Pancreatic tissue grafts are reinnervated by neuro-peptidergic and cholinergic nerves within five days of transplantation

The reinnervation process is crucial for the survival and functioning of cell, tissue or organ transplants. This study was designed to examine the exact time of reinnervation of intraocular pancreatic tissue transplants in rats. The rate of survival of neuropeptide-containing cells in pancreatic tissue grafts was also investigated. Calcitonin gene-related peptide (CGRP), galanin (GAL), neuropeptide Y (NPY) were observed in the surviving nerve cell bodies of the grafts. The iridal nerves reinnervating the pancreatic grafts expressed CGRP, GAL, NPY and choline-acetyl-transferase (ChAT) on day 5, and tyrosine hydroxylase (TH) and nitric oxide synthase (bNOS) on day 6 of the transplantation period. The expression of CGRP in the reinnervating nerves was more consistent when compared to GAL, NPY, ChAT, TH and bNOS. Although all of the three neuropeptides (CGRP, GAL, NPY) were present in the surviving nerve cell bodies of the pancreatic tissue graft up to the end (day 9) of the transplantation period, the number of CGRP-immunopositive cells was consistently higher throughout the transplantation period. Hence, the number of CGRP-positive cells in the pancreatic tissue graft was significantly (P < 0.05) higher than that of GAL and NPY. In conclusion, pancreatic fragments were reinnervated by neuropeptidergic (CGRP, NPY) and cholinergic (ChAT) nerves within the first 5 days of transplantation. In addition to the reinnervation of pancreatic tissue grafts, the intrinsic neurones of the grafts also survived after transplantation. The rate of survival of CGRP-containing cells in the pancreatic tissue grafts was more consistent compared to that of NPY and GAL.     

Reinnervation of syngeneic mouse pancreatic islets transplanted into renal subcapsular space.

A syngeneic transplantation of 150 islets into the subcapsular renal space was performed on normoglycemic or alloxan-induced diabetic male C57BL/6 mice. Six, 8, 14, or 20-21 wk after transplantation, the graft-bearing kidney was removed and processed for microscopical examinations with indirect immunofluorescence for neuropeptides and tyrosine hydroxylase, and with acetylcholinesterase staining to visualize nerve fibers within the graft. Six weeks after implantation, only a few scattered nerve fibers were observed within the grafts. A progressive increase in the number of nerves was observed until 14 wk after transplantation, after which, a stable level was reached. Alloxan-induced diabetic mice showed quantitatively and qualitatively similar reinnervation to normoglycemic mice 20 wk after transplantation. The findings demonstrate the presence of sympathetic nerve fibers (containing tyrosine hydroxylase and neuropeptide Y), mainly accompanying ingrowing blood vessels; parasympathetic nerve fibers (containing acetylcholinesterase and vasoactive intestinal peptide), possibly reaching the graft from the adjacent renal capsule; and afferent nerve fibers (containing substance P and calcitonin gene-related peptide), which were less numerous. The data suggest that transplanted islets become reinnervated by ingrowth of nerve fibers from the implantation organ and that several types of nerves are present.     

NPY hyperinnervation in Hirschsprung's disease: both adrenergic and nonadrenergic fibers contribute.

In Hirschsprung's disease, the aganglionic bowel is characterized by an absence of ganglion cells and an increased number of adrenergic and presumed cholinergic nerve fibers. In addition, a severe derangement of peptide-containing nerve fibers is encountered including a hyperinnervation of neuropeptide Y (NPY)-containing fibers. Using immunochemical and immunocytochemical methods, we examined the nature of the NPY-containing nerve fibers contributing to the hyperinnervation. The concentration of NPY was markedly increased in the aganglionic segment. Coexistence of NPY, vasoactive intestinal peptide (VIP), and the adrenergic enzyme tyrosine hydroxylase (TH) showed small populations of nerve fibers containing NPY/TH, NPY/VIP, or TH alone in ganglionic intestine. Numerous nerve fibers stored VIP but lacked NPY. These fibers did not contain TH, indicating that all VIP-containing fibers are nonadrenergic. In the aganglionic intestine there was a marked increase in the number of nerve fibers storing NPY/TH and NPY/VIP, whereas the fibers storing VIP alone were reduced in number. A small number of nerve fibers storing NPY alone occurred in the hypertrophic nerve bundles. NPY/VIP-containing nerve fibers were particularly numerous in the mucosa in aganglionic intestine, which may be of interest in the diagnosis of Hirschsprung's disease allowing the use of mucosal biopsy specimens. Thus, the proliferating NPY-containing nerve fibers in the aganglionic intestine seem to comprise three different populations, one adrenergic and two nonadrenergic, one of which contains in addition VIP.     

Immunohistochemical study of intralaryngeal ganglia in the cat.

To study the mechanism of autonomic regulation in the larynx, intralaryngeal local ganglia of the cat were investigated using immunohistochemical techniques. Small intralaryngeal ganglia were found in the peripheral portions of internal branches of the superior laryngeal nerve. Ninety-one percent of the ganglionic neurons were immunoreactive (IR) to vasoactive intestinal polypeptide (VIP), and 10% of the VIP-IR cells were also immunoreactive to enkephalin (ENK) and/or substance P (SP). The immunoreactivity of neuronal cell bodies remained unchanged even after denervation of the bilateral superior and recurrent laryngeal nerves. A dense distribution of calcitonin gene-related peptide (CGRP)-IR nerve fibers was found around almost all neuronal cells in the intralaryngeal ganglia. A few VIP-IR, ENK-IR, and SP-IR nerve fibers were also observed. Only the CGRP-IR fibers disappeared after the denervation experiments. In the laryngeal glands and mucosal arterioles, VIP-IR nerve terminals were found that were also immunoreactive to ENK and/or SP. However, these immunoreactive nerve endings in the glands and arterioles remained after the denervation experiments. The results of our study indicate that laryngeal exocrine secretion and blood flow are regulated by postganglionic autonomic parasympathetic fibers from intralaryngeal ganglia that contain VIP alone or VIP with ENK and/or SP, and that these ganglionic neurons may be innervated by CGRP-IR extrinsic nerve fibers.     

Reinnervation after nerve suture in rat groin flaps

Regeneration of sensory and adrenergic nerves in the skin was studied in rats. The aim was to investigate the effect of reanastomosing the cut nerve ends of the nerve trunk leading to the microvascular groin flap. Reinnervation was demonstrated immunohistochemically using calcitonin gene-related peptide (CGRP) as marker for sensory nerves, neuropeptide Y (NPY) and tyrosine hydroxylase (TH) as markers for adrenergic nerves and Protein Gene Product 9.5 (PGP 9.5) as general neuronal marker. It was demonstrated that reanastomosing of the nerve trunk was favourable for both the sensory and sympathetic reinnervation of microsurgical flaps. © 1998 Wiley-Liss, Inc. MICROSURGERY 18:1–5, 1998.     

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.     

Xenografting human T2 sympathetic ganglion from hyperhidrotic patients provides short-term restoration of catecholaminergic functions in hemiparkinsonian athymic rats

Previous studies have suggested that allografting peripheral sympathetic ganglia, such as superior cervical ganglia, partially relieves clinical or behavioral deficits in parkinsonian patients and animals. However, removal of these ganglia can cause Homer's syndrome, which limits the utilization of this approach. Hyperhidrosis, a disease of excessive sweating, is commonly seen in young Orientals. Treatment of hyperhidrosis often involves surgical removal of the second thoracic sympathetic ganglia (T2G), which contain catecholaminergic neurons. The purpose of our study was to investigate behavioral responses and tyrosine hydroxylase (TH) immunoreactivity in hemiparkinsonian rats at different time points after transplantation of human T2G from hyperhidrotic patients. Athymic Fisher 344 rats were injected unilaterally with 6-hydroxydopamine into the medial forebrain bundle to destroy the nigrostriatal dopaminergic (DA) pathway. The effectiveness of lesions was tested by measuring methamphetamine (MA)-induced rotations. These unilaterally lesioned rats were later transplanted with T2G or T2 fiber tract (T2F) obtained from adult hyperhidrotic patients. Animals grafted with T2G showed a reduction in MA-induced rotation by 2 weeks; however, rotation returned to the pregrafting levels by 3 months. Animals receiving T2F grafts did not show any reduction of rotation over a 3-month period. Animals were later sacrificed for TH immunostaining at different time points. Tyrosine hydroxylase-positive [TH(+)] cell bodies and fibers were found in the lesioned striatum 2-4 weeks after T2G grafting, suggesting the survival of transplants. Two to 3 months after grafting, TH(+) fibers were still found in almost all the recipients. However, TH(+) cell bodies were found in only three of seven rats studied. Animals receiving T2F grafting did not show any TH immunoreactivity in the lesioned striatum over the 3-month period. These data indicate that T2G transplants from adult hyperhidrotic patients can survive and provide transient normalization of the motor behavior in the hemiparkinsonian athymic rats. Because of the short-term improvement in behavior after grafting, the use of T2G in human trials should be cautious at the present time. Further laboratory research is required.     

Neuropeptide Y, calcitonin gene-related peptide, and galanin in Hirschsprung's disease: an immunocytochemical study

The aganglionic intestinal segment in Hirschsprung's disease is known to contain a reduced number of nerve fibers storing vasoactive intestinal peptide (VIP), substance P (SP), enkephalin, and gastrin-releasing peptide (GRP). In this study, nerves containing three newly described neuropeptides: neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), and galanin were examined using immunocytochemistry. Nerve fibers displaying NPY immunoreactivity were found to be more frequent in the aganglionic than in nonafflicted ganglionic intestine. Nerve fibers storing CGRP and galanin on the other hand were roughly equally frequent but the distribution pattern differed in that the bulk of fibers in the aganglionic intestine was localized to large nerve trunks not seen in the ganglionic segment. The functional significance of these changes has yet to be defined.     

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

Regeneration of adrenergic nerves was studied sequentially in segments of the supradiaphragmatic inferior vena cava transplanted from one rat into the abdominal aorta of another of the same inbred strain. Nontransplanted vein segments were examined as controls. The adrenergic nerves were demonstrated by using the glyoxylic acid-induced fluorescence method and the formaldehyde-induced fluorescence method for the demonstration of cathecholamines. A total of 16 syngeneic grafts were examined 3, 5, 8, 16, 27, and 32 weeks after transplantation. Three additional grafts were wrapped inside a silicone tube during the operation to prevent regeneration of nerves from the surroundings. These rats were then killed 8 weeks after the operation. In the control specimens the supradiaphragmatic inferior vena cava was innervated with a rather dense plexus of adrenergic nerves showing varicosities. No nerves were seen in the 3-week-old grafts. In the 5-week-old grafts some sparsely distributed solitary nerve fibres were seen. In the 8-week-old grafts sparsely distributed regenerated nerves reinnervating the vasa vasorum and nerve plexa were observed. The 16-, 27-, and 32-week-old grafts showed rather poorly innervated areas and areas where the nerve plexus was dense. One graft showed very dense reinnervation with morphologically abnormal adrenergic nerves forming “droplet fibers” and showing dense accumulations of catecholamines. The grafts surrounded with silicone tubes had no adrenergic nerves and showed only some regenerated nerves crossing the suture line. The results of the present study indicated that syngeneic inferior vena cava transplanted into the abdominal aorta will be reinnervated with adrenergic nerves. The pattern of the nerve regeneration remains incomplete and patchy.     

Autonomic neuropeptides in the interface membrane of aseptic loose hip prostheses.

We analyzed the presence of autonomic nerve fibers in the interface membranes (n = 9) surrounding aseptic loosened hip prostheses by immunohistochemistry. The study focused on the autonomic messengers neuropeptide Y (NPY), tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of noradrenaline (NA), and vasoactive intestinal polypeptide (VIP). Protein gene product (PGP) 9.5, a general marker of peripheral nerve fibers, was also analyzed to establish the neuronal character of the immunoreactive structures. PGP 9.5-positive and NPY-positive nerve fibers were identified in all 9 samples, and VIP-immunoreactive and TH-immunoreactive fibers were found in 7. There was a difference in the distribution of nerve fibers both between and within the samples. Among the neuropeptides analyzed, NPY was most abundant. NPY-positive and TH-positive fibers were predominantly found around the blood vessel walls forming varicose nerve terminals. VIP-positive fibers were mainly observed as thin varicose nerve terminals with no relationship to blood vessels. Autonomic neuropeptides exert not only vasoactive and immunoregulatory effects, but also have been found to have direct effects on bone tissue. Moreover, the autonomic nervous system has been strongly implicated in nociception and inflammation. Neuronal NPY, TH, and VIP in the interface membrane may prove to contribute to the pathologic mechanisms leading to aseptic loosening of hip prostheses.     

Immunohistochemical determination of the sympathetic pathway in the orbit via the cranial nerves in humans

OBJECT: The present study was undertaken to elucidate the extent and precise distribution of the postganglionic sympathetic fibers in the cranial nerves projecting to the orbit and to reconstruct sympathetic routes in the orbit in humans. For this purpose, the authors made an immunohistochemical determination of the sympathetic fibers by using an antibody against norepinephrine-synthetic enzyme, tyrosine hydroxylase (TH). METHODS: Specimens containing the orbit and the cavernous sinus were obtained from formalin-fixed human cadavers. First, it was confirmed that the superior cervical ganglion contained strongly immunostained TH-positive neuronal cell bodies and fibers. After careful dissection of the cranial nerves projecting to the orbit, different segments of each cranial nerve were processed for immunohistochemical analysis for TH. All of the intraorbital cranial nerves contained TH-positive sympathetic fibers, although the amounts were very different in each cranial nerve. At the proximal site of the common tendinous ring, TH-positive fibers were found mainly in the abducent and trochlear nerves. At the distal site of this ring, TH-positive fibers were lost or markedly reduced in number in the abducent and trochlear nerves and were distributed mostly in the ophthalmic and oculomotor nerves. Among the cranial nerves projecting to the orbit, the ophthalmic nerve and its bifurcated nerves--frontal, lacrimal, and nasociliary--contained numerous TH-positive fibers. CONCLUSIONS: The authors conclude that the postganglionic sympathetic fibers are distributed to all cranial nerves projecting to the orbit and that the ophthalmic nerve provides a major sympathetic route in the orbital cavity in humans.     

Nerve reinnervation and itch behavior in a rat burn wound model

In this study, we investigated whether postburn itch in rats, after a full thickness burn, is correlated to the nervous reinnervation of the burn wound area. For this purpose, we determined scratching duration (expressed as second/hour) at 24 hours, 2, 4, 8, and 12 weeks postburn and combined this with immunohistochemistry for protein gene product 9.5 (PGP9.5) to identify all nerve fibers, calcitonin gene related peptide (CGRP) to identify peptidergic fibers, tyrosine hydroxylase (TH) for sympathetic fibers, and growth‐associated protein 43 (GAP‐43) for regrowing fibers. We found a modest, but highly significant, increase in scratching duration of all burn wound rats from 3 to 12 weeks postburn (maximally 63 ± 9.5 second/hour compared to sham 3.1 ± 1.4 second/hour at 9 weeks). At 24 hours postburn, all nerve fibers had disappeared from the burn area. Around 4 weeks postburn PGP 9.5‐ and CGRP‐immunoreactive nerve fibers returned to control levels. TH‐ and GAP‐43‐IR nerve fibers, which we found to be almost completely colocalized, did not regrow. No correlation was found between scratching duration and nervous reinnervation of the skin. The present results suggest that in rat, like in human, burn wound healing will induce increased scratching, which is not correlated to the appearance of nervous reinnervation.     

Noradrenergic and cholinergic reinnervation of islet grafts in diabetic rats

Grafted islets become denervated due to the islet transplantation procedure. The aim of the present study was 1) to examine whether islet grafts in the liver, the spleen, and under the kidney capsule in rats become reinnervated following the transplantation and experimental procedures used in our laboratory, 2) whether there is any difference in reinnervation at these different sites, and 3) how these results relate to previous physiological experiments. Isogeneic isolated islets were transplanted into diabetic Albino Oxford rats, resulting in normoglycaemia. After at least 5 wk, graft-receiving organs were removed and several antibodies were employed to detect insulin, neuronspecific proteins, and cholinergic and noradrenergic nerve fibers. Islets in all three receiving organs contained viable insulin-positive B-cells. Neuron-specific enolase (NSE) as well as the growth-associated protein B-50 was observed at all sites. The cholinergic marker choline acetyltransferase (ChAT) was localized in islets grafts at all sites, but with the lowest density in the spleen. Staining for the noradrenergic markers tyrosine hydroxylase (TH) and dopamine-phydroxylase (DBH) was observed in islet grafts at all sites with the lowest density in grafts under the kidney capsule. All these neurochemical substances were most frequently observed in fibers associated with blood vessels, which may be the route along which nerves grow into the graft. It can be concluded that 1) islet grafts in the liver, in the spleen and under the kidney capsule become reinnervated; 2) the innervation pattern of the islet grafts differs only slightly from that in the control pancreatic islets; and 3) in combination with our previously physiological data, we can conclude that these nerve fibers are, at least partly, functionally active.     

Changes in vasoconstrictor and vasodilator neurotransmitters in nerves supplying arterioles in developing colorectal polyps

OBJECTIVE: To examine the changes that occur in the immunohistochemistry of vasoconstrictor and vasodilator transmitters in nerves supplying early and advanced colorectal polyps. SUBJECTS AND METHODS: We studied the perivascular innervation of submucosal arterioles of colorectal polyps (n = 18) and the innervation of the epithelial layer of polyps compared to normal controls (n=8), using immunohistochemical markers for the neurotransmitters; noradrenaline (NA) (marker used; tyrosine hydroxylase (TH)), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP), and calcitonin gene-related polypeptide (CGRP). (Advanced polyps; villous adenomas>1.5 cm, polyps with severe dysplasia or partial carcinoma). RESULTS: In submucosal arterioles there was a progressive decrease from controls through early polyps to advanced polyps in TH and NPY perivascular immunoreactivity (P<0.015 for both). VIP and SP immunoreactivity was higher in early polyps compared to controls, but markedly reduced in advanced polyps (P<0.05 for VIP). Sparse CGRP immunoreactivity was present in polyps only. Neural VIP and SP immunoreactivity in the lamina propria of polyp mucosa was more intense than in controls. CONCLUSION: There is a decrease in vasoconstrictor neurotransmitters NPY and NA (shown by TH) around submucosal arterioles of both early and advanced polyps, but an increase in the vasodilator neurotransmitters, particularly VIP, in early colorectal polyps. These results suggest a predominantly vasodilatory neural influence in early polyps, perhaps indicating a mechanism that maintains polyp growth.     

改良颈7神经移位术后支配肌功能恢复的实验研究

目的 观察利用C7神经同时修复2组受损神经后的功能恢复情况。方法 建立大鼠C7改良移位术（同时修复2组受损神经）动物模型，并与传统单组移位术后神经再支配肌的功能进行比较。结果 术后早期（2～6周）C7改良移位组肌肉功能劣于同时间段单组移位组，但术后8周起逐渐接近单组移位和正常对照组。结论 大鼠C7改良移位术不仅能达到传统C7单组移位术的功能恢复效果，又能同时多恢复1组受损神经的功能。     

Autonomic innervation of tendons, ligaments and joint capsules. A morphologic and quantitative study in the rat.

We analyzed the neuronal occurrence of autonomic transmitters; noradrenaline (NA), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), in the Achilles tendon, medial and lateral collateral ligaments and knee joint capsule in the rat--by immunohistochemistry (IHC). In addition, the tissue concentrations of the sympathetic neuropeptide, NPY, and the parasympathetic peptide, VIP, were determined by radioimmunoassay (RIA). IHC demonstrated nerve fibers containing sympathetic vasoconstrictors--NA and NPY--and the parasympathetic vasodilator, VIP, in all tissues. NPY- and NA-positive nerve fibers were predominantly observed in larger blood vessels, whereas, nerve fibers immunoreactive to VIP were found in smaller vessels. In many nerve fibers a co-localization of the transmitters was seen. RIA showed that the concentration of NPY compared to VIP was 15-times higher in ligaments and twice as high in tendons and capsules. The differences noted may reflect a difference in vulnerability to degenerative conditions. In pathological conditions, dysregulation of autonomic transmitters in hypovascularized tissues subjected to repetitive mechanical load may contribute to tissue hypoxia leading to degeneration and rupture of tendons and ligaments.     

Glycerol injection to the rat trigeminal nerve: Histological and immunohistochemical studies

Summary The effect of topical glycerol injection into the rat trigeminal nerves was investigated histologically and immunohistochemically. Anhydrous glycerol was injected into the preganglionic portion of the trigeminal nerves via a ventral approach. Extensive myelin swelling and axonolysis were observed in the rats killed 1 and 2 weeks after glycerol injection. Numerous inflammatory cells were seen especially in the animals sacrificed 1 week after surgery. Myelin disintegration continued up to 4 weeks after glycerol injection. In normal and saline injected sham operated nerves, calcitonin gene-related peptide (CGRP)- and substance P(SP)-like immunoreactivities were densely localized in the nerve fibers. A marked decrease in both CGRP- and SP-like immunofluorescence was seen in the nerves after glycerol injection. The remaining nerve fibers often had blunt endings with increased fluorescence. Swollen and winding structures were also found. These immunohistochemical changes were observed in the rats killed 1 and 2 weeks following surgery. A similar change but of lesser degree was seen in the 4-week-animal. The present study suggests that topical glycerol injection into the trigeminal nerve induces degeneration of the nerves immunoreactive to CGRP and SP. These changes emphasize the putative functional implications of the peptides in relieving the pain of trigeminal neuralgia after topical glycerol injection.     

Peptide-containing nerves in human urinary bladder

Nerves containing immunoreactive vasoactive intestinal polypeptide (VIP), substance P and two newly discovered peptides, neuropeptide tyrosine (NPY) and PHI (peptide having N-terminal histidine and C-terminal isoleucine), have been found in the human urinary bladder by immunocytochemistry and radioimmunoassay. Somatostatin immunoreactivity was detected by radio-immunoassay. The VIP-immunoreactive nerves were widely distributed in all regions, but were particularly dense beneath the epithelium and in the muscle layer. Scattered intramural ganglia were found to be reactive to VIP antiserum. Higher concentrations of extractable VIP were detected in the trigone than in the dome. VIP- and PHI-immunoreactive nerves were similarly distributed, the latter being less numerous. NPY immunoreactive nerves were seen mainly in the muscle layer, particularly in the trigonal area. The distribution patterns of VIP- and NPY-immunoreactive nerves resembled those of the previously reported cholinergic and adrenergic nerves, respectively. Many blood vessels were found to be innervated by both types of immunoreactive nerves. Scattered substance P-immunoreactive fibers were occasionally seen, being present in the submucosa and around the detrusor muscles. The significance of these nerves remains to be elucidated.     

转化生长因子β1质粒联合反复冻融移植物减轻大鼠神经移植后的排斥反应

目的 探讨局部注射含转化生长因子β1(TGF-β1)基因的质粒联合预先反复冻融处理移植神经对大鼠异体神经移植后排斥反应的影响.方法 SD大鼠分为4组进行实验,每组20只.TGF-β1组大鼠移植Wistar大鼠的冷冻坐骨神经,局部注射含TGF-β1基因的质粒;空质粒组大鼠移植Wistar大鼠的冷冻坐骨神经,注射空质粒;新鲜异体神经移植组大鼠接受Wistar大鼠的新鲜坐骨神经移植,不注射质粒;自体移植组SD大鼠移植自体的冷冻坐骨神经,不注射质粒.移植后用Western印迹法检查移植神经中TGF-β1的表达,并进行混合淋巴细胞反应(MLC),测量运动神经的传导速度.各组移植神经样本分别在光镜和投射电镜下进行观察.结果 TGF-β1组中TGF-β1的表达明显较高.移植后12周时,自体移植组、TGF-β1组、空质粒组和新鲜异体神经移植组的MLC结果(吸光度值)分别为0.312±0.009、0.213±0.006、0.521±0.018和0.928±0.032.TGF-β1组各个时间点的运动神经传导速度均高于空质粒组和新鲜异体神经移植组,TGF-β1组与自体神经移植组相比较,差异无统计学意义(P＞0.05).光镜下可见,TGF-β1组的神经纤维结构破坏不明显.结论 局部注射含TGF-β1基因的质粒联合反复冻融处理异体移植神经可以减轻神经移植后的排斥反应.     

Radiation-Induced Changes in Neuropeptides in the Rat Urinary Bladder

Purpose

To determine whether there was a change in innervation in the rat urinary bladder following x-ray irradiation.

Materials and Methods

The urinary bladders were obtained from rats irradiated 6 months previously with single doses of 15 Gy and 25 Gy x-radiation, and from nonirradiated (control) animals. They were examined immunohistochemically to localize neuropeptide Y (NPY), substance P (SP), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), met-enkephalin (m-ENK), leu-enkephalin (l-ENK), somatostatin (SOM) and the enzyme, tyrosine-hydroxylase (TH). Computer assisted image analysis was used to assess the density of immunoreactive nerve fibres.

Results

The greatest density of nerves observed in the bladder from control animals contained NPY, followed (in decreasing order) by CGRP, VIP, SP and TH. The nerves appeared to run predominantly along the longitudinal axis of the circular and longitudinal muscle fibres. SP-, CGRP-, TH- and occasionally VIP-immunoreactive nerves were observed in the lamina propria, at the base of the urothelium. Perivascular nerves containing neuropeptides and TH were observed throughout the bladder wall.There was an absence of m-ENK-, l-ENK- and SOM-immunoreactive nerves in the control and irradiated rat urinary bladders.In the rat urinary bladder irradiated with 25 Gy x-radiation, there was a significant increase (P less than 0.05) in the density of NPY-, TH- and SP- but not CGRP- and VIP-immunoreactive nerves. There were regional differences within the bladder, that is, there was an increase in VIP-, CGRP- and SP-immunoreactive nerves around and within the urothelium. NPY-immunoreactive nerves were seen in the connective tissue and elastic fibres of the lamina propria for the first time. An increase in the density or fluorescence intensity of perivascular TH- but not neuropeptide-containing nerves was observed.

Conclusion

The increase in the density of NPY-, SP- and TH-immunoreactive nerves in the irradiated bladders may be due to axonal sprouting which contributes to the symptoms of radiation injury.