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.
Abstract: Objective To investigate the effect of autologous bone marrow mesenchymal stem cells (MSCs) transplantation on cardiac function and their proliferation and differentiation in the post-infarct myocardium in rabbits. Methods Twenty New Zealand rabbits were randomly divided into two groups, the autologous bone marrow mesenchymal stem cells group (MSCs group,n=10) and control group (n=10). Myocardial infarct model was set up by ligation of the left anterior descending (LAD), two weeks after establishment of the infarct model,either 400μl of cell suspension (total cells 1×106) labled by 1,1’-dioctadecyl3,3,3’,3’-tetramethyl indocarbocyanine perchlorate (Dil) or a comparable volume of L-DMEM medium were autologously transplanted into several different points of the periphery of the scar respectively. To evaluate the heart function, echocardiography were performed before modeling,two weeks after modeling, 2 and 4 weeks after the cells transplantation for asurements of left ventricular end systolic diameter (LVESD) and left ventricular end diastolic diameter (LVEDD), tocalculate left ventricular eject fraction(LVEF) and left ventricular fractional shortening (LVFS). Meanwhile the myocardial contrast echocardiography (MCE) were performed for evaluating the blood perfusion of the post-infarct myocardium. Eight weeks after the transplantation, the animalswere undergoing euthanasia, specimens were acquired for pathology. Results Echocardiography indicated that:The LVEF and LVFS between two groups were fundamentally the same before modeling,two weeks after modeling respectively (0.72±0.08 vs. 0.71±0.04,0.56±0.11 vs. 0.55±0.09; 0.35±0.06 vs. 0.35±0.04, 0.24±0.08 vs. 0.23±0.03, Pgt;0.05), but those were improved significantly in group MSCs when compared with control group at two weeks and four weeks after the cells transplantation(0.71±0.05 vs. 0.60±0.05，0.72±0.07 vs. 0.62±0.08 and 0.34±0.03 vs. 0.29±0.01， 0.35±0.06 vs. 0.27±0.05 respectively,Plt;0.05). There were no differences in LVESD and LVEDD between two groups in any time points(Pgt;0.05). MCE showed the blood perfusion of the infarct myocardium were improved two and four weeks after the cell transplantation. Pathology indicated that Dil positive cells were survived in MSCs transplanted hearts, stained positively for αsarcomeric actin and desmin eight weeks after cell transplantation, HE slides indicated that the capillary density in all the cells transplanted hearts were much higher when compared with control group (38.6±7.6/mm2 vs. 21.4±3.9/mm2，Plt;0.05). ConclusionMSCs can differentiate into cardiomyocytes, improve myocardial perfusion and cardiac function when transplanted into ischemic myocardium.
To investigate the objective method for electrophysiological examination in evaluating the functional recovery in motor nerve regeneration, 30 rabbits were divided into 5 groups randomly. The common peroneal nerve on left side of every rabbit was sectioned and repaired by epineural suture, while that of the right side was left intact as control. In 2nd, 4th, 8th, 12th and 16th week after operation, the muscle power and the changes of the electrophysiological parameters of the nerve and the muscle were determined dynamically. The linear correlation analysis was used to assess their relationship. The results showed that the electrophysiological parameters and muscle contractibility revealed signs of recovery in parallel. There was a significant linear relationship among the amplitude of the muscle action potential, velocity of nerve-muscle conductivity and muscle contractibility. The conclusion was that the electrophysiological examination of motor nerve and muscle could be used to assess the regeneration of the motor nerve, and it would also reflect the recovery of muscle contactibility in the early stage.
OBJECTIVE Following the delayed repair of peripheral nerve injury, the cell number of anterior horn of the spinal cord and its ultrastructural changes, motorneuron and its electrophysiological changes were investigated. METHODS In 16 rabbits the common peroneal nerves of both sides being transected one year later were divided into four groups randomly: the degeneration group and regeneration of 1, 3 and 5 months groups. Another 4 rabbits were used for control. All transected common peroneal nerves underwent epineural suture except for the degeneration group the electrophysiological examination was carried out at 1, 3 and 5 months postoperatively. Retrograde labelling of the anterior horn cells was demonstrated and the cells were observed under light and electronmicroscope. RESULTS 1. The number of labelled anterior horn cell in the spinal cord was 45% of the normal population after denervation for one year (P lt; 0.01). The number of labelled cells increased steadily from 48% to 57% and 68% of normal values at 1, 3 and 5 months following delayed nerve repair (P lt; 0.01). 2. The ultrastructure of the anterior horn cells of the recover gradually after repair. 3. With the progress of regeneration the latency become shortened, the conduction velocity was increased, the amplitude of action potential was increased. CONCLUSION Following delayed repair of injury of peripheral nerve, the morphology of anterior horn cells of spinal cord and electrophysiological display all revealed evidence of regeneration, thus the late repair of injury of peripheral nerve was valid.
Objective To study the effect of recombinant growth hormone (rhGH) on improvement of liver function and liver regeneration in animal and patients after hepatectomy. Methods The liver cirrhosis model of SD species mouse was set up, then the mouse were randomly divided into experiment group and control group, then 30%-40% liver of all the models were resected, rhGH was used by hypodermic injection (0.2-0.4ml/100g) in experimental group, and the equal dose of N.S. were given in control group every day. Then liver function, arterial blood ketone body ratio(AKBR), and the regenerated liver/body weight ratio (RL/W) were determined, histopathology of the cirrhosis with microscope and electron microscope and the mitotic index (MI) of liver cell on 7, 14 and 28th day after operation were observed. Clinically,39 hepatectomized patients were randomly divided into experiment group and control group, liver function, PA， Glu, RI and AKBR were measured preoperatively and on 1, 7,14th day after operation. Postoperative clinical course were also compared between the two groups. Results In the animal experiment group, as compared with the control group, AKBR was obviously higher (P＜0.01), seruim level of total protein and PA were increased faster (P＜0.05), and RL/W was higher. The mitotic index of liver cell was increased faster on 14th day, the numbers of regenerated liver cell with double nucleus and rough endoplasmic reticulum were higher in 14 and 28th day. In the clinical experiment group, as compared with the control group, serum total bilirubin, alanine aminotransferase and aspartate aminotransferase were lower on 7 and 14th postoperative day (P＜0.05). Serum albumin, PA, Glu, RI and AKBR were higher on 7, 14th postoperative day (P＜0.05). Conclusion Both experimental and clinical study show that the rhGH can promote liver regeneration and improve liver function after hepatectomy.
Objective To investigate the effect of repairing bone defect with tissue engineered bone seeded with the autologous red bone marrow (ARBM) and wrapped by the pedicled fascial flap and provide experimental foundation for cl inicalappl ication. Methods Thirty-two New Zealand white rabbits (male and/or female) aged 4-5 months old and weighing2.0-2.5 kg were used to make the experimental model of bilateral 2 cm defect of the long bone and the periosteum in the radius. The tissue engineered bone was prepared by seeding the ARBM obtained from the rabbits on the osteoinductive absorbing material containing BMP. The left side of the experimental model underwent the implantation of autologous tissue engineered bone serving as the control group (group A). While the right side was designed as the experimental group (group B), one 5 cm × 3 cm fascial flap pedicled on the nameless blood vessel along with its capillary network adjacent to the bone defect was prepared using microsurgical technology, and the autologous tissue engineered bone wrapped by the fascial flap was used to fill the bone defect. At 4, 8, 12, and 16 weeks after operation, X-ray exam, absorbance (A) value test, gross morphology and histology observation, morphology quantitative analysis of bone in the reparative area, vascular image analysis on the boundary area were conducted. Results X-ray films, gross morphology observation, and histology observation: group B was superior to group A in terms of the growth of blood vessel into the implant, the quantity and the speed of the bone trabecula and the cartilage tissue formation, the development of mature bone structure, the remolding of shaft structure, the reopen of marrow cavity, and the absorbance and degradation of the implant. A value: there was significant difference between two groups 8, 12, and 16 weeks after operation (P lt; 0.05), and there were significant differences among those three time points in groups A and B (P lt; 0.05). For the ratio of neonatal trabecula area to the total reparative area, there were significant differences between two groups 4, 8, 12, and 16 weeks after operation (P lt; 0.05), and there were significant differences among those four time points in group B (P lt; 0.05).For the vascular regenerative area in per unit area of the junctional zone, group B was superior to group A 4, 8, 12, and 16 weeks after operation (P lt; 0.05). Conclusion Tissue engineered bone, seeded with the ARBM and wrapped by the pedicled fascial flap, has a sound reparative effect on bone defect due to its dual role of constructing vascularization and inducing membrane guided tissue regeneration.
【Abstract】 Objective To study liver regeneration of the non-ligated liver lobes following portal branch ligation (PBL). Methods Sixty male Wistar rats were randomly divided into PBL group and sham operation (SO) group. Under ether anesthesia, the rats were subjected to PBL and sham operation, respectively. The animals were sacrificed on the 1st, 2nd, 3rd, 7th and 14th day respectively. The blood sample was collected from heart and the livers were harvested to determine serum alanine aminotransferase (ALT) levels and total liver weight, respectively. The hepatic histopathology was studied through light microscopy. The number of liver cell nuclear mitosis index was counted. The number of proliferative cell nuclear antigen (PCNA) index was counted by immunohistochemistry. The hepatic ultrastructural changes were studied under electron microscope. Results ①Elevated serum ALT level was observed in the first postoperative day in PBL group compared with SO group (P＜0.01)， but began to recover in the second day. ②No significant total liver weight change in PBL group and SO group were found. ③Liver cell nuclear mitosis index and PCNA index were markedly increased in PBL group compared with SO group in day 1-3 postoperative day (P＜0.01). It reached the peak in the second day and decreased slightly in the 3rd day, but still higher than SO group, then gradually return to normal lately. Conclusion The ligation of left portal branch can induce active regeneration of hepatic cell of non-ligated liver lobes in rats. The regeneration of non-ligated liver lobes may restore previous total liver weight. The ligation of 75% portal branch does not affect liver function and may be safely performed. The portal branch ligation in rats may be used as an animal model in study of liver regeneration.
ObjectiveTo investigate the effects of bone marrow mesenchymal stem cells (BMSCs) transplantation for treating spinal cord injury (SCI) in rat and the cytokine expression changes in the local injury tissues.
MethodsBMSCs were separated from Sprague Dawley (SD) rat and cultured with the whole bone marrow culture method. rAd-EGFP was used to transfect the 5th generation BMSCs for green fluorescent protein (GFP) label. Twelve SD rats were randomly divided into experimental group (n=6) and control group (n=6). After the T10 SCI model was established with Allen's impact device in 2 groups, 1×106 GFP-labeled BMSCs and PBS were administered by subarachnoid injection in situ in experimental group and control group, respectively. Basso-Beattie-Bresnahan (BBB) score was used to detect the motor function at immediat, 1, 2, 3, 4, and 5 weeks after SCI. At 5 weeks, the spinal cord tissues were harvested for the histological and immunofluorescent staining examinations to measure the expressions of neural marker molecules, including Nestin, glial fibrillary acidic protein (GFAP), and neuron-specific nuclear protein (NeuN). Cytokine was analyzed with antibody array.
ResultsAt 5 weeks, 2 rats died of urinary tract infection in 2 groups respectively, the other rats survived to the end of experiment. BBB score of experimental group was significantly higher than that of control group at 1, 2, 3, 4, and 5 weeks (P < 0.05). At 5 weeks, histological results showed that there were many cells with regular arrangement in the experimental group; there were less cells with irregular arrangement in the control group. Compared with the control group, Nestin and NeuN expressions significantly increased (P < 0.05), and GFAP expression significantly decreased (P < 0.05) in the experimental group. Leptin and ciliary neurotrophic factor levels were higher in the experimental group than the control group, but granulocyte-macrophage colony-stimulating factor, tumor necrosis factorα, interleukin 1β, and tissue inhibitor of metalloproteinases 1 levels were lower in the experimental group than the control group.
ConclusionBMSCs transplantation can improve survival and regeneration of nerve cells and enhances the recovery of nerve function by regulating secretion of cytokines from grafted BMSCs.
Objective To give a prel iminary experimental evidence and to prove chitosan and allogeneic morsel ized bone as potential bone substitutions in repairing rabbit radius segmental defect. Methods Chitosan and allogeneic morsel ized bone were mixed with various ratios (1 ∶ 5, 1 ∶ 10, 1 ∶ 25, 1 ∶ 50, and 1 ∶ 100). After preparation, the physicaland chemical properties of the composites were prel iminary detected; the composites at the ratios of 1 ∶ 50 and 1 ∶ 25 had good physical and chemical properties and were used for the animal experiment. The radius segmental defects of 15 mm in length were made in 50 adult New Zealand white rabbits (weighing 2.5-3.0 kg), then the animals were divided into 2 groups. In groups A and B, chitosan/allogeneic morsel ized bone composites were implanted at the ratio of 1 ∶ 50 and 1 ∶ 25, respectively. After 1, 2, 4, 8, and 12 weeks of operation, the gross, histological, immunohistochemical observations were performed. Before the rabbits were sacrified, X-ray films were taken; the serum calcium and alkal ine phosphatase (ALP) concentration were measured; and the biomechanical measurement was carried out at 12 weeks. Results The results of gross observation were essentially consistent with those of the X-ray films. The histological observation showed that the bone formation was earl ier in group A than in group B; the amount of new bone formation in group A was more than that in group B; and the bone forming area in group A was bigger than that in group B (P lt; 0.05) at 4 and 8 weeks after operation. The immunohistochemical staining showed that vascular endothel ial growth factor and insul in-l ike growth factor receptor II proteins expressed in the cytoplasm of 2 groups after 4 and 8 weeks, and the expression in group A was higher than that in group B (P lt; 0.05). There was no significant difference in the serum calcium concentration between 2 groups at each time point (P gt; 0.05). After 4 and 8 weeks, the ALP concentration in group A was significantly higher than that in group B (P lt; 0.05). After 12 weeks, the radius maximum bending loads of groups A and B were (299.75 ± 27.69) N and (278.54 ± 17.09) N, respectively, showing significant difference (t=4.045,P=0.002). Conclusion The composite of chitosan and allogeneic morsel ized bone has good osteogeneic activity and can beused as a bone tissue engineering scaffold, and the optimum ratio of chitosan to allogeneic morsel ized bone was 1 ∶ 50.
Objective To evaluate the feasibil ity of direct anastomosis in the rat model of the brachial plexus extravertebral foramen nerve root division of C5-7. Methods Forty-eight SD rats (male or female) aging 4-6 months and weighing 250-300 g were selected to make the model of extravertebral foramen nerve root division of C5-7. The left C5-7 nerve roots, as the experimental sides, were separated to the brachial plexus nerve trunk and the transected roots were sutured to theproximal stump immediately after cutting off the brachial plexus extravertebral foramen nerve root division. The right C5-7nerve roots, as the control sides, received no operation. The general condition of rats after operation was observed. The gross observation, the histological observation and BDA nerve tracing technology were adopted to observe the wet weight of musculus biceps brachii, the cross section of biceps brachii and the spinal cord and distal nerve trunk at 3 weeks, 3 months and 6 months after operation. Results All rats survived well after operation. Claudication and unfold claw reflex were observed in the experimental sides, and the unfold claw reflex disappeared 3 months later. Comparatively, the control sides were normal. Nerve adhesion aggravated gradually and the neural stems were shriveled within 6 months after operation in the experimental sides. Comparatively, the control sides were normal. The wet weight of biceps brachii in the experimental sides and the control sides at 3 weeks, 3 months and 6 months after operation was (0.28 ± 0.12), (1.37 ± 0.33), (0.58 ± 0.10), (1.36 ± 0.35), (1.39 ± 0.31), (1.37 ± 0.38) g, respectively, indicating significant differences between two sides at 3 weeks and 3 months (P lt; 0.05), but no significant difference at 6 months (P gt; 0.05). The modified Marsland and the LFB staining of spinal cord and superior trunk of brachial plexus showed that the number of neurons, cell nuclear and Nissl body decreased and cell bodies changed from swell ing to shrinkage, dyeing nerve fibers increased, neural axone was thin and myel in sheath was sl ightly stained at each time point in experimental side. The number of motor neurons in cornu anterius medullae spinal is in the experimental side was 84.5% ± 3.2%, 74.4% ± 4.5%, 73.7% ± 3.8% of that in the control side at each time point, respectively. HE staining of biceps brachii detected thatthe muscle denaturation was very serious at 3 months after operation and then recovered. Neural tracing used BDA showed that the closer to the proximal of nerve trunk, the more obviously stained it was of myel in sheath and the more massive of axon at 6 months after operation. And there was almost no myel in and axon stained in musculocutaneous nerve. Conclusion In the rat model of brachial plexus extravertebral foramen nerve root division, the motoneuron in cornu anterius medullae spinal is necrosis rate reaches 20%-30%, and most of the residual neurons are pathologic. The regenerated fibers manifest as insufficient dynamic power and incomplete development, making no sense for the recovery of end organ function. Therefore, the exact mechanism of the recovery of biceps brachial muscle demands further study.
