帕金森病大鼠颈动脉体球细胞移植后TH、GFAP和TNF-α表达的动态变化

目的：探讨帕金森病（PD）大鼠颈动脉体球细胞移植后细胞存活状况和宿主的反应。方法：立体定位注射6－羟多巴胺（6－OHDA）制备偏侧PD大鼠模型，右侧纹状体内分别移植入自、导体颈动脉体和胚胎大鼠中脑组织块，移植后2、4、8和12周用免疫组织化学法同步检测酪氨酸羟化酶（TH）阳性细胞数、移植区周围胶质纤维酸性蛋白（GFAP）和肿瘤坏死因子－α（TNF－α）表达水平的改变。结果：移植后12周与胚胎中脑组织移植组比较，自、异体颈动脉体球细胞移植组存活TH^ 细胞显著增多（P＜0．05），但自、异体颈动脉体球细胞移植组间比较差异无显著性。各移植组移植后2周，移植区内GFAP和TNF－α阳性数显著增高，移植后12周时下降，但仍高于健侧，差异仍有极显著性（P＜0．01）。结论：颈动脉体球细胞块移植治疗可显著提高TH^ 细胞存活率，而移植区GFAP和TNF－α的表达对其有不利影响。     

慢传输型便秘大鼠结肠肌间神经丛内VIP能神经、SP能神经的免疫组化研究

目的　探讨结肠肌间神经丛血管活性肠肽 (VIP)能神经及P物质 (SP)能神经在慢传输型便秘发病中的作用。方法　建立大鼠慢传输型便秘模型 ,利用铺片技术制作结肠肌间神经丛标本 ,采用免疫组织化学法在铺片上显示便秘大鼠结肠肌间神经丛内VIP能神经及SP能神经的分布及形态学改变。结果　与正常大鼠比较 ,便秘大鼠结肠肌间神经丛VIP能神经节及节间束细小 ,节内神经元数量明显减少 ,节间束内神经纤维分布稀疏 (平均面密度值 2 3.0 8± 1.82vs 19.35± 1 13,P <0 0 1) ;便秘大鼠结肠肌间神经丛SP能神经节明显增大 ,神经束增粗 ,神经元和神经纤维明显增多 (平均面密度值 15.4 5± 1 0 5vs18.14±1 18,P <0 .0 1)。结论　慢传输型便秘大鼠结肠壁内存在明显的神经病理学改变 ,结肠传输功能障碍可能与结肠肌间神经丛VIP和SP能神经病理改变和 或功能障碍有关。     

粒细胞集落刺激因子对大剂量盐酸戊乙奎醚所致大鼠认知功能障碍的影响

目的观察粒细胞集落刺激因子(G-CSF)对大剂量盐酸戊乙奎醚所致大鼠认知功能障碍的影响。方法 24只14个月龄Wistar成年雄性大鼠随机分为三组:空白组(N组)、盐酸戊乙奎醚组(P组)和G-CSF治疗组(G组),每组8只。N组腹腔注射生理盐水1ml,P组腹腔注射盐酸戊乙奎醚2.56mg/kg,G组腹腔注射盐酸戊乙奎醚2.56mg/kg后皮下注射G-CSF 50μg/kg,连续5d。第1、3、5天给药后用Morris水迷宫测试其空间学习记忆能力的改变,末次水迷宫实验后用免疫组化法观察海马胆碱乙酰转移酶(ChAT)阳性细胞表达个数。结果与第1天比较,第3天和第5天三组潜伏期和游泳距离均明显缩短(P<0.05),且第5天N组和G组明显短于P组(P<0.05)。与P组比较,N组和G组海马区ChAT阳性细胞个数均明显增多(P<0.05)。结论 G-CSF可以改善大剂量盐酸戊乙奎醚引起的大鼠认知功能障碍。     

周围神经损伤后脊髓神经元形态及神经活性物质变化的研究

为观测周围神经损伤，尤其是晚期损伤后，脊髓神经元形态、神经活性物质变化及其功能状态，采用切断大鼠坐骨神经，通过辣根过氧化物酶逆行示踪、免疫组化，并结合计算机图像分析等方法，对脊髓前角运动神经元形态及降钙素基因相关肽、胆碱乙酰转移酶和脊髓后角Ｐ物质变化进行定量分析。结果：①前角细胞胞体伤后３周肿胀，６周恢复正常，１２周萎缩２７％。树突持续性收缩，至１２周萎缩５３％。胞体和树突２４周与１２周相似，均无进一步变化。②降钙素基因相关肽神经损伤１周后，即在脊髓前角运动神经元中升至最高并持续４周，８周恢复正常，观察２４周无明显变化；胆碱乙酰转移酶在脊髓前角运动神经元中无明显变化；Ｐ物质在脊髓后角于损伤２～６周下降至最低，１６周恢复正常；而降钙素基因相关肽在脊髓后角则无明显变化。认为，晚期周围神经损伤，从形态学及神经活性物质的变化来看，脊髓神经元的功能活动仍保持在一定水平，具有修复价值。     

脓毒症大鼠脾脏非神经性胆碱能系统活性与肿瘤坏死因子-α含量变化的关系

目的:观察脓毒症大鼠脾脏胆碱能系统酶活性变化与致炎细胞因子TNF-α含量变化的关系,初步探讨脾脏胆碱能系统对炎症反应的调控作用.方法:采用盲肠结扎穿孔(CLP)方法制备大鼠脓毒症模型.动物随机分为对照组及CLP6、12和24 h 4组.于术后相应时间点取脾脏,测定脾脏TNF-α含量及胆碱乙酰基转移酶(chAT)、乙酰胆...     

氨基胍对饥饿大鼠小肠肌间神经丛神经元诱导型一氧化氮合酶表达的影响

目的 评价氨基胍对饥饿大鼠小肠肌间神经丛神经元诱导型一氧化氮合酶(iNOS)表达的影响.方法 雄性SD大鼠90只,体重230～270 g,采用随机数字表法,将其随机分为3组:正常对照组(C组,n=10)、饥饿组(S组,n=40)和氨基胍+饥饿组(A组,n=40),S组和A组无饲料供应,自由摄水,A组腹腔注射氨基胍150 mg·kg-1·d-1.S组和A组分别于饥饿3、5、7、9d时取10只动物,采用葡聚糖蓝染色法测定小肠传输比率,然后处死动物,取小肠组织,采用NADPH-黄递酶组织化学染色法测定回肠肌间神经丛神经元iNOS表达.结果 与C组比较,S组和A组各时间点小肠传输比率降低,回肠肌间神经丛神经元iNOS表达上调(P＜0.05或0.01);与S组比较,A组于饥饿3、5、7d时小肠传输比率升高,回肠肌间神经丛神经元iNOS表达下调(P＜0.05或0.01).结论 氨基胍改善饥饿大鼠小肠运动功能的机制与下调小肠肌间神经丛神经元iNOS表达有关.     

大鼠盆神经节胆碱乙酰转移酶和一氧化氮合酶阳性神经元对尿道外括约肌的支配

目的 观察大鼠盆神经节(MPG)中胆碱乙酰转移酶(ChAT)、一氧化氮合酶(NOS)阳性神经元对尿道外括约肌(EUS)的支配.方法 24只成年SD大鼠随机分为A组(n=12)和B组(n=12).应用辣根过氧化物酶(HRP)逆行示踪技术结合免疫组织化学和酶组织化学双标技术,观察A组大鼠MPG中ChAT阳性神经元和还原型辅酶Ⅱ(NADPH,NOS标志物)阳性神经元对EUS的支配.B组大鼠MPG内注射麦芽凝集素-辣根过氧化物酶(WGA-HRP)进行顺行神经追踪.结果 A组大鼠HRP标记神经元散在分布于MPG内.HRP标记神经元中43.3%(155/358)为ChAT免疫反应阳性,14.5%(49/339)为NADPH组化反应阳性.B组大鼠EUS内有HRP阳性神经末梢.结论 大鼠盆神经节中的神经元对EUS有直接支配,其神经递质有乙酰胆碱和一氧化氮.     

多能成体祖细胞移植治疗帕金森病及其作用机制

目的 探讨骨髓间质分离的多能成体祖细胞(MAPCs)移植后在大鼠脑组织内神经细胞分化及修复神经功能的作用机制.方法 制作帕金森病大鼠模型,将在体外纯化、增殖和已用5-溴-2脱氧尿苷(BrdUrd)处理过的MAPCs注入帕金森病大鼠脑内.3个月后,对移植大鼠采用免疫组织化学技术、逆转录-聚合酶链反应(RT-PCR)、行为学测试等方法鉴定和分析MAPCs在大鼠脑组织内神经元样细胞分化和修复神经功能的作用机制.结果 6-羟多巴诱导的大鼠行为学变化MAPCs组明显好于对照组;细胞移植后第4、8、12周时,移植的PD大鼠1h内的旋转圈数为对照组(687.8±2.7、754.1±13.4、763.2±14.8)和MAPCs组(528.0±12.7、440.5±12.0、435.0±7.1).MAPCs在中脑黑质和纹状体区分化为神经元样细胞和多巴胺能神经元;PCR检查发现多巴胺-β-羟化酶、多巴胺转运体和神经生长因子表达水平MAPCs组(分别为0.510±0.028、0.620±0.046、0.850±0.051)明显比对照组(分别为0.310±0.072、0.400±0.044、0.480±0.057)升高(P＜0.05).结论 骨髓间质分离的MAPCs移植后能在大鼠脑组织微环境中自主分化为多巴胺能神经细胞并有效地修复6-羟多巴诱导的神经功能缺损.     

Increased expression of Semaphorin 3A in the endothelin receptor-B null mouse model of Hirschsprung disease

Purpose

Semaphorins are guidance cues for developing neurons, implicated in the determination of the migratory pathway of neural crest-derived neural precursors during enteric nervous system development. Recently, it has been reported that Semaphorin 3A (SEMA3A) expression is up-regulated in the aganglionic colon in Hirschsprung disease (HD) patients, suggesting that increased SEMA3A expression may be a risk factor for HD. Thus, the aim of our study was to determine the expression of SEMA3A using Sox10-Venus mice gut.

Colonic dysmotility in postsurgical patients with Hirschsprung's disease. Potential significance of abnormalities in the interstitial cells of Cajal and the enteric nervous system

Purpose

Normal gut muscular function depends on the coordinated activity of both the enteric nervous system (ENS) and the interstitial cells of Cajal (ICC). Hirschsprung's disease (HD) has long been considered a purely neuronal deficit but recent data point to abnormalities in ICC in the proximal ganglionated HD colon. We examined the labeling of ICC and neuronal cells in the proximal ganglionated colon in patients with HD to determine whether abnormalities of ICC and ENS might be associated with a poor clinical outcome.

Methods

Tissue from 11 patients with HD was studied using immunohistochemistry for ICC and neuronal identification in comparison to control tissue from patients without HD. Image data were evaluated quantitatively and interpreted relative to clinical outcome.

Results

Interstitial cells of Cajal in the ganglionated colon of the HD group did not differ from the control group, but nerve cells/fibers were decreased 40%. Paired decreases in both nerve fibers and ICC in individual patients were associated with normal bowel function. Poor postoperative outcome was observed in a patient with normal innervation but with a profound decrease in ICC in the ganglionated colon.

Conclusions

Nerve fibers are decreased in the proximal ganglionated colon in patients with HD without associated gut dysmotility. Poor clinical outcome was noted only in a patient with normal innervation and markedly decreased ICC. Collection of data from a much larger number of patients with poor clinical outcome will be necessary to determine the significance of this imbalance of ICC and innervation.     

Murine model of Hirschsprung-associated enterocolitis. I: Phenotypic characterization with development of a histopathologic grading system

Purpose

The aim of the study was to characterize enterocolitis in the Ednrb-null (Ednrb−/−) mouse with aganglionosis of the colon and to develop and validate a semiquantitative histopathologic grading system to assess enterocolitis.

Methods

We isolated colon and ileal specimens of Ednrb−/− and control mice (Ednrb+/+) and performed histochemical staining (H&E) on tissue sections. After establishing inflammation grading criteria, 2 blinded pathologists independently assessed the severity and depth of inflammation of proximal colon segments on 2 separate occasions. Interclass correlations (ICCs) and coefficient of variation (CV) were calculated to determine interrater and intrarater agreement. We then prospectively applied the enterocolitis grading system to Ednrb−/− mice that became clinically ill. A cohort of Ednrb−/− mice were observed until they developed clinical illness, at which time they were euthanized and had multiple organ homogenates cultured for bacteria, and colon and small bowel were histopathologically graded for enterocolitis. Spearman's rank correlations comparing enterocolitis scores with level of bacteremia were performed.

Results

Intra- and interrater ICCs of the histologic scoring system were satisfactory (0.61 and 0.94, respectively), as were intra- and interrater CVs (18% and 9%, respectively). Of the Ednrb−/− mice, 65% developed bacteremia. Those with bacteremia had significantly higher enterocolitis scores than those without bacteremia (P < .01). Ednrb−/− mice that developed bacteremia showed a strong positive correlation between total enterocolitis scores and number of bacterial colony forming units in peritoneal lavage, liver, kidney, and aerobic spleen.

Conclusions

The Ednrb−/− mouse with aganglionosis develops enterocolitis and has features similar to Hirschsprung-associated enterocolitis in humans. Our grading system is a reliable way to assess enterocolitis. By performing microsurgical pull-through, we can now perform controlled, hypothesis-driven, mechanistic studies to evaluate etiologic factors affecting enterocolitis in the Ednrb−/− mouse.     

Controversies concerning diagnostic guidelines for anomalies of the enteric nervous system: a report from the fourth International Symposium on Hirschsprung's disease and related neurocristopathies

Intestinal Dysganglionoses (IDs) represent a heterogeneous group of Enteric Nervous System anomalies including Hirschsprung's disease (HD), Intestinal Neuronal Dysplasia (IND), Internal Anal Sphincter Neurogenic Achalasia (IASNA) and Hypoganglionosis. At present HD is the only recognised clinico-pathological entity, whereas the others are not yet worldwide accepted and diagnosed. This report describes the areas of agreement and disagreement regarding definition, diagnosis, and management of IDs as discussed at the workshop of the fourth International Meeting on “Hirschsprung's disease and related neurochristopathies.”The gold standards in the preoperative diagnosis of IDs are described, enlighting the importance of rectal suction biopsy in the diagnostic workup. The most important diagnostic features of HD are the combination of hypertrophic nerve trunks and aganglionosis in adequate specimens. Acetylcholinesterase staining is the best diagnostic technique to demonstrate hypertrophic nerve trunks in lamina propia mucosae, but many pathologist from different centers still use H&E staining effectively. Moreover, the importance of an adequate intraoperative pathological evaluation of the extent of IDs to avoid postoperative complications is stressed. Although it is not clear whether IND is a separate entity or some sort of secondary acquired condition, it is concluded that both IND and IASNA do exist. Other interesting conclusions are provided as well as detailed results of the discussion. Further investigation is needed to resolve the many controversies concerning IDs. The fourth International Conference in Sestri Levante stimulated discussion regarding these entities and led to the International guidelines to serve the best interest of our patients.     

Is Hirschsprung disease a purely neurological condition? A study of the Actin G2 smooth muscle gene in Hirschsprung disease

BackgroundHirschsprung disease is a functional obstruction of the gastrointestinal tract due to the congenital absence of ganglion cells in the intermyenteric plexuses of the distal bowel. Gastrointestinal motility requires intact muscular layers as well as neural network connection to function properly. The Actin G2 gene is the main gene encoding actin gamma 2; a smooth muscle actin found in enteric tissues.AimThis study of the Actin G2 gene in patients with Hirschsprung disease explores a possible molecular basis abnormal muscle function and post-surgical pseudo-obstruction in a group of patients. As far as the authors are aware, this is the first report confirming structural muscle deficits in Hirschsprung disease.Patients and methodsEthical permission and informed consent were obtained. DNA was extracted from whole blood samples in 10 patients with histologically proven HSCR patients. PCR amplification of the ACTG2 gene, were subjected to semi-automated bi-directional sequencing analysis. Sequencing results were analyzed using FinchTV Sequence Alignment Software (http:/en.biosoft.net) to read chromatogram files. Further predicting bioinformatic investigation was obtained by PolyPhen 2 software to evaluate the significance of the observed amino acid changes.ResultsTen new patients with similar HSCR phenotypes were prospectively investigated for variation in the Actin G2 gamma gene (ACTG2) variations. The results of ACTG2 gene analysis showing variation in exons 5, 8 and 10 of the ACTG2 gene in 7 of them (64%). The c.109C > G S345 L was the most frequent occurring in 6 of the 10 patients (54%), the c.171 A > A K119E in 2 and the significant c.108 T > G W357G variation in exon 10 (1 patient) Four patients had a combination of different variants in different exons which were less significant. Allele frequency on a control sample of the South African population showed no comparable pathology link scores (http://gnomad.broadinstitute.org/). Bioinformatic in silico modeling showed that the residue replacements in both variants (Lys to Glu and Trp to Gly) are highly non-conservative and variation can alter interactions within the protein conformation.ConclusionsThe Actin smooth muscle gene showed variation in 64% of samples, indicating a reason for abnormal functioning muscle in many HSCR patients. Hirschsprung disease is part of a complex spectrum which also includes smooth muscle.Level of EvidenceVI     

Changes in intestinal motility and in the myenteric plexus in a rat model of intestinal ischemia-reperfusion

Background/Purpose

Early histologic changes induced by intestinal ischemia-reperfusion injury (IIRI) have been extensively studied using animal models. However, information regarding late effects on intestinal motility is lacking. The aim of this study was to investigate the late effects of IIRI on myenteric plexus histology and intestinal motility.

Materials and Methods

Thirty-two postweaning male mice weighing between 58 and 103 g were divided randomly into 4 groups: Control (unoperated), Sham (celiotomy), 30-minute ischemia (celiotomy and superior mesenteric artery ischemia for 30 minutes), and 45-minute ischemia (celiotomy and superior mesenteric artery ischemia for 45 minutes). Postoperative intestinal motility was assessed by weighing total fecal output for 24 hours on the 3rd, 7th, 14th, and 21st day after surgery. Segments of duodenum, jejunum, and ileum were examined at light microscopy for changes in the myenteric plexus.

Results

Three weeks after IIRI, the ganglion cells from the myenteric plexus appeared in light microscopy, spongy or foamy, containing many vacuoles in their cytoplasm. The neuronal nucleus became irregular, with degenerative signs. These alterations did not occur among animals from the control or sham groups. Although the animals of the 45-minute ischemia group showed a significant drop in fecal output in the 21st postoperative day, this appeared to have no effect on weight gain.

Conclusions

The results suggest that intestinal ischemia-reperfusion causes late neuronal damage. These changes resulted in alterations of intestinal motility, which, within the conditions of the present study, had no repercussion on general weight gain.