【Abstract】ObjectiveTo investigate the protective effects and the impact on the expression of Bcl-2 and Caspase-3 mRNA by Panax notoginseng saponins (PNS) preconditioning in rat liver transplantation. MethodsMale Sprague-Dawley rats were used as donors and recipients of orthotopic liver transplantation (OLT) and were divided into PNS preconditioning group (PNS group) and NS control group (NS group) randomly according to whether PNS was injected by venous (50 mg/kg) 1 h before liver grafts harvesting. The animals were respectively killed 2 h, 6 h and 24 h after reperfusion. Plasma samples were collected for ALT and AST test. Liver tissues were collected to detect histological changes, apoptosis and the expression of Bcl-2, Caspase-3 mRNA. ResultsThe serum levels of ALT and AST and the apoptosis index (AI) of liver tissue in PNS group were lower than those in NS group’s significantly (P＜0.05) at 2 h, 6 h and 24 h after reperfusion. The expression of Bcl-2 mRNA was enhanced significantly in PNS group at 6 h, 24 h after reperfusion and the expression of Caspase-3 mRNA was decreased significantly in PNS group at 2 h, 6 h after reperfusion as compared with NS group’s(P＜0.05). ConclusionPNS preconditioning can attenuate liver grafts ischemia/reperfusion injury and apoptosis of hepatocytes. Affecting expression of Bcl-2 and Caspase-3 genes may be one of the mechanisms of PNS antiapoptotic effects.
Objective To simplify surgical technique andincrease postoperative survival rate, sleeve anastomosis technique combined cuff technique was used in developing the model of cervical heart transplantation in rats. Methods In this model, the hearts from 25 male SD rats were transplanted into the neck of Wistar rats by anastomosing the donor innominate artery to the recipient right common carotid artery by use of sleeve technique, and the donor pulmonary artery to the recipient right external jugular vein by use of cuff technique. After operation,the rats were treated with cyclosporine A (1.5mg/kg, q.d.), transplanted hearts were followed by daily inspection or palpation and the allograft survival time was more than 3 days as the standard of successful operation. Results The mean operative time was (48.7±3.4) min, with a successful rate of 88%(22/25). Complications were anastomotic hemorrhage( 1 case) and thrombosis(2 cases). During the followup period, 6 rats died of pulmonary infection, abscess in the neck,liver or bladder tumor. The remaining 16 transplanted hearts survived more than3 months. Conclusion The modified operation have advantages ofless operative procedure, shorter operation and ischemia time and easier monitoring of graft function.
Objective To provide theoretical evidence for clinical application of the epidermal stem cells after an investigation on changes of the epidermal stem cells during the survival process after the fullthickness skin autograft. Methods On the backs of 42 Wistar rats, orthotopic transplantation models (1.5 cm×1.5 cm) of the fullthickness skin autograft were made. According to the time of the specimen taking, at 1, 3, 5, 7, 14, 21 and 30 days after operation, the rats were randomly divided in 7 groups (Groups 1-7). Specimens taken in each group before operation were used as controls. At each time point, the gross observation was made on the transplanted skin flaps, from which the skin tissues were harvested at each time point before and after operation. The routine pathological and the immunohistochemical examinations were performed on the specimens, which were stained by HE and were observed for immunohistochemical changes and the changes in the cells positive for integrinβ-1 and p63. Results All the fullthickness skin autografts survived 3 days after operation except the skin autograft in 1 rat in both Group 5 and Group 6, which was infected around the transplanted skin flap. In Groups 1-4, cell edema, inflammatory cell infiltration, and increased fibrocytes were observed. In Groups 5-7, the maturity degree of the epithelial cells became higher and higher, and the fibrocyte proportion was lowered. In each group the cell positivity rate for integrin β1 was lower than the cell positivity rate for p63. The positive cells were arranged in disorder, distributed into the layers of the epidermis and gradually concentrated in the basal layer of the epidermis and the bulge of the folliculus pili. The positive cells were also found in the other layers of the epidermis.The positive cells were gradually decreased in number, and reached the lowest level in Group 2. There was a significant difference in the above variables in Groups 1,2,3,5,6 and 7 between before and after operations (P＜0.05). Conclusion During the survival process of the fullthickness skin autograft, the proportion of theepidermal stem cells is gradually decreased at first; Then, the proportion isgradually increased, even beyond the normal level; finally, the proportion is decreased again. The distribution of the epidermal stem cells appear in disorder, almost distributed in the layers of the epidermis; finally, the almost normal distribution can be found.
Objective To investigate the effects of ecdysterone on the survival of the dorsal random-pattern skin flap with large length-to-width ratio in rats and its possible mechanisms. Methods Twenty-four healthy adult SD rats (male and/or female) weighing 200-250 g were randomly divided into the experimental group and the control group (n=12 per group).A caudally based dorsal random pattern skin flap, measuring 8 cm × 2 cm, was symmetrically raised. Ecdysterone (5 mg/kg) and normal sal ine (5 mg/kg) were injected into the abdominal cavity of rats in the experimental group and the control group at 10 minutes before operation and from the first to the fifth day after operation, respectively. The general condition of the rats was observed after operation. At 7 days after operation, the survival rate of the flap was detected, the superoxide dismutase (SOD) activity and the malonyldialdehyde (MDA) level were tested, HE and immunohistochemistry staining observation of the flap were performed. VIII factor dried microvessels in the middle part of the flap (4 cm far away from pedicle) were counted. Results All the rats survived until the end of the experiment. At 7 days after operation, the survival rate of the flap was 62.323% ± 7.046% in the experimental group and 47.753% ± 2.952% in the control group (P lt; 0.001); SOD activity was (54.560 ± 4.535) U/mgprot in the experimental group and (23.962 ± 3.985) U/mgprot in the control group (P lt; 0.001); MDA level was (8.445 ± 0.992) nmol/mgprot in the experimental group and (14.983 ± 0.929) nmol/mgprot in the control group (P lt; 0.001). Histology observation: compared with the control group, the inflammatory cells infiltration was less and the hyperplasia of fibers was more obvious in the experimental group. The microvessel counting in the middle part of the flap was 17.817 ± 2.420 in the experimental group and 8.967 ± 2.000 in the control group (P lt; 0.001). Conclusion Perioperative intraperitoneal injection of ecdysterone can promote the survival of the random-pattern skin flaps with large length-to-width ratio. Its mechanism may be related to its effects of improving SOD activity, decreasing l ipid peroxidation, and promoting angiogenesis of skin flaps.
Objective To explore the possible mechanism of nerve growth factor (NGF) mixed insul in on the angiogenesis of burn wounds and the effect on the expressions of Bcl-2 and Bax in diabetic rats. Methods A total of 75 SPF male Wistar rats, weighing 200-220 g, were selected randomly and divided into nomal control (group A, n=15), the rats with diabetic control (group B, n=15), insul in treatment (group C, n=15), NGF treatment (group D, n=15), NGF and insul in treatment (group E, n=15) groups. In groups B, C, D, and E, streptozotocin was given by intraperitoneal injection at dose of 10 mg/kg on the 1st day and 50 mg/kg on the 3rd day to prepare the diabetic rat models. In group A, citric acid buffer at the samedose was given. After 1 month of diabetic models, second degree scald was made on the back of the rats, and then wounds were treated with 3-layer normal sal ine gauze in groups A and B, with 3-layer gauze containing 5 U Novol in 30R and subcutaneous injection of Novol in 30R (4-6 U/kg) everyday in group C, with 3-layer gauze containing 5 mL NGF (25 U/mL) in group D, and with a combination of groups C and D in group E. At 7, 11, 15, and 21 days, the wound heal ing rate was calculated; at 3, 7, 11, 15, and 21 days, the expressions of Bcl-2, Bax, and CD34 were determined and the microvascular density was measured by immunohistochemistry staining. Results All rats survived till experiment was finished. The area of wounds became smaller gradually with time. Group E was better than other groups in the wound heal ing rate (P lt; 0.05), the skin keratosis, the hair growth, and the granulation tissue and collagen fibers growth. With time, the expressions of CD34 and Bcl-2 increased gradually, reached the peak at 15 days and decreased at 21 days; the expression was ber in group E than in other groups (P lt; 0.05). At 3 days, Bax did not express; at 7 days, Bax began to express in new vascular endothel ial cells and the expression increased gradually with time; the expression was weaker in group E than in other groups (P lt; 0.05). Conclusion A combination of NGF and insul in local appl ication can enhance the angiogenesis of the burn wound in diabetic rats and accelerate wound heal ing by increasing the expression of Bcl-2 and decreasing the expression of Bax and restraining apoptosis of the wounds vascular endothel ial cells of diabetic rats.
Objective To investigate the expression and significance of growth-associated protein 43 (GAP-43) in the dorsal root ganglion (DRG) and intervertebral disc in the rat model of intervertebral disc inflammation. Methods A total of 103 adult male Sprague Dawley rats (weighing, 200-250 g) were randomly divided into the experimental group (n=48), the control group (n=48), and the blank control group (n=7). Fluoro-gold (F-G) as tracer was injected into the L5, 6 intervertebral disc of 3 groups; after 7 days of F-G injection, complete Freund’s adjuvant (50 μL) and the same volume of saline were injected in the experimental group (to prepare the model of intervertebral disc inflammation) and the control group, respectively, and the blank control group had no further treatment. After 1, 3, 7, and 14 days, T13-L6 DRG and L5, 6 intervertebral disc of experimental group and control group were harvested to detect the GAP-43 by using fluorescent immunohistochemistry, in situ hybridization, and RT-PCR. The DRG and intervertebral disc of blank control group were also harvested after 8 days of F-G injection. Results Fluorescent immunohistochemistry results showed that the number of F-G-labeled GAP-43 immunoreaction (GAP-43-IR) cells of the DRGs in the experimental group was significantly higher than that in the control group (P lt; 0.05) at 3 days, and no significant difference was found at the other time points (P gt; 0.05). There was no significant difference in the cross-sectional area of F-G-labeled GAP-43-IR cells between the experimental group and the control group at each time point (P gt; 0.05). The co-expression of GAP-43 with calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4)-binding glycoprotein exhibited that the expression of CGRP was 91.4% ± 7.4% in the control group and was 87.6% ± 7.8% in the experimental group, showing no significant difference between 2 groups (P gt; 0.05). There was no IB4-binding glycoprotein expression in GAP-43-IR cells of the DRGs in 2 groups. The expressions of GAP-43, CGRP, and IB4-positive nerve fibers in the intervertebral disc exhibited that the GAP-43-IR nerve fibers in the experimental group were significantly more than that in the control group (P lt; 0.05), but no significant difference was found in the expression of CGRP between 2 groups (P gt; 0.05); and there was no IB4-binding glycoprotein expression in GAP-43-IR nerve fibers of the intervertebral disc in 2 group. In situ hybridization and RT-PCR detection showed that the positive expression cells ratio of GAP-43 mRNA and the level of GAP- 43 mRNA were significantly higher in the experimental group than in the control group at 1 day (P lt; 0.05), and no significant difference was found at the other time points (P gt; 0.05). Conclusion Intradiscal inflammatory environment may induce the expression of GAP-43, and potentially promote the nerve fiber ingrowth of rat.
Objective To study three-dimensional culturing methods of neonatal rat cardiac myocytes in simulated microgravity. Methods Neonatal rat primary cardiac myocytes were separated and seeded into polylactic acid scaffolds, stirredin spinner flasks for 24 hours, and then moved into rotary cell culture system for three-dimensional culture. The growth of cardiac myocytes was observed underinverted phase contrast microscope, scanning electron microscope and transmission electron microscope, and metabolic assay was assessed by MTT assay. Results Cardiac myocytes with sustained metabolic activity attached to the polylactic acid scaffolds, extended and confluenced. Pulsations of PLAcardiac myocytes was found in some areas. Conclusion The rotary cell culture system is suitable to develop neonatal rat cardiac myocytes culturing for three-dimensional modeling.
A acute partial obstructive hepatocholangitis model by selective ligation and injection of E coli into left hepatic bile duct was successfully founded in rat. Using parameters including mortality, mitochondrial glutamic oxalacetic transaminase and ornithine carbamoytransferase activity, pathological observation and blood culture of bacteria, we evaluated the model. The authors emphasize that this models is superior to the wole-bile-duct-challenged cholangitis model, which is characterized by liver injury.
ObjectiveTo observe the dynamic changes of neuroglobin (NGB) expression in hippocampus after status epilepticus(SE) in rats, and to explore the role of NGB in epileptic seizures.Methods40 healthy male Sprague Dawley rats were randomly divided into two group according to random number table method:control group (n=5) and epilepsy model group(n=35).Epilepsy model group according to observation time was divided into:0h, 1h, 3h, 12h, 24h, 10d and 30d.Intraperitoneal injection Lithium-pilocarpine (20 mg/kg~127 mg/kg, Li-PC) to establish the rat model of SE.Observe the behavioral changes in rats with epilepsy.Nissl staining was used to detect the neuronal damage in hippocampus. Streptavidin-biotin-peroxidase complex immunohistochemical method was used to detect the expression level of NGB in hippocampus;ResultsAfter SE, the neurons in hippocampus were severely damaged with the progress of epileptic seizures, the number of surviving neurons in CA1, CA3 regions showed a near linear decline.Among them, the number of surviving neurons in (12h, 24h, 10d, 30d)CA1, (0h, 12h, 24h, 10d, 30d)CA3 and(12h, 24h, 10d, 30d) DG area were significantly lower than that of the control group (P < 0.05).The expression level of NGB in CA1, CA3 and DG region of hippocampus were increased after SE, and both of CA1 and DG were reached peak in 24h after SE, but was still higher than the control group.And the CA3 area showed a continue rising trend.Among them, CA1(24h, 10d, 30d), CA3(24h, 10d, 30d) and DG(12h, 24h, 10d, 30d) were higher than that of control group significantly (P < 0.05).In addition, it was found that there was a positive correlation between the number of surviving neurons in CA3 area and the expression level of NGB (R=0.306, P=0.011).ConclusionUp-regulation of NGB expression in hippocampus after status epilepticus, and was positively correlated with the number of neurons in the CA3 area, suggesting that up regulation of NGB expression may be a compensatory protective mechanism of ischemic injury induced by seizures, and participate in the protection of epilepsy related neuronal damage.
Objective To investigate effect of the removal of epithelium and mixed glands from the tracheal allografts on the graftimmunosuppression. Methods Fresh untreated tracheal allografts， cryopreserved tracheal allografts, and 10 off-epithelium tracheal allografts were obtained from 25 male SD rats. Fresh untreated tracheal allografts(40) were divided into 4 groups and dipped respectively in the solution of protease ⅩⅣ in 0, 0.1, 0.3 and 0.5 mg/ml at 4℃ for 12 hours. Thirty recipient male SD rats were randomly and equally divided into group A (fresh untreated tracheal allografts), group B(cryopreserved tracheal allografts), and group C(offepithelium tracheal allografts). The transplanted allografts were implanted into the abdominal cavity of other rats by being embedded in the greater omentum. Twenty-one days after transplantation, the tracheal graft segments were surgically removed, and then were initially fixed in cold 10% neutral buffered formalin solution for hematoxylineosin staining. Histological observation and lymphocyte infiltration were performed on the grafts to evaluate rejection. Results The 0.3 mg/ml protease ⅩⅣ could remove the epithelium and mixed glands of the grafts completely, but did no damage to cartilage. The cartilages of each group all survived and were revascularized. The lumens of group A were filled with granulation and necrosis tissue. In contrast, group B was filled with a few granulation tissues and group C was not at all. The number of lymphocyte infiltration in group A, B, and C was 29.16±2.69/HP, 15.17±2.19/HP, and 11.56±0.87/HP respectively. There was significant difference between group A and both group B and group C (Plt;0.05), and there was significant difference between group B and group C (Plt;0.05). Therefore, the grade of graftrejectionwas group Agt;group Bgt;group C. Conclusion The 0.3 mg/ml protease ⅩⅣ can completely remove the epithelium and mixed glands of grafts at 4℃ for 12 hours, and it preserves the normal structure of cartilage. The antigenicity of tracheal grafts can be greatly reduced by removing the epithelium and by the cryopreservation. The prior tracheal allograft in the omentum is feasible for the revascularization of the grafts.