E, n=3). Under the DSA, the anatomic relationships of the abdominal aorta, bilateral external il iac arteries, bilateral internal il iac arteries, and median sacral artery were observed before embol ization. The survival dogs were observed and the specimens of bladder, rectum, sciatic nerve, and gluteal muscles were harvested for the general and histological observations at 3 days after embolization. Results In dogs, there was no common il iac artery; bilateral external il iac arteries originated from the abdominal aorta and the starting of the median sacral artery had variation. Seven dogs (3 in group A, 3 in group C, and 1 in group D) died within 2 days after embolization, and the others survived to the end of the experiment. In the dead dogs of groups A, C, and D, the darkening and necrosis of the rectum were observed; the bladder presented lamellar obfuscation and focal hemorrhage and edema; and the median urinary volume in bladder was 270.6 mL. In survival dogs, no obvious change was observed in the rectum; the bladder only manifested l ight edema; and the median urinary volume in bladder was 137.0, 220.5, and 28.0 mL, respectively in groups B, D, and E.The rectum and bladder of dead dogs in groups A, C, and D manifested the disrupted cells, generous inflammatory cells infiltration, and desquamation of epithel ial cells; the rectum and bladder of survival dogs in groups B, D, and E manifested l ight inflammatory cells infiltration and edema; the embol ized artery mainly focused on the arterioles whose diameter was 100-200 μm. The sciatic nerve and gluteal muscles of each group had no obvious change except for l ight edema. Conclusion When the internal il iac artery and median sacral artery are embol ized with gelfoam particle with a diameter of 50-150 μm, to ensure the safeness of pelvic organs, the embol ized artery can not exceed the first branch when the 3 arteries are embol ized at the same time, or reserve at least unilateral internal il iac artery when embol ized to the trunk , or it will result in pelvic organ necrosis and perforation.
Objective To explore the construction of a canine model of vascularized allogeneic spinal cord transplantation (vASCT) and preliminarily evaluate its therapeutic efficacy for spinal cord injury (SCI). Methods Sixteen female Beagle dogs aged 8-12 months were randomly selected, with 8 dogs serving as donors for the harvesting of spinal cord tissue with a vascular pedicle [dorsal intercostal artery (DIA) at the T10 level and accompanying vein]. The remaining 8 dogs underwent a 1.5-cm-length spinal cord defect at the T10 level, followed by transplantation of the donor spinal cord tissue for repair. Polyethylene glycol (PEG) was applied to both ends to spinal cord graft; then, using a random number table method, the dogs were divided into an experimental group (n=4) and a control group (n=4). The experimental group received immunosuppressive intervention with oral tacrolimus [0.1 mg/(kg∙d)] postoperatively, while the control group received no treatment. The operation time and ischemia-reperfusion time of two groups were recorded. The recovery of hind limb function was estimated by Olby score within 2 months after operation; the motor evoked potentials (MEP) was measured through neuroelectrophysiological examination, and the spinal cord integrity was observed through MRI. ResultsThere was no significant difference in the operation time and ischemia-reperfusion time between the two groups (P>0.05). All dogs survived until the completion of the experiment. Within 2 months after operation, all dogs in the control group failed to regain the movement function of hind limbs, and Olby scores were all 0. In the experimental group, the movement and weight-bearing, as well as walking abilities of the hind limbs gradually recovered, and the Olby scores also showed a gradually increasing trend. There was a significant difference between the two groups from 3 to 8 weeks after operation (P<0.05). Neuroelectrophysiological examination indicated that the electrical signals of the experimental group passed through the transplanted area, and the latency was shortened compared to that at 1 month after operation (P<0.05), showing continuous improvement, but the amplitude did not show significant improvement (P>0.05). The control group was unable to detect any MEP changes after operation. MRI examination showed that the transplanted spinal cord in the experimental group survived and had good continuity with normal spinal cord tissue, while no relevant change was observed in the control group. ConclusionThe vASCT model of dogs was successfully constructed. This surgical procedure can restore the continuity of the spinal cord. The combination of tacrolimus anti-immunity is a key factor for the success of transplantation.
Objective To establish and evaluate a hydrocephalus model in dogs. Methods Twelve healthy adult male mongrel dogs (weight, 10-15 kg) were randomly divided into the control group (n=6) and the experimental group (n=6). All the dogs were given CT and neurological examination to exclude congenital ventricular enlargement and neurological abnormity before they received hydrocephalus induction. Surgical procedures included the exposing of the foramen magnum area, the opening of the atlantooccipita anadesma, and the injecting of silicone oil (0.3 ml/kg) into the fourth ventricle through a silicone tube. Normal saline was injected in the control group. The Tarlov neurological fitness assessment and the Evan’s ratio were used to evaluatethe degree of hydrocephalus at 3, 14 and 56 days after operation. Results In the experimental group, the dogs were dull and unsteady in walking,and they drank and ate less. The lateral ventricle began to expand 3 days afteroperation, and then the temple horn of the lateral ventricle and the third ventricle were also affected 14 days after operation. The ventricles were enlarged progressively after operation. The Tarlov scores measured at 3, 14 and 56 days afteroperation had a significant difference at the same time point between the control group(5.83±0.75，6.50±0.55，6.00±0.63) and the experimental group (4.00±0.89，4.83±1.17，4.50±1.05，P＜0.01), but had no significant difference within the same group at different time points (P＞0.05). The Evan’s ratios measured at 3, 14 and 56 days after operation were 0.33±0.04,0.39±006，0.44±0.03，respectively,in the experimental group; and were 0.27±0.06，0.25±0.09, 0.26±0.05,respectively,in the control group. There was a significant difference atthe same time point between the two groups, and at different time points within the experimental group (P＜0.05).Conclusion The dog model of hydrocephalus induced by the injecting of silicone oil into the fourth ventricle has a highsuccess rate, and the model is appropriate for the studies on diagnosis and therapy of hydrocephalus.
Objective To explore an effective method of culturing the canine bladder smooth muscle cells, observe the morphological characteristics of the bladder smooth muscle cells growing on acellular small intestinal submucosa（SIS） and offer an experimental basis for reconstruction of the bladder smooth muscle structure by the tissue engineering techniques. Methods The enzymetreatment method and the explant method were respectively used to isolate and harvest the canine bladder smooth muscle cells, and then a primary culture of these cells was performed. The canine bladder smooth musclecells were seeded on the SIS scaffold, and the composite of the bladder smooth muscle cells and the SIS scaffold were co cultured for a further observation. At 5,7 and 9 days of the co culture, the specimens were taken; the bladder smooth muscle cells growing on the SIS scaffold were observed by the hematoxylin staining, the HE staining, and the scanning electron microscopy. The composite of the bladder smooth muscle cells on the SIS scaffold was used as the experimental group, and the bladder smooth muscle cells with no SIS were used as the control group. In each group, 9 holes were chosen for the seeded bladder smooth muscle cells, and then the cells were collected at 3, 5 and 7 days for the cell counting after the enzyme treatment. Morphological characteristics of the cells were observed under the phase contrast microscope and the transmission electron microscope. Expression of the cell specific marker protein was assessed by the immunohistochemical examinaiton. The proliferation of the cells was assessed by the cell counting after the seeding on the SIS scaffold. Results The primary bladder smooth muscle cells that had been harvested by the enzyme treatment method were rapidly proliferated, and the cells had good morphological characteristics. After the primary culture in vitrofor 5 days, the bladder smooth muscle cells grew in confluence. When the bladder smooth muscle cells were seeded by the explant method, a small amount of the spindleshaped bladder smooth muscle cells emigrated from the explant at 3 days. The cells were characterized by the welldeveloped actin filaments inthe cytoplasm and the dense patches in the cell membrane under the transmissionelectron microscope. The immunohistochemical staining showed the canine bladdersmooth muscle cells with positive reacting α actin antibodies. The bladder smooth muscle cells adhered to the surface of the SIS scaffold, growing and proliferating there. After the culture in vitro for 5 days, the smooth muscle cells covered all the surface of the scaffold, showing a singlelayer cellular structure. The cell counts at 3, 5 and 7 days in the experimental group were（16.85±0.79）×105，（39.74±2.16）×105 and （37.15±2.02）×105, respectively. Thecell counts in the control group were（19.43±0.54）×105，（34.50±1.85）×105 and （33.07±1.31）×105, respectively. There was a significant difference between the two groups at 5 days （P＜0.05）. ConclusionWith the enzyme treatment method, the primarily cultured canine bladder smooth muscle cells can produce a great amount of good and active cells in vitro. The acellular SIS can offer an excellent bio scaffold to support the bladder smooth muscle cells to adhere and grow, which has provided the technical foundation for a further experiment on the tissue engineered bladder reconstruction.
ObjectiveTo study the preservation effect of true bone ceramics (TBC) prepared by high-temperature calcination of bovine bone on alveolar ridge of canine extraction socket.MethodsSix healthy Beagle dogs (aged 1.5-2 years) were selected to extract the second and fourth premolars of both mandibles and the second premolars of the maxilla. The left extraction socket was implanted with TBC as the experimental group, and the right side was implanted with the calcined bovine bone (CBB) as the control group, to observe the alveolar ridge preservation effect. Three dogs were euthanized after general observation at 1 and 6 months after operation respectively. After separating the maxilla and mandible, cone beam CT (CBCT) was performed to measure the average gray value of the graft site and the adjacent reference area (the area between the roots of the adjacent third premolar) and calculate the gray scale ratio between the bone graft site and the reference area. Histological observation was made on the bone graft site to evaluate the new bone formation.ResultsGeneral observation showed that the wounds of both groups were basically healed at 2 weeks after operation, and the bone graft materials were not exposed. The wounds healed well at 1 and 6 months after operation without swelling. The results of CBCT showed that the residual material was found in both groups at 1 month after operation, and no significant residual material was found in both groups at 6 months after operation, and the alveolar ridge height of the bone graft area was not significantly reduced. There was no significant difference in the bone mineral density between the experimental group and the control group. The gray scale ratios of the experimental group at 1 month and 6 months after operation were 0.97±0.14 and 0.93±0.06, respectively, and were 0.99±0.16 and 0.94±0.05 in control group, showing no significant difference between the two groups (t=−1.030, P=0.333; t=−0.770, P=0.466). HE staining observation showed that a large number of bone graft materials did not degrade and new bone formed around the grafts in both groups at 1 month after operation; the bone graft materials were absorbed and a large number of new bones were formed in both groups at 6 months after operation.ConclusionTBC can maintain bone mineral density and have good osteoconductivity in the alveolar ridge site preservation experiment of dogs, and can be used for alveolar ridge site preservation.
To find the relation between the damage of gastric remnant mucosal barrier and the precancerous lesion of gastric remnant mucosa, in the process of the canine gastric remnant precarcinogenesis induced by N-methyN’-nitro-N-nitrosoguanidine (MNNG), we performed regularly the esophagogastroscopy and the mucosal biopsy.At the same time, we also measured gastric transmucosal potential difference and intracellular DNA content of remnant mucosa.We found that the more severe the damage of gastric remnant mucosal barrier was , the greater the malignant capacity of gastric remnant mucosal was.Our study suggests that the damage of gastric remnant mucosal barrier plays an important role in the gastric remnant mucosal precarcinogenesis.