Objective To investigate the rule of the morphological changes of the detrusor muscle and its neuromuscular junction after the medullary cone injury in rats. Methods Forty-eight SDadult rats were divided into 6 groups randomly, each of which was 8. There werenormal control group(group A), 4 weeks group(group B), 6 weeks group(group C), 8 weeks group(group D), 10 weeks group(group E) and 12 weeks group(group F) after the medullary cone injury respectively. The medullary cone injury was completed in the level of L4,5 with a sharp and transsectional way. The HE dyeing of the detrusor muscle was performed firstly to observe the changes of the section areas of muscle fibers. And the electron microscopic samples of the detrusor muscle were made to investigate the rules of the ultrastructructral changes in the detrusor muscle and its neuromuscular junction. Finally, the Masson trichromatic dyeing of the detrusor muscles was performed to calculate the percentages of the smooth muscle and the connective tissue.Results The HE dyeing of the detrusor muscle indicated the section areas of muscle fibers in groups E, F was significantly less than that in group A (P＜0.05). The gradually aggravated ultrastructructral changes of detrusor cells in groups B-F were observed in atonic bladders，such as various shape and size，malalignment, wide separation between musclecells, abundant collagen fibrils and irregular dense structures between individual cells, obvious rough endoplasmic reticulum widen and mitochondrial edema were noted．And the ultrastructructral changes of the neuromuscular junctions manifested that the similar structures in group A and the reduction of the mitochondria and synaptic vesicles was seen in groups B, C and D, the conspicuous degenerative neuromuscular junction and the obvious reduction of the synaptic vesiclesand the mitochondria was observed in group E,and the deteriorative degenerativeneuromuscular junction and the obvious reduction or disappearance of the synaptic vesicles and the mitochondria even to the degenerative corpuscle was noted in group F. The Masson trichromatic dyeing in the detrusor muscles indicated that there were significant differences in the percentage of the connective tissue in the detrusor muscles between groups E,F and group A, and between group E and group F respectively (P＜0.05). Conclusion The irreversible changes of the detrusor muscle and its neuromuscular junction canbe seen in the 10th week after medullary cone injury in rat. And the nerverepairing procedures should be performed before this.
【Abstract】ObjectiveTo establish an efficient, effective hepatocyte isolation technique in order to increase cell production and decrease the prime cost. Methods The inferior vena cava below diaphragm was dissected and ligatured, and the inferior vena cava below liver was separated. Subsequently, the liver was perfused with EGTA through the portal vein while the inferior vena cava below liver was opened, and then the liver was harvested. The liver tissue was cut into 1 mm×1 mm×1 mm and digested at 37 ℃ water bath with Ⅳ collagenase for 30-40 minutes, then the hepatocytes were purified and cultured in CO2 incubator. The production and function of hepatocytes were assessed. ResultsThe isolated hepatocytes using this technique were more than 95% among the all isolated cells. No statistic difference was found in cell production and cell function comparing with traditional technique. But this technique was simplified and more economically. ConclusionThis modified hepatocyte isolation technique is efficient and effective. It can ensure the amount of production and purity of hepatocytes.
ObjectiveTo explore the effect and mechanisms of bone marrow mesenchymal stem cells (BMSCs) on healing quality of acetic acid-induced gastric ulcer. MethodsForty-eight clean grade male Wistar rats were used to establish the model of gastric ulcer with acetic acid and were randomly divided into 3 groups after 3 days of modeling, 16 rats each group. After the abdominal cavity was open and stomach was pulled out, no treatment was given in group A, 150 μL phosphate buffered saline (PBS) and 150 μL BMSCs at passage 4+PBS (1×108 cells/100 μL) were injected into the gastric wall surrounding the ulcer at 5 different points in groups B and C respectively. After 10 days, the ulcer area was measured, the mucosal thickness and the number of dilated glands were tested in the regenerative mucosa by histological method. And the expression of vascular endothelial growth factor (VEGF) was detected at ulcerative margin by immunohistochemical method. ResultsThe ulcer area in group C was significantly smaller than that of groups A and B (P<0.01), but no significant difference was found between groups A and B (P>0.05). HE staining showed that group C had thicker regenerative gastric mucosa, less dilated glands, and more regular mucosal structure than groups A and B, showing significant differences in regenerative gastric mucosa thickness and dilated glands number (P<0.01), but no significant difference between groups A and B (P>0.05). Immunohistochemical staining showed that the positive expression of VEGF in the ulcer margin mucosa of group C was significantly higher than that of groups A and B. The integral absorbance (IA) value of VEGF expression in group C was significantly higher than that in groups A and B (P<0.01), but no significant difference between groups A and B (P>0.05). ConclusionBMSCs can accelerate ulcer healing by the secretion of VEGF, and improve the quality of ulcer healing.
Objective To investigate tissue engineered spinal cord which was constructed of bone marrow mesenchymal stem cells (BMSCs) seeded on the chitosan-alginate scaffolds bridging the both stumps of hemi-transection spinal cord injury (SCI) in rats to repair the acute SCI. Methods BMSCs were separated and cultured from adult male SD rat. Chitosan-alginate scaffold was produced via freeze drying, of which the structure was observed by scanning electron microscope (SEM) and the toxicity was determined through leaching l iquor test. Tissue engineered spinal cord was constructed by seeding second passage BMSCs on the chitosan-alginate scaffolds (1 × 106/mL) in vitro and its biocompatibil ity was observed under SEM at 1, 3, and 5 days. Moreover, 40 adult female SD rats were made SCI models by hemi-transecting at T9 level, and were randomly divided into 4 groups (each group, n=10). Tissue engineered spinal cord or chitosan-alginate scaffolds or BMSCs were implanted in groups A, B, and C, respectively. Group D was blank control whose spinal dura mater was sutured directly. After 1, 2, 4, and 6 weeks of surgery, the functional recovery of the hindl imbs was evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating score. Other indexes were tested by wheat germ agglutinin-horseradish peroxidase (WGA-HRP) retrograde tracing, HE staining and immunofluorescence staining after 6 weeks of surgery. Results Chitosan-alginate scaffold showed three-dimensional porous sponge structure under SEM. The cells adhered to and grew on the surface of scaffold, arranging in a directional manner after 3 days of co-culture. The cytotoxicity of chitosan-alginate scaffold was in grade 0-1. At 2, 4, and 6 weeks after operation, the BBB score was higher in group A than in other groups and was lower in group D than in other groups; showing significant differences (P lt; 0.05). At 4 and 6 weeks, the BBB score was higher in group B than in group C (P lt; 0.05). After 6 weeks of operation, WGA-HRP retrograde tracing indicated that there was no regenerated nerve fiber through the both stumps of SCI in each group. HE and immunofluorescence staining revealed that host spinal cord and tissue engineering spinal cord l inked much compactly, no scar tissue grew, and a large number of neurofilament 200 (NF-200) positive fibers and neuron specitic enolase (NSE) positive cells were detected in the lesioned area in group A. In group B, a small quantity of scar tissue intruded into non-degradative chitosan-alginate scaffold at the lesion area edge, and a few of NSE flourescence or NF-200 flourescence was observed at the junctional zone. The both stumps of SCI in group C or group D were filled with a large number of scar tissue, and NSE positive cells or NF-200 positive cells were not detected. Otherwise, there were obviously porosis at the SCI of group D. Conclusion The tissue engineered spinal cord constructed by multi-channel chitosan-alginate bioscaffolds and BMSCs would repair the acute SCI of rat. It would be widely appl ied as the matrix material in the future.
Objective To observe the plastic changes of sensory nerve in terms of structure and function when targetorgan changed through making the rat model of nerve regeneration by anastomosing the proximal end of sensory nerve and the distal end of motor nerve. Methods Thirty adult SD rats (male or female), weighing 200-250 g, were randomized into three groups (n=10 per group). The left upper l imb of the each rat was used as the experimental side, while the right upper l imb as the control side. In group A, the medial antebrachial cutaneous nerve was cut 5 mm away from its origin and its proximal end was anastomozed end-to-end to the distal end of musculocutaneous nerve. In group B, the musculocutaneous nerve was cut 5 mm away from its nerve entry point and the proximal end of medial antebrachial cutaneous nerve were anastomozed end-to-end to the distal end of musculocutaneous nerve. In group C, medial antebrachial cutaneous nerve and musculocutaneous nerve were cut, without further anastomosis. Twenty-four weeks after operation, the general condition and the motion of the elbow joint of rats, the wet weight and muscle fiber cross-section area of the biceps brachii as well as the latent period and the ampl itude of the evoked potential were observed and the acetylchol inesterase (AchE) staining of nerve of proximal end of anastomosis was conducted. Results All the rats survived for 24 weeks with good general condition and without wound infection. The rats in groups A, B and C were lost the active flexion of left elbow joint after operation. The rats in groups A and B got recovered to some degree at 24 weeks. The behavioral evaluation showed that there were 7 l imbs in group A and 5 l imbs in group B scoredas 4-5 points, there was a significant difference when compared with group C (P lt; 0.05), but there was no significant difference between group A and group B (P gt; 0.05). Group A and group B were superior to group C in terms of the wet weight and the muscle fiber cross-section area of the biceps brachii (P lt; 0.05), but no significant difference between group A and group B was detected (P gt; 0.05). The evoked potential of the biceps brachii and motor nerve fibers in proximal end of anastomosis could be detected in both group A and group B. But there was no significant difference between group A and group B with respects of function recovery of elbow joint, the latent period and the ampl itude of the evoked potential of the biceps brachii and the quantity of motor nerve fiber in proximal end of anastomosis (P gt; 0.05). Conclusion The change of target organ leads to the sensory nerve plasticity structurally and functionally, which may provide a new approach for peripheral nerve repair.
Objective To establish a reliable rats model of orthotopic liver transplantation with non-heart beating donors. Methods The model was established with modified double-cuff method. According to obtain pre-liver warm ischemia time experiencing non-heart-beat the rats were divided into 3 groups: 10 min (R10 group), 20 min (R20 group) and 30 min (R30 group), then one week survival after operation was compared in rats. Results The operative time of donor was 30 min approximately except warm ischemia time and the cold preservation time of donor liver was 1 h. The anastomotic time for suprahepatic vena cava was 12-22 min (mean 15 min). The anastomotic time for portal vein and infrahepatic vena cava was about 2 min and 1 min, respectively. The anhepatic phase sustained 14-24 min (mean 19 min). The operative time of receptor was 50-65 min (mean 60 min). Twelve rats died at 24 h after operation, which was considered as operative failure. The success rates of operation in R10 group, R20 group, and R30 group were 95% (19/20), 80% (16/20), and 65% (13/20), respectively. After one week the survival rate was 95% (18/19), 81% (13/16), and 54% (7/13), respectively. Conclusions Improved non-heart donor liver transplantation model of rat on the basis of Kamada’s “twocuff technique” acts as a good simulation in clinical non-heart-donor liver transplantation. This study showes that rat liver can tolerate warm ischemia time less than 30 min, the short-term survival after transplantation can reach satisfactory results. However, long-term survival requires further study.
Objective To investigate the effect of cold ischemia on the development of transplant arteriosclerosis (TA) in rat aortic isografts. Methods Aorta grafts from SD and Wister rats were stored in a cold perfusion solution for 0.5 hours and 4 hours respectively before being orthotopically transplanted to Wister recipients. After observation times ranging from 15 to 60 days, the grafts were examined by using histological and electron microscopy techniques. Regional changes in the lumen, intima and media layers were measured by using an image analysis system. Results Partial intima thickings were showed in control isografts at 60 day posttransplantation. Pronounced intima thickings were seen in experimental isografts and control allografts at the same time. The thicking neointimas consist mainly of monocyte/macrophage and smooth muscle cells (SMC). The broken interior elastic lamina (IEL) and necrosis SMC in media were detected in allogenic grafts. Conclusion The damage due to prolonged cold ischemia time is sufficient to cause pronouced graft arteriosclerosis.
Objective To investigate the effects of icariin and mixed prescri ption of icariin, radix hedysari polysaccharide, and l iquid extracted from earthworm on peri pheral nerve regeneration. Methods Twenty male SD rats weighing (200 ± 10) g were selected and randomized into four groups (n=5 per group): sham operated group (group A), model group (group B), icariin group (group C), and mixed l iquid group (group D). In group A, the left sciatic nerves of the rats were only exposed, and treated at fixed time from the following day with the NS (2 mL/d). In groups B, C, D, the models were made by clamping sciatic nerve and treated with NS, icariin and mixed l iquid, respectively (2 mL/d). The general state ofanimals was observed after the treatment daily. The nerve function index, motor nerve conductive velocity and the morphous and number of myel inated sciatic nerve fibers were measured at 21 days. Results Animals in various groups were all in good state. After 21 days, the weights of rats in groups A, B, C and D were (366.9 ± 14.0), (370.1 ± 16.3), (373.3 ± 19.6) and (374.0 ± 11.4) g, respectively, and there was no significant difference among these groups (P gt; 0.05). For sciatic function index, there was no significant difference between group A and group D (P gt; 0.05), between group B and group C (P gt; 0.05), while there was significant difference between group B and group D (P lt; 0.05). For tibial function index, there was significant difference between group A and groups B, C, D (Plt; 0.05), there was no significant difference between group B and groups C, D (Pgt; 0.05). For peroneal function index, there was no significant difference between group A and groups C, D (P gt; 0.05), between group B and groups C, D (P gt; 0.05). The sciatic motor nerve conductive velocities of group A, B, C and D were (45.0 ± 2.9), (8.0 ± 2.6), (13.4 ± 6.8), and (19.6 ± 9.3) m/s, respectively, there was no significant difference between group B and group C (P gt; 0.05), and there was significant difference between group A and groups B, C, D and between group B and group D (P lt; 0.05). The size of individual myel inated sciatic nerve fibers of regenerated nerves in groups B, C, and D was significantly smaller than that in group A. Comparing with group A, the number of myel inated sciatic nerve fibers in groups B, C, and D was 93.3% ± 35.6%, 90.6% ± 37.1%, and 115.4% ± 40.6%, respectively, but there was no significant difference among four groups (P gt; 0.05). Conclusion Icariin and mixed prescription are safe. The improving peripheral nerve regeneration effect of mixed prescription is more obvious than that of icariin, indicating the comprehensive study of modified formula radixhedysari is necessary to find the effective part or mixture of effective compounds with fixed percentage.
ObjectiveTo investigate the main influence factors of microbubble-enhanced sono-thrombolysis by an orthogonal array experimental design (OAD) and to confirm the optimal parameters of microbubble-enhanced sono-thrombolysis in vitro. Methods The peripheral blood was collected from 50 female Sprague Dawley rats to prepare the standard plasma, and then 100 μL standard plasma and 25 μL thrombin (0.15 U/μL) were mixed and incubated in 37℃ water bath for 3, 6, 12, and 24 hours respectively to prepare corresponding standardized thrombus. The physical parameters for the designed experiments included transmit powers of ultrasound (factor A: 5%, 25%, 50%, and 100%), microbubble volume (factor B: 50, 100, 200, and 400 μL), urokinase (UK) concentration (factor C: 100, 200, 400, and 800 U/mL), and thrombolysis time (factor D: 10, 20, 30, and 40 minutes), respectively. Then an OAD based on four parameters and four levels [L16(45)] was employed to optimize the thrombolysis conditions. The ultrasound frequency was 1.82 MHz. HE staining and scanning electron microscope (SEM) were used to observe the clots before and after thrombolysis. The thrombolysis rate was measured. ResultsHE staining and SEM observation showed that the fibrin was dissolved after thrombolysis. According to the OAD, the optimal parameter combination was C4-D4-A1-B4, indicating UK concentration 800 U/mL, thrombolysis time 40 minutes, transmit power of ultrasound 5%, and microbubble volume 400 μL, respectively. The four factors above had significant influence on thrombolysis (P lt; 0.05), and UK concentration was the most significant. There were significant differences in thrombolysis between different thrombolysis time (P lt; 0.05). ConclusionUnder the condition of fixed ultrasound frequency, microbubble-enhanced sono-thrombolysis efficiency is better in lower transmit power of ultrasound, higher UK concentration, longer thrombolysis time, higher microbubble volume, and shorter thrombolysis time
Objective To observe the effect of BMSCs on the cardiac function in diabetes mellitus (DM) rats through injecting BMSCs into the ventricular wall of the diabetic rats and investigate its mechanism. Methods BMSCs isolated from male SD rats (3-4 months old) were cultured in vitro, and the cells at passage 5 underwent DAPI label ing. Thirty clean grade SD inbred strain male rats weighing about 250 g were randomized into the normal control group (group A), the DM group (group B), and the cell transplantation group (group C). The rats in groups B and C received high fat forage for 4 weeks and the intraperitoneal injection of 30 mg/kg streptozotocin to made the experimental model of type II DM. PBS and DAPI-labeledpassage 5 BMSCs (1 × 105/μL, 160 μL) were injected into the ventricular wall of the rats in groups B and C, respectively. After feeding those rats with high fat forage for another 8 weeks, the apoptosis of myocardial cells was detected by TUNEL, the cardiac function was evaluated with multi-channel physiology recorder, the myocardium APPL1 protein expression was detected by Western blot and immunohistochemistry test, and the NO content was detected by nitrate reductase method. Group C underwent all those tests 16 weeks after taking basic forage. Results In group A, the apoptosis rate was 6.14% ± 0.02%, the AAPL1 level was 2.79 ± 0.32, left ventricular -dP/dt (LV-dP/dt) was (613.27 ± 125.36) mm Hg/s (1 mm Hg=0.133 kPa), the left ventricular end-diastol ic pressure (LVEDP) was (10.06 ± 3.24) mm Hg, and the NO content was (91.54 ± 6.15) nmol/mL. In group B, the apoptosis rate was 45.71% ± 0.04%, the AAPL1 level 1.08 ± 0.24 decreased significantly when compared with group A, the LVdP/ dt was (437.58 ± 117.58) mm Hg/s, the LVEDP was (17.89 ± 2.35) mm Hg, and the NO content was (38.91±8.67) nmol/mL. In group C, the apoptosis rate was 27.43% ± 0.03%, the APPL1 expression level was 2.03 ± 0.22, the LV -dP/dt was (559.38 ± 97.37) mm Hg/ s, the LVEDP was (12.55 ± 2.87) mm Hg, and the NO content was (138.79 ± 7.23) nmol/ mL. For the above mentioned parameters, there was significant difference between group A and group B (P lt; 0.05), and between group B and group C (P lt; 0.05). Conclusion BMSCs transplantation can improve the cardiac function of diabetic rats. Its possible mechanismmay be related to the activation of APPL1 signaling pathway and the increase of NO content.