Innervation of syngeneic vein grafts in the rat. The regeneration of cholinesterase positive nerves

The regeneration of cholinesterase positive nerves into segments of the supradiaphragmatic inferior vena cava transplanted from one rat into the abdominal aorta of another rat of the same inbred strain and sex were studied, using a histochemical thiocholine method for the demonstration of cholinesterase. Iso-OMPA and BW-284 C 51 were used as inhibitors for nonspecific cholinesterase (nsChE) and acetylcholinesterase (AChE), respectively. A total of 12 grafts were examined 3, 5, 8, 16, 27 and 32 weeks after transplantation. One single regenerating nsChE reactive nerve was seen near the suture line in a 3-week-old graft. Some solitary regenerating nerves were regularly seen in the grafts at 5 weeks. Eight weeks after grafting some solitary sparsely distributed nerves were found in the outer layers of the graft. In the 16-, 27-, and 32-week-old grafts, nsChE reactive nerves and small nerve bundles were running either solitary or forming nerve plexa in addition to nerves in the vicinity of vasa vasorum. These regenerated nsChE reactive nerves are probably vasomotor in nature. The AChE reaction showed a clear activity in the intimal layer of the grafted veins. No AChE reactive nerves were seen in the 3-, 5-, and 8-week-old grafts. Only occasional AChE positive nerves were observed 16, 28 and 32 weeks after transplantation. These nerves are probably sympathetic cholinergic nerves.     

Connective tissue changes in syngeneic aortal vein grafts in rats

The supradiaphragmatic inferior vena cava was syngeneically transplanted into the infrarenal part of the abdominal aorta in rats of inbred strains. The initial histologic changes coincided with the changes usually seen in wound healing during the inflammatory phase and the phase of fibroplasia. Later the reparative process occurred predominantly in the luminal part of the vein wall, and was usually seen as intimal hyperplasia. In some grafts this process caused a narrowing of the lumen. In this particular experimental model the thickening of the intimal layer was mainly due to organization of fibrin layering thrombus. This was shown by detection of lamellar fibrin deposits and a high concentration of fibronectin in this layer. A large amount of fibronectin was detected in and around the proliferating fibroblasts and in thrombi. This explains the high concentration of fibronectin in the proliferating intimal layer of the graft.     

Autogenous vein and nerve grafts: a comparative study of nerve regeneration in the rat

A new experimental model using the rat sciatic nerve has been devised and used in ten rats to compare the results of using a vein and a nerve graft in different fascicles of the same sciatic nerve, a third fascicle being preserved intact as a control. The results, judged clinically, electrophysiologically and histologically, show persistence of the vein graft as a conduit even six months after the operation and a similar pattern of nerve regeneration in the two different grafts.     

GDNF-transduced Schwann cell grafts enhance regeneration of erectile nerves

Background

Schwann cell–seeded guidance tubes have been shown to promote cavernous nerve regeneration, and the local delivery of neurotrophic factors may additionally enhance nerve regenerative capacity. The present study evaluates whether the transplantation of GDNF-overexpressing Schwann cells may enhance regeneration of bilaterally transected erectile nerves in rats.

Methods

Silicon tubes seeded with either GDNF-overexpressing or GFP-expressing Schwann cells were implanted into the gaps between transected cavernous nerve endings. Six (10 study nerves) or 12 wk (20 study nerves) postoperatively, erectile function was evaluated by relaparotomy, electrical nerve stimulation, and intracavernous pressure recording, followed by ultrastructural evaluation of reconstructed nerves employing bright-field and electron microscopy. Additional animals were either sham-operated (positive control; 20 study nerves) or received bilateral nerve transection without nerve reconstruction (negative control; 20 study nerves).

Results

The combination of GDNF delivery and Schwann cell application promoted an intact erectile response in 90% (9 of 10) of grafted nerves after 6 wk and in 95% (19 of 20) after 12 wk, versus 50% (5 of 10) and 80% (16 of 20) of GFP-expressing Schwann cell grafts (p = 0.02). The functional recovery was paralleled by enhanced axonal regeneration in GDNF-overexpressing Schwann cell grafts, as indicated by larger cross-sectional areas and a significantly higher percentage of neural tissue compared with GFP-transduced controls.

Conclusions

These findings demonstrate that the time required to elicit functional recovery of erectile nerves can be reduced by local delivery of GDNF. In terms of clinical application, this enhanced nerve repair might be critical for timely reinnervation of the corpus cavernosum as a prerequisite for functional recovery in men.     

自体移植静脉中sonic hedgehog表达水平的动物实验研究

目的 研究细胞周期相关因子sonic hedgehog(SHH)在大鼠自体移植静脉中的表达变化及其与内膜增生的关系.方法 雄性Wistar大鼠24只,8周龄,体质量140 g.建立颈静脉-腹主动脉移植模型,分别于术后14 d、28 d各处死12只大鼠,取材移植血管,免疫组化检测SHH蛋白在移植血管新生内膜中的表达,Western blot法观察SHH及细胞增殖核抗原(PCNA)的表达,Real-time PCR定量检测SHH mRNA的表达;以对侧颈静脉作为对照.结果 免疫组化结果显示,正常静脉、术后14 d和术后28 d移植静脉SHH+细胞数比例分别为(2.0±0.5)%、(39.4±0.4)%和(63.0±0.3)%,正常静脉与移植静脉差异有统计学意义(P<0.01).Western blot法显示术后SHH表达水平增加并与PCNA表达呈正相关(r=0.808,P<0.01).Real-time PCR结果显示术后14 d组和术后28 d组的SHH mRNA表达增加,分别为对照组含量的9.5和23.8倍.结论 自体静脉移植术后,SHH在新生内膜中高表达并可能与细胞增殖相关.     

Tissue specificity in rat peripheral nerve regeneration through combined skeletal muscle and vein conduit grafts

Diffusible factors from the distal stumps of transected peripheral nerves exert a neurotropic effect on regenerating nerves in vivo (specificity). This morphological study was designed to investigate the existence of tissue specificity in peripheral nerve fiber regeneration through a graft of vein filled with fresh skeletal muscle. This tubulization technique demonstrated experimental and clinical results similar to those obtained with traditional autologous nerve grafts. Specifically, we used Y-shaped grafts to assess the orientation pattern of regenerating axons in the distal stump tissue. Animal models were divided into four experimental groups. The proximal part of the Y-shaped conduit was sutured to a severed tibial nerve in all experiments. The two distal stumps were sutured to different targets: group A to two intact nerves (tibial and peroneal), group B to an intact nerve and an unvascularized tendon, group C to an intact nerve and a vascularized tendon, and group D to a nerve graft and an unvascularized tendon. Morphological evaluation by light and electron microscopy was conducted in the distal forks of the Y-shaped tube. Data showed that almost all regenerating nerve fibers spontaneously oriented towards the nerve tissue (attached or not to the peripheral innervation field), showing a good morphological pattern of regeneration in both the early and late phases of regeneration. When the distal choice was represented by a tendon (vascularized or not), very few nerve fibers were detected in the corresponding distal fork of the Y-shaped graft. These results show that, using the muscle-vein-combined grafting technique, regenerating axons are able to correctly grow and orientate within the basement membranes of the graft guided by the neurotropic lure of the distal nerve stump.     

Disappearance of catecholamine fluorescence from the adrenergic nerves in arterial grafts in rats: an experimental fluorescence histochemical study.

The disappearance of catecholamine fluorescence from the noradrenaline-containing sympathetic nerve fibres after arterial transplantation was studied using a femoral artery graft sutured to rat carotid artery. Glyoxylic acid-induced fluorescence was used to demonstrate adrenergic nerves histochemically. At six hours the network of fibres had started to degenerate, and catecholamine fluorescence from the adrenergic nerves had almost completely disappeared within 24 hours of grafting. Control specimens from normal femoral arteries showed a dense network of fluorescent adrenergic nerves. Based on observations of the relatively rapid liberation of catecholamines from the degenerating adrenergic nerves, we suggest that catecholamines liberated from degenerating adrenergic nerves may have an important role in early vasospasm in microvascular and coronary bypass surgery.     

Innervation of the rat urinary bladder. I. The morphological changes of intrinsic nerves of the urinary bladder after pelvic ganglionectomy

The dual autonomic innervation of the urinary bladder of the female rat was studied using histochemical and light and electron microscopic methods. The bladder body in the normal state had a rich supply of cholinergic nerve fibers with a uniformly reticular pattern, but it was sparsely supplied with adrenergic nerves mainly associated with the blood vessels rather than the detrusor muscles. Seven days after the unilateral extirpation of the pelvic ganglion, most cholinergic and adrenergic fibers on the ipsilateral side of the bladder body disappeared markedly with the use of enzymatic and fluorescent histochemical techniques. The acetylcholinesterase activity of cholinergic fibers and the intensity of fluorescence of adrenergic fibers were somewhat restored 14 days after the operation. Seventy days postoperatively, it was found that the former had recovered in the relatively fine nerve fibers, while the latter had been almost completely restored, compared with the normal adrenergic innervation of the bladder body.     

Innervation of skin grafts over free muscle flaps.

Skin grafts regain their sensory innervation from the graft bed by the regeneration of nerve endings. Although some clinical studies report sensory recovery in skin grafts implanted on free muscle flaps, the mechanism of recovery is obscure. The purpose of this study was to investigate nerve regeneration in experimental skin grafts on free muscle flaps to elucidate this phenomenon. Thirty-eight male Sprague-Dawley rats, weighing 450-550 g were used in the study. The rat gracilis muscle flap was the free flap model transferred from one groin to the other using microvascular anastomoses. Full-thickness skin grafts harvested from the abdomen were used to cover the free muscle flaps after transfer. Four study groups were formed: Group I (n = 10): Free muscle flaps were transferred without any nerve anastomosis; Group II (n = 10): Free flaps transferred with the anastomosis of the muscle's motor nerve to a sensory nerve at the recipient site; Group III (n = 10): Free flaps transferred with the anastomosis of the muscle's motor nerve to a motor nerve at the recipient site; Group IV (n = 8): Skin grafts were placed directly on the fascia layer over the medial hindlimb muscles and served as controls. The specimens were harvested for histologic examination after 12 weeks. Histologic examination was performed to visualise regenerating nerve endings using H&E, S100, Luxol Fast Blue and tyrosine hydroxylase staining. The specimens were categorically scored according to the staining pattern of neural structures around pilosebaceous units and statistical comparisons were performed by using paired t-test. Skin grafts in both Group II and Group III markedly received tyrosine hydroxylase at the base of their pilosebaceous units in many of the specimens and functional nerve twigs could also be traced from the muscle layer to the overlying skin graft. In contrast, the skin grafts in Group I did not show any nerve function in the central parts. The overall staining scores of Groups II, III and IV were significantly higher than Group I (P < 0.05; P < 0.001; P < 0.05, respectively). There was no statistically significant difference between other groups. No myelinated nerve fibres could be detected in any of the skin grafts with Luxol Fast Blue technique. It was concluded in the present study that skin grafts over reinnervated free muscle flaps can develop significantly better innervation than skin grafts over non-innervated muscle flaps. However, the activity in skin appendages indicating nerve regeneration may only imply a gross sensation and in the absence of any myelinated nerve fibres transmission of finer sensation cannot be expected in any of the study groups.     

Locally-applied collagenase and regeneration of transsected and repaired rat sciatic nerves.

The possible effects of collagenase on peripheral nerve regeneration were evaluated after epineurial repair of rat sciatic nerves. In the control group the repair site was covered by fibrin adhesive and infused with isotonic saline and in the experimental group collagenase was infused into the fibrin adhesive. In the short term study the regeneration distance was measured by a pinch test four, six, or eight days postoperatively. In the long term study the evaluation of nerve regeneration and recovery of motor function was made by testing the tetanic contraction force of the anterior tibial muscle three months postoperatively. There were no significant differences between the two groups in either the short or long term. We conclude that locally-applied collagenase had no effect on peripheral nerve regeneration.     

The influence of bacterial collagenase on regeneration of severed rat sciatic nerves

Summary Regeneration of peripheral nerve fibers is impeded by the formation of scar tissue at the site of injury. The possible beneficial effect of collagenase on nerve regeneration was studied using clinical, neurophysiological (evoked potentials) and histological (nerve fiber counts) methods.The sciatic nerves of rats were transected and the severed ends abutted and sewn together. In one series, the area about the lesion was covered with fibrin adhesive and infused with either isotonic saline (controls) or collagenase (treatment group). In the other series, the severed ends of the nerve were inserted into a silicone tube and separated by a collagen plug, which was infused with either saline or collagenase. Compared to the controls, the treated animals showed a significant improvement of clinical and neurophysiological parameters. After 3 months of observation, the collagen content of the transection site was reduced, and in the silicone series, the total number of myelinated axons 5mm distal to the site of transection was increased, while the fiber diameter distribution was unchanged.Supported by a grant from the Deutsche Forschungsgemeinschaft (We 1248).     

Reinnervation of syngeneic pancreatico-duodenal grafts in rats

BACKGROUND: Knowledge on the reinnervation of transplanted organs is scarce, and the aim of the study was therefore to evaluate to what degree syngeneic pancreas grafts were reinnervated in rats. METHODS: Syngeneic pancreatico-duodenal transplantations were performed in normoglycemic Wistar-Furth rats. Native and transplanted pancreas and duodenum were removed 4 or 40 weeks after implantation, and processed for indirect immunofluorescence using antibodies directed against vasoactive intestinal peptide, substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), tyrosine hydroxylase (TH), or the general neuronal marker protein gene product 9.5. RESULTS: Four weeks after transplantation a moderate to rich number of protein gene product 9.5-positive nerve fibers were found homogeneously distributed through the pancreas, probably representing the intrapancreatic nervous system, because the grafted pancreas lacked both a sympathetic (TH/NPY) and sensory (SP/CGRP) innervation 4 weeks after implantation. In a few of the animals there was a marked increase in SP-immunoreactive nerves (lacking CGRP), most conspicuous in the duodenal portion, both 4 and 40 weeks after transplantation probably secondary to a chronic pancreatitis. The fibers seemed to emanate from intrapancreatic ganglia and possibly also from enteric neurons in adjacent parts of the duodenum. A few scattered vasoactive intestinal peptide-containing nerve fibers probably also emanating from local ganglia could be seen throughout the grafted pancreas both 4 and 40 weeks after transplantation. At 40 weeks after transplantation sympathetic (TH- and NPY-positive) nerve fibers were regularly seen, whereas CGRP-positive nerve fibers were still virtually lacking in the pancreas. To trace the origin of the ingrowing nerve fibers, the tracer True Blue was injected into the grafted pancreas of some rats 38 weeks after transplantation, i.e., 2 weeks before killing. True Blue-labeled nerve cell bodies were numerous in the celiac ganglion (presumably sympathetic nerves) and few in dorsal root ganglia (sensory nerves). CONCLUSIONS: The data suggest that the transplanted rat pancreas becomes reinnervated by mainly sympathetic nerve fibers.     

Endothelialization of microporous polytetrafluoroethylene grafts in the infrarenal aorta and caval vein of the rat.

To study healing and endothelialization of vascular grafts, microporous polytetrafluoroethylene (PTFE) prostheses 2 mm in inner diameter and 5 mm long were implanted into the infrarenal aorta or caval vein of the rat. Patency was assessed in six rats from each group at days 3, 7, 14, 28, and 56 after implantation. Four grafts were occluded, two in the aorta (56 days) and two in the caval vein (3 and 14 days). The prostheses were examined via scanning electron and light microscopy for evaluation of endothelialization. At 3 days, the inner surface of the aortic grafts was covered by a plasma proteinaceous layer and that of the caval vein grafts by a thin mural thrombus. Endothelial cells then migrated from aorta/caval vein edges over the graft. At 14 days, the caval vein grafts were completely re-endothelialized, and, at 28 days, the mural thrombus in these grafts was replaced by neointima. In contrast, endothelialization of the aortic grafts had advanced only 1 mm at about 56 days, never forming a complete endothelial layer. We conclude that endothelialization of microporous PTFE prostheses is more rapid and complete in the caval vein than in the aorta of the rat.     

门静脉动脉化对大鼠肝脏再生的影响

目的探讨门静脉动脉化重建肝血流后对肝脏再生的影响。方法建立门静脉动脉化重建肝脏血流加半肝切除(43%)的大鼠实验模型,分别在术后3 d和10 d取出肝脏烘干称重、光镜下计数进入有丝分裂期的肝细胞和分离肝细胞进行流式细胞仪分析,以观察肝脏再生的情况。结果实验组术后3 d和10 d测定的肝脏干重分别为(67.56±3.70)%(、78.76±5.68)%,与对照组(71.66±3.24)%(、82.38±4.86)%相比无显著性差异(P>0.05);进入有丝分裂期的肝细胞计数(708.4±68.21、239.6±24.50)与对照组(724.8±69.99、216.2±23.81)相比无显著性差异(P>0.05);流式细胞仪测得的进入G2和M期的肝细胞的DNA含量[(25.72±4.78)%、(15.60±2.52)%]与对照组[(28.78±3.37)%、(13.34±2.88)%]相比无显著性差异(P>0.05)。结论行门静脉动脉化重建肝血流不影响肝脏的再生。