In vivo complete neural isolation of the rat jejunoileum: a simple model to study denervation sequelae of intestinal transplantation

BACKGROUND: Our aim was to develop and validate a technically easy, reliable, and reproducible method of complete jejunoileal denervation in the rat to allow study of the physiologic effects of intestinal transplantation devoid of immunologic phenomena and ischemia/reperfusion injury. MATERIALS AND METHODS: Six adult Sprague-Dawley rats underwent transection and reanastomosis of the proximal jejunum and proximal colon, transection of all neurolymphatic tissues at the base of the mesentery, stripping adventitia off the superior mesenteric artery and vein, and radial transection of the intervening mesenteries, thereby denervating the jejunoileum in situ without disrupting blood flow. Three rats each were sacrificed 1 and 6 months later. Intestinal smooth muscle from the still-innervated duodenum and the denervated jejunum, mid-small bowel, and ileum was compared to corresponding tissues from a normal rat for tyrosine hydroxylase immunohistochemistry, a marker of extrinsic innervation. RESULTS: One and six months after denervation, all duodenal samples demonstrated normal tyrosine hydroxylase immunostaining. In contrast, tyrosine hydroxylase immunoreactivity was undetectable in jejunum, mid-small bowel, or ileum of rats at 1 month and 2 of the 3 rats at 6 months; 1 rat at 6 months had low levels of tyrosine hydroxylase immunoreactivity at the mesenteric border of jejunum and mid small bowel. CONCLUSION: This simple technique of in situ neural isolation effectively and reproducibly achieves complete extrinsic denervation of the entire rat jejunoileum. Low levels of neural regeneration may be present 6 months after denervation.     

Cajal间质细胞在先天性巨结肠和巨结肠同源病结肠中的分布研究

目的研究先天性巨结肠（Hirschsprung’s disease，HD）和巨结肠同源病（allied Hirschsprung’s disease，AHD）肠壁内Cajal间质细胞（interstitial cells of Cajal，ICCs）的分布状态，探讨HD和AHD的发病机制。方法选择确诊为HD和AHD的患者各20例，取巨结肠根治术吻合口远端的全层肠壁作为实验组，另取16例正常结肠标本作为对照组。用鼠抗人c—kit单克隆抗体（CD117）标记ICCs，Image Pro-Plus图像分析系统检测ICCs。结果对照组中大量ICCs分布在肌间神经丛周围和环纵肌层内，ICCs包绕神经丛周围，肌层间ICCs连续分布；HD组远端肠管中肌层间和各肌层内ICCs明显减少甚至缺如，与对照组比较差异有统计学意义，P〈0．01；AHD组远端肠管中神经丛大小不一，ICCs分布差异大，大多数神经丛区ICCs减少，环肌层内ICCs明显减少，与AHD组和对照组比较差异有统计学意义，P〈0.01。HD组远端结肠中ICCs减少比AHD组显著，差异有统计学意义，P〈0．01。结论HD、AHD病变肠管中除了神经节细胞的异常外，同时存在ICCs异常；ICCs在HD和AHD的分布不同可能与两者临床症状差异有关；肠管中ICCs数量可能与临床症状及预后有一定关系。     

感觉神经保护延缓失神经性肌萎缩的实验研究

目的研究感觉神经延缓失神经肌萎缩的作用。方法成年雄性SD大鼠40只,随机分成两组,实验组(n=20)即感觉神经移植保护组,对照组(n=20)即纯失神经组。第一阶段:对照组仅横断肌皮神经。实验组完成近端锁骨上神经和远端肌皮神经吻合。第二阶段:第一阶段术后6个月,实验组及对照组完成胸小肌运动神经分支与肌皮神经远端的吻合。第二阶段术后4周及8周内,分别测定大鼠理毛实验、运动单位电位、肌肉湿重、肌肉HE染色、运动终板染色计数等。结果实验组术后第4及8周运动单位电位波幅明显高于对照组,电流刺激敏感度明显优于对照组。二头肌湿重恢复率、肌纤维直径、肌纤维截面积恢复率、运动终板数明显优于对照组。理毛实验于术后第2周时优势明显。结论近端感觉神经与远端运动神经端端吻合可有效延缓失神经肌肉萎缩。     

神经干细胞移植再支配失神经骨骼肌的实验研究

目的 探讨采用神经干细胞移植的方法使失神经骨骼肌重获神经再支配的可行性。方法 将108只SD大鼠按注射药物的不同随机分为3组，每组36只大鼠。切断右侧胫神经建立腓肠肌失神经实验模型。实验组：将神经干细胞悬液注射到切断胫神经的远端。对照组：注射等量的细胞培养液。损伤组：注射等量的生理盐水。术后8、10、12周采用免疫组织化学、HRP逆行示踪技术和神经形态学的方法，检测失神经骨骼肌是否重新获得移植神经干细胞的再支配。结果 术后8周局部即可检测到分化的神经元和神经胶质细胞，其再生的轴突与靶肌肉已经建立神经突触连接。术后10、12周时，再生的神经纤维进一步增多。结论 周围神经断伤后局部用神经干细胞移植能够使远端失神经骨骼肌重新获得神经再支配。     

Induction and recovery of colonic motility/defecatory disorders after extrinsic denervation of the colon and rectum in rats

BACKGROUND: Anterior resection for rectal disease is associated with extrinsic autonomic denervation of the neorectum, which may influence the myenteric plexus, and subsequently the motility/defecatory status after operation. METHODS: A rat model with denervated neorectum was constructed. Colonic contractile activity in vivo, the amount of generic neuron marker (PGP 9.5) and nitric oxide synthase (NOS) were measured periodically. The responses of the muscle strip in each period to electrical field stimulation were evaluated using various neurotransmitters. RESULTS: In rats with denervated neorectum, giant migrating contractions (GMCs) of the distal colon, the number of fecal lumps per day and their small size, significantly increased in the early phase postoperatively, although both recovered in the late-phase period. The contractile response of the muscle strip of the denervated colon to acetylcholine was reduced throughout the period; however, contraction of the denervated colon under the addition of NO inhibitor (l-NAME) was enhanced significantly in the late-phase period, and recovered to the control level by atropine. Neuronal NOS, but not PGP 9.5 concentration, in the myenteric plexus at the distal denervated colon, significantly increased in the late-phase period. None of the above items differed from the control at other colonic portions throughout the period. CONCLUSIONS: Extrinsic autonomic denervation causes abnormal hyper-motility in the neorectum, which may be associated with multiple evacuations in the early phase postoperatively. Increased acetylcholine and the subsequent increase of neuronal NOS in the myenteric plexus may be an adaptive mechanism to compensate for such abnormal colonic motility after extrinsic denervation.     

Continuous inferior mesenteric ganglion block for the control of abdominal pain.

BACKGROUND AND OBJECTIVES: A 71-year-old woman was referred for control of intractable left-sided abdominal pain and constipation caused by stage 2B rectosigmoid colon cancer. She was treated with an intravenous morphine sulfate infusion at 4 mg/h which made her drowsy and lethargic. Because the distal colon is innervated by the inferior mesenteric sympathetic ganglion, it was hypothesized that a continuous block of this ganglion would provide both pain control and increased intestinal motility. METHODS: The patient was placed in a prone position, and a Tuohy needle was placed at an entry site 7 cm lateral to the L3 spinous process. The needle was advanced 2 cm anterior to the L3 vertebral body with fluoroscopic guidance. An 18-gauge indwelling catheter was placed through the needle and a continuous infusion with 0.1% bupivacaine was maintained for 4 days. RESULTS: The patient obtained immediate pain relief and bowel motility. She remained awake and comfortable throughout the duration of therapy. She was discharged home 3 days after stopping the infusion and required a minimal amount of opioids. CONCLUSION: Continuous inferior mesenteric ganglion block may be an option in providing relief for pain caused by distal colon pathology.     

Innervation of the rat urinary bladder. II. Effects of prostaglandins on the denervated detrusor muscle after bilateral pelvic ganglionectomy

The effects of prostaglandin (PG) F2 alpha and E2 on the denervated smooth muscle of the urinary bladder in female rats were studied in vivo by histochemistry and electron microscopy. The urinary bladder denervated by bilateral removal of the pelvic ganglion was markedly distended, being filled with urine. Daily intravenous administration of PGF2 alpha or PGE2 for 6 days following the operation showed that rats receiving PGE2 urinated remarkably more than those receiving PGF2 alpha. But the ultrastructural changes on the smooth muscle cells, such as dilated tubules of rough endoplasmic reticulum and large Golgi vacuoles, were more prominent in the PGF2 alpha administrated urinary bladders than in PGE2 administrated ones. On occasion, cholinergic ganglion cells happened to be encountered in the muscular layer of a rat urinary bladder. These intramural ganglion cells and the cholinergic nerve fibers surrounding the cells displayed strong acetylcholinesterase activity, unaffected by bilateral pelvic ganglionectomy.     

Motor neurotization by segmental epineurectomy and implantation: lateral muscular neurotization

In this study, we aimed to develop a new muscular neurotization technique, in which transection of the donor nerve is avoided. We investigated the outcome of insertion of the donor nerve within the denervated muscle following segmentary epineurectomy (lateral muscular neurotization). Thirty-five male Wistar rats were evaluated in five groups, including sham control, denervation control, denervation combined with segmentary epineurectomy, direct muscular neurotization, and lateral muscular neurotization. Electromyography, muscle weight measurements, and histological evaluations were performed at postoperative months 2 and 3. The denervation group was statistically successful for denervation as compared with the sham control group for all parameters. The lateral neurotization group was successful in preventing muscle atrophy and gaining reinnervation in electromyographic, histological, and weight parameters. The direct neurotization group was also successful in histological and weight parameters. Lateral muscular neurotization is promising because it does not interrupt the fascicular integrity and is successful in reinnervation; therefore, it seems to be a good alternative for direct muscular neurotization.     

盆神经节海绵体内移植对双侧海绵体神经损伤后大鼠阴茎勃起功能的影响

目的　研究双侧海绵体神经离断后 ,自体盆神经节海绵体内移植对大鼠阴茎勃起功能的影响。方法　将 2 6只雄性SD大鼠 (3～ 6个月 ,30 0～ 4 0 0g 只 )随机分为 2组 :(1)实验组 (n =16 )行双侧海绵体神经离断及左盆神经节移植入左侧阴茎脚内 ;(2 )对照组 (n =10 )仅离断双侧海绵体神经。1个月及 3个月后阿朴吗啡皮下注射检测勃起功能 ,电刺激左阴茎脚移植区域或右侧盆神经节比较海绵体内压的变化 ,NADPH染色检测海绵体内一氧化氮合酶 (NOS)阳性神经纤维 ,透射电镜观察盆神经节移植后的存活情况。结果　两组大鼠 1个月及 3个月后注射阿朴吗啡后均无勃起反应 ;电刺激左阴茎脚盆神经节移植区域后亦不能导致勃起。 1个月后电刺激两组左阴茎脚后的海绵体内压变化分别为 (9 4 1± 3 2 0 )、(4 16± 2 5 8)cmH2 O(t=4 76 9,P <0 0 5 ) ,3个月后分别为 (13 6 7± 4 18)、(5 0 9± 2 74 )cmH2 O(t=9 18,P <0 0 5 )。两组大鼠左阴茎脚区域的NOS阳性神经纤维数目 1个月后分别为 (2 18 7± 2 4 5 )、(18 0± 3 7) (t=14 77,P <0 0 5 ) ,3个月后分别为 (183 2± 19 7)、(19 0± 3 8) (t =12 18,P <0 0 5 ) ;透射电镜证实了神经节移植后的存活。结论　盆神经节海绵体内移植后可以存     

选择性结肠切断旷置术治疗重度结肠慢传输性便秘的探讨

目的：探讨一种可行的重度结肠慢传输性便秘的外科手术方式。方法：有选择性地保留近端结肠肠段，将远端结肠之口侧封闭后旷置。用保留的肠段行结-乙或结-直吻合。结果：28例中痊愈25（89．3％），缓解3例（10．6％）。结论：选择性结肠切断旷置术治疗重度结肠慢传输性便秘术式简化，术程缩短，安全有效，有较大『临床应用价值。     

Experimental studies on sympathetic innervation of lower urinary tract. Quantitation of nerve terminals and urethral pressure response after hypogastric denervation

On 6 female mongrel dogs (denervated group), bilateral hypogastric nerves were cut distally to the inferior mesenteric ganglion. Five dogs were kept intact as a control group. After 2-5 months, urethral pressure response to continuous noradrenaline infusion (0.1 microgram/kg/min) was monitored. The urethral pressure rose significantly after noradrenaline loading in each group, however there was no significant difference in the degree of the response between the denervated group and control group. Subsequently, the bladder and the urethra were extirpated to determine the intrinsic noradrenaline content. The tissue noradrenaline concentration was highest in the posterior urethra, intermediate in the bladder base and lowest in the bladder dome. Although these values tended to be lower in the denervated group than in the control group, no significant difference was obtained between the groups in each portion. These results suggest that the majority of sympathetic components which consists in the hypogastric nerve may involve short adrenergic neurons. Thus, chronic hypogastric denervation alone does not induce sympathetic denervation supersensitivity. Simultaneous decentralization of the pelvic nerve may be necessary for inducing sympathetic denervation supersensitivity.     

Muscle flap mass preservation with end-to-side neurorrhaphy: an experimental study

The authors examined the preservation of rat gracilis muscle flap mass after motor and sensory end-to-side neurorrhaphy. The rat gracilis muscle flap model was designed based on a previous study. Twenty-four Sprague-Dawley rats were divided into three groups. In Group 1 (n = 8), the flap was denervated by transecting the obturator nerve. In Group 2 (n = 8), the flap was reinnervated by coapting the proximal saphenous nerve to the distal obturator nerve. In Group 3 (n = 8), the flap was reinnervated by coapting the motor branch of the femoral nerve to the distal stump of the obturator nerve. At 6 months postoperatively, the gracilis muscle flaps were examined, harvested, and weighed individually. Results showed that the flaps with motor nerve reinnervation retained good bulk, with a weight of 634.0 +/- 65.1 gm, which was statistically significantly higher than the denervated group (457.5 +/- 125.3 gm, p < 0.01). However, muscle mass preservation in the sensory reinnervated group (606.9 +/- 209.1 gm) was not significantly different, compared to the denervated group. Histology revealed atrophic changes in the denervated group, compared to the sensory and motor-reinnervated groups. The authors concluded that muscle mass can be preserved by end-to-side nerve repair. Motor nerve reinnervation is able to better arrest atrophic changes of the muscle flaps.     

Segmental aganglionosis of the appendix

Ignoring the pathologist's dictum that the appendix is not reliable for the identification of ganglion cells in suspected cases of total colonic aganglionosis, the author (K.D.A.) performed an ileostomy in a child whose appendix contained no ganglion cells. The ascending colon was later found to be ganglionic. The appendices from 36 cases of Hirschsprung's disease were examined and the number of ganglion cells in 10 high power fields was compared with 10 appendices from patients who did not have Hirschsprung's disease. There were no ganglion cells in any of the patients with total aganglionosis of the colon. Twenty-two patients with Hirschsprung's disease involving the sigmoid colon had an average of 3.0 ganglion cells per high power field (range 0.5 to 5.1). Five patients with longer segment aganglionosis had 2.2 cells per high power field (range 0 to 4.1). The index case fell into this group and was serially sectioned without finding any ganglion cells. The control group averaged 3.41 cells per high power field (range 1.4 to 5.9). There was no significant difference between the control group and the groups with less than total colonic aganglionosis. It is speculated that this case represents segmental Hirschsprung's disease with the skip area in the ascending colon.     

Absence of the interstitial cells of Cajal in a child with chronic pseudoobstruction

Absence or altered distribution of the interstitial cells of Cajal (ICCs) has been described in association with intestinal pseudoobstruction in adults. We report the first pediatric case with regional absence of ICCs in the distal small bowel and colon associated with intestinal pseudoobstruction. This report highlights that abnormalities of the ICCs in intestinal pseudoobstruction should be considered early in the diagnostic workup of children with intestinal pseudoobstruction.     

神经干细胞移植延缓失神经肌萎的作用机制

目的 研究脊髓神经干细胞移植到外周神经后延缓失神经肌萎的作用及机制.方法 取孕10～12 d的SD孕鼠,提取、培养及纯化脊髓神经干细胞,传2～3代后,将分散成单细胞的神经干细胞(106/μl×5μl)移植到胫神经切断模型的远端;设立对照组,移植5μl干细胞培养液.术后分别于3、5个月取材,观察移植神经干细胞的分化;比较干细胞移植能否延缓失神经肌萎;观察靶肌肉中突触(神经肌肉接头)的变化情况.结果 移植后3、5个月,发现移植组小腿三头肌萎缩较对照组减轻(P<0.05);移植组中的突触后膜退变萎缩明显好于对照组,术后5个月出现了新的突触结构.结论 神经干细胞移植可以延缓失神经肌萎的程度,有效保护失神经肌肉突触后膜的结构,并能形成新的突触.     

Correction of postoperative fecal incontinence in children

The results of surgical treatment of II-III degree incontinence of feces in 6 patients previously operated on for anorectal anomaly are presented. Clinico-functional examination permitted topical verification of damage to rectal sphincter apparatus. Sphincterolevatoroplasty with the use of a posterior sagittal approach was performed. The given approach permitted maximum use of the preserved muscular elements of the obturator apparatus. In a number of cases sphincterolevatoroplasty was combined with smooth muscle plasty of the internal anal sphincter from the distal colon. The functional results followed in 5 patients were considered as good and satisfactory ones.     

Development of a Simple Model of Extrinsic Denervation of the Small Bowel in Mouse

Small bowel transplantation (SBT) is associated with poorly understood enteric dysfunction. The study of SBT in mice is hindered by the technical difficulty of orthotopic SBT in the mouse. Our aim was to develop an easy preparation of extrinsic denervation of the entire jejunoileum in mice as a model of orthotopic SBT. All neurolymphatic tissues accompanying the superior mesenteric artery (SMA) and vein (SMV) were ligated just distal to the middle colic vessels. The SMA and SMV were then stripped of investing adventitia, and the mesentery to jejunum and colon were transected radially. Jejunum and colon were not transected and reanastomosed. To confirm extrinsic denervation 1, 3, and 6 months later, segments of small bowel were stained for protein gene product 9.5 (PGP9.5) and tyrosine hydroxylase (TH). Tyrosine hydroxylase immunoreactive intensity was then quantified using a semiquantitative analysis. Immunohistochemical fluorescence showed persistence of PGP9.5 immunoreactivity confirming enteric nerves in jejunoileum; however, there was no TH immunoreactivity in jejunoileum in denervated mice despite the expected preservation of TH immunoreactivity in the still-innervated duodenum at 1 month. At 3 months, sparse immunoreactivity for TH was present, and by 6 months, reinnervation of TH-containing nerves appeared similar to controls. Quantification of intensity at each time-point further confirmed this trend. This technique in the mouse accomplishes a complete extrinsic denervation of jejunoileum early postoperatively (1 and 3 months); reinnervation occurs by 6 months. This is an easily learned murine model of orthotopic SBT. Presented at the American Gastroenterological Association during Digestive Disease Week in Los Angeles, CA, as a poster presentation on May 23 2006. Abstract published in GastroenterologyE 2006; 130:A604.     

Effects of intraluminal infusion of short-chain fatty acids on the healing of colonic anastomosis in the rat

The effects of an intracolonic infusion of short-chain fatty acids (SCFA) on the healing of colonic anastomosis in the rat were investigated. Thirty-three Sprague-Dawley rats underwent transection and anastomosis of the descending colon and transection and diversion of the ascending colon. The proximal limb of the ascending colon was exteriorized as an end colostomy, and the distal (defunctionalized) limb was cannulated for continuous infusion. Rats received either no infusion (N = 11) or an infusion of either electrolytes (N = 11) or SCFA (acetate, propionate, and butyrate; N = 11) into the defunctionalized colonic segment. On the sixth postoperative day bursting pressure (BP) and bowel wall tension (BWT) were determined. The occurrence of spontaneous anastomotic dehiscence was significantly less for the SCFA group (0/11) compared with the no infusion group (5/11, p less than 0.01). The anastomotic suture line burst in significantly fewer colons from the SCFA group (1/11) than either the electrolyte infusion (8/11, p less than 0.003) or the no infusion (6/6, p less than 0.001) groups. BP and BWT were significantly higher for the SCFA group (BP: 147 +/- 10 mm Hg; BWT: 59 +/- 1.0 dyne 10(3)/cm) than for either the electrolyte (BP: 99 +/- 30 mm Hg, p less than 0.002; BWT: 45 +/- 19, p less than 0.03) or no infusion (BP: 111 +/- 42, p less than 0.02; BWT 36 +/- 15, p less than 0.007) groups. The results of this study indicate that intracolonic infusion of SCFA resulted in significantly stronger colonic anastomosis in the rat.     

Traumatic transection of the pancreas: a report of four cases

Four patients with transection of the pancreas by blunt trauma are reported. All were treated by distal pancreatectomy and splenectomy. The three patients who survived were free from exocrine or endocrine deficiency at the time of discharge from hospital. One patient, whose transection was missed at the initial laparotomy, was operated upon later when in renal failure, and died from sepsis. Distal pancreatectomy with splenectomy seems the appropriate because other operations have high complication rates, and extirpation of the distal pancreas is not debilitating.     

Immunocolocalization of the heme oxygenase-2 and interstitial cells of Cajal in normal and aganglionic colon

Purpose: Interstitial cells of Cajal (ICCs) are pacemaker cells that play an important role in the control of gut motility. Carbon monoxide (CO) has been proposed as an endogenous messenger molecule between ICC and smooth muscle cells in the gastrointestinal tract (GIT). Heme oxygenase-2 (HO-2) is the main physiologic mechanism for generating CO in human cells. The aim of this study was to investigate the immunocolocalization of the HO-2 and ICCs in normal and aganglionic bowel of Hirschsprung's disease (HD). Methods: Full-thickness specimens were obtained from aganglionic colon during pull-through operation from 10 patients diagnosed as having HD. Normal control large bowel specimens were collected from 4 patients during bladder augmentation procedures. Double immunostaining was carried out using c-kit and HO-2 antibodies. Immunolocalization was detected by means of confocal laser scanning microscopy. Results: HO-2 immunoreactivity (IR) was found in many ICCs present around the myenteric plexus, within the longitudinal and circular muscle layers and at the innermost part of the circular muscle layer in normal colon. In the aganglionic colon there was absence of HO-2 IR in the sparsely found ICCs. In the transitional zone of HD bowel the colocalization of HO-2 IR and ICCs was much reduced compared with controls. Conclusions: The results of this study provide the first evidence for the presence of HO-2 immunoreactivity in the ICCs in normal human colon and absence of HO-2 immunoreactivity in sparsely appearing ICCs in the bowel of HD patients. The lack of HO-2 in the ICCs in the bowel of HD patients may result in impaired intracellular communication between ICCs and SMCs causing motility dysfunction. J Pediatr Surg 38:73-77.