Embryonic development of the ganglion plexuses and the concentric layer structure of human gut: a topographical study

In this study, we performed a detailed topographical study on the development of ganglion plexuses and the smooth muscle layers of human embryonic and fetal gut. Neuron and glia differentiation was investigated with anti-PGP9.5 and anti-S100 antibodies respectively. The differentiation of smooth muscle and interstitial cells of Cajal (ICC) was studied with anti-smooth muscle -actin and anti-C-Kit antibodies respectively. By week 7, rostro-caudal neural crest cell (NCC) colonization of the gut was complete, and NCCs have differentiated into neurons and glia. At the foregut, neurons and glia were aggregated into ganglion plexus in the myenteric region, and the longitudinal and circular muscle layers have started to differentiate; however, neurons and glia were not found in the submucosa. At the hindgut, neurons and glia were dispersed within the mesenchyme. Myenteric plexus, longitudinal and circular muscle layers formed along the entire gut by week 9. Scattered and individual neurons and glia, and small ganglion plexuses were detected in the foregut and midgut submucosa by week 12. Ganglion plexus was not seen in the hindgut submucosa until week 14. Muscularis mucosae was formed at the foregut and midgut by week 12 but was only discernible at the hindgut 2 weeks later. As the gut wall developed, ganglion plexus increased in size with more neurons and glia, and the formation of intra-plexus nerve fascicle. ICCs were localized in the ganglion plexus as early as week 7. ICCs were initially dispersed in the plexus and were preferentially localized at the periphery of the plexus by week 20. The specification of the annular layers of human embryonic and fetal gut follows a strict spatio-temporal pattern in a rostro-caudal and centripetal manner suggesting that interaction between (1) homotypic and/or heterotypic cells; and (2) cells and the extracellular matrix is critical for the embryonic development of the gut mesenchyme and the enteric nervous system.     

Spatiotemporal expression of BMP7 in the development of anorectal malformations in fetal rats

The aim of this study was to determine the expression patterns of bone morphogenetic protein 7 (BMP7) during anorectal development in normal rat embryos and in embryos with anorectal malformations (ARM), and to investigate the possible role of BMP7 in the pathogenesis of ARM. ARM was induced by treating rat embryos with ethylenethiourea on the 10th gestational day (GD10). Embryos were harvested by Cesarean delivery and the spatiotemporal expression of BMP7 was evaluated in normal (n=168) and ARM embryos (n=171) from GD13 to GD16 using immunohistochemistry staining and western blot analysis. Immunohistochemical staining in normal embryos revealed that BMP7 was abundantly expressed on the epithelium of the urorectal septum (URS) and the hindgut on GD13, and BMP7-immunopositive cells were extensively detected in the URS, hindgut, and cloacal membrane by GD14. Increased positive tissue staining was noted on the fused tissue of the URS and the thin anal membrane on GD15. In ARM embryos, the epithelium of the cloaca, URS, and anorectum were negatively or only faintly immunostained for BMP7. BMP7 protein expression showed time-dependent changes in the developing hindgut according to western blotting, and reached a peak on GD15 during anus formation. BMP7 expression levels from GD14 to GD15 were significantly lower in the ARM group compared with the normal group (P<0.05). Spatiotemporal expression of BMP7 was disrupted in ARM embryos during anorectal morphogenesis from GD13 to GD16. These results suggest that downregulation of BMP7 at the time of cloacal separation into the primitive rectum and UGS might be related to the development of ARM.     

Spatiotemporal expression of Wnt5a during the development of the striated muscle complex in rats with anorectal malformations

Fecal incontinence and constipation after procedures for anorectal malformations (ARMs) are closely related to the maldevelopment of the striated muscle complex (SMC). Previous studies have demonstrated that myogenic regulatory factors (MRFs) play a significant role in muscle development. Wnt signal pathway is extremely important for MRFs regulation. This study was designed to investigate the spatiotemporal expression pattern of Wnt5a in SMC in ARMs rat embryos. Materials and Methods: Anorectal malformation embryos were induced by ethylene thiourea on embryonic day 10 (E10). Expression levels of protein and mRNA of Wnt5a were confirmed by immunohistochemistry staining, western blot and quantitative real-time PCR (qRT-PCR) between normal rat embryos and embryos with ARMs. Results: Immunostaining revealed that, on embryonic day 17 (E17), the Wnt5a protein was initially expressed in the SMC in normal embryos. With the growth of pregnancy, the positive staining cells gradually increased. The same time-dependent changes of Wnt5a protein were detected in ARMs embryos. Besides, immunostaining showed that Wnt5a had a significant increase in normal embryos compared with ARMs embryos. Similarly, in Western blot and qRT-PCR, the higher expression of Wnt5a protein and mRNA were remarkable in normal embryos during the SMC development, relatively. Conclusion: Our study demonstrated that the downregulation of Wnt5a at the time of SMC development might partly be related to the dysplasia of SMC in ARMs.     

Adaptive remodelling of smooth muscle in the neo-intima of vein-to-artery grafts in rats: a detailed morphometric analysis

Summary End-to-end autogenous vein-to-artery grafts in rats have been used extensively as a model for neointimal thickening (hyperplasia), which develops over the first 6 weeks after grafting. This study employed computerised morphometric techniques to analyse 16 grafts, in order to quantitate precisely how the neointima develops. Two important features were described that have not been identified previously, due to the extensive variation in neo-intimal thickness inherent in vein grafts. Firstly, the proximal region of the graft was significantly thicker than the distal region, up until 6 months after grafting. The smooth muscle cells in the graft may have developed more rapidly in the proximal region, due to the altered haemodynamics within the graft. Secondly, within the central region of the graft the characteristic focal nature of neo-intimal hyperplasia was evident throughout the period of the study, but by 6 months the neo-intima tended to be distrubuted more evenly. By 6 months remodelling of smooth muscle throughout the graft neo-intima had occurred, and the neo-intima had matured to a thickness equivalent to that of the intima plus media of the adjacent iliac artery.     

Tin mesoporphyrin in conjunction with retinoic acid reverses the retinoic acid induced enhancement of phospholipase A(2) activity in vivo in rats

Retinol (Vitamin A) is known to destabilize membranes. The effect of one of its natural derivatives, Retinoic Acid, was therefore studied on membrane stability, by inversely correlating this stability with Phospholipase A(2) activity. Lipid metalloporphyrin interactions have been the object of intense research in recent times. Our results revealed that the administration of Retinoic Acid (50,000 I.U.) caused a tremendous induction of hepatic Phospholipase A(2) activity in Wistar rats. However when Retinoic Acid and Tin-mesoporphyrin (50 micromol/kgbw) were co-administered, hepatic Phospholipase A(2) activity was inhibited. This result clearly reveals a possible therapeutic application of this metalloporphyrin in alleviating membrane destabilization, connected with Retinoic acid administration.     

Abnormal innervation patterns in the anorectum of ETU-induced fetal rats with anorectal malformations

Valproate (VPA) is commonly used in the treatment of bipolar disorder and epilepsy. The mechanism underlying its clinical efficacy is complicated, including its ability to inhibit histone deacetylase (HDAC). Here, we show that VPA promoted endoplasmic reticulum (ER) chaperone expression and attenuated ER-induced apoptosis after ischemia/reperfusion (I/R) injury in retina. Male Wistar rats were randomly divided into four groups: sham (group A), sham + VPA (group B), I/R + vehicle (group C), and I/R + VPA (group D). VPA was administered subcutaneously at 300 mg/kg twice daily before insult. Morphological changes were analyzed on stained histological sections and flat-mounted retinas labeled by Fluoro-gold. Western blot analysis was used to determine protein levels of GRP78, CHOP, caspase-12 and acetylation of histone H3 in each group. In group C, the severe retinal damage was shown in histological sections, however, the damage was reduced by VPA in group D. Significant loss of retinal ganglion cells (RGCs) was observed in group C, whereas, the density of RGCs was significantly higher in group D at 7 days post-insult. VPA increased GRP78 expression and acetylation of histone H3, attenuated upregulation of CHOP and activation of caspase-12 in group D. Our results suggest that VPA can protect ischemic retinas from ER stress-induced apoptosis by mechanisms that may involve HDAC inhibition.     

Differentiation of smooth muscle in the genital tract of the female mouse and its temporal relation with the development of the Wolffian nerve

Summary The development of the smooth muscle in the genital tract of the female mouse was studied by light and electron microscopy before and after birth. These studies showed that: a) between 13 days of fetal development and 2 days after birth the cells surrounding the Mullerian duct were undifferentiated and showed a fibroblast-like appearance; b) between 3 and 10 days after birth the cells acquired several characteristics of smooth muscle but they did not seen fully mature; c) between 30 and 180 days after birth the cells acquired a mature appearance; and d) the Wolffian nerve reached the Mullerian duct surrounding tissue before the start of smooth muscle differentiation.     

Enhancement of muscular performance by a coformulation of propionyl-L-carnitine,coenzyme Q10, nicotinamide,riboflavin and pantothenic acid in the rat

A coformulation of essential factors, i.e. propionyl-L-carnitine (PLC), coenzyme Q10 (CoQ10), nicotinamide (NAM), riboflavin and pantothenic acid, was administered orally to Wistar rats for 7 weeks and its efficacy was tested through in vivo and in vitro techniques in improving motor functions of striated, cardiac and smooth musculature of the rat. In vivo experiments showed that long-term supplementation significantly improved horizontal locomotor activity by about 19% in male and 26% in female rats. Maximum values of shortening velocity, work and power were significantly increased (P<.05) in papillary muscle isolated from treated rats. A positive inotropic effect was also observed on colonic smooth muscle strips upon treatment. Work was the most affected parameter and it increased by 160% in smooth muscle from treated animals. The present results indicate that supplementation with the combination of the above mentioned substances elicits positive functional changes on motor performance of skeletal, cardiac and smooth muscle of the rat.     

热处理延缓维甲酸诱导P19细胞的生长及向神经元分化

研究热环境对P19细胞生长和RA诱导的向神经元分化的影响,为探讨热环境刺激对神经系统发育的影响提供参考。成熟的P19细胞在热环境的条件下培养1h后,消化接种至培养皿中正常培养,于不同时间点取样分析细胞数目。热环境处理P19细胞后用维甲酸诱导分化4d,随后接种至培养皿中,参照神经元的原代培养方法培养12d,用神经元抗体免疫鉴定,计数不同培养时间NSE、NF阳性神经元在总体细胞中的比例。结果发现接受热处理的P19细胞贴壁不良,生长始终缓慢,说明热环境刺激阻止细胞生长分裂。P19细胞经RA诱导分化后出现类似神经细胞的突起和纤维网络,而热环境导致P19细胞向神经元分化明显迟缓,NF阳性细胞比例增加。     

Spectral analysis of spontaneous contractions of the isolated portal vein of rats

Summary The spontaneous contractions of segments of rat portal veins have been examined in vitro under isotonic and isometric conditions. The power density spectra of recorded time series lasting 10–60 min were calculated. The spectra usually consist of harmonic frequency components. Only during shorter periods of analysis (10 min time series) we sometimes found additional non-harmonic components. All frequency components are proportionally shifted by changes of the bath temperature according to an average Q₁₀ of 2.0. Increase of the load decreases the frequency of the contractions.The results of the spectral analysis, indicating a preponderance of a single source of periodicity, were supported by direct evidence of a pacemaker region. By recording contractions after systematic dissections of the portal vein segment, we found that spontaneous activity is generated at the central end of the segment.This work was supported by the Austrian Research Fund     

咪达普利对实验性骨质疏松大鼠的骨代谢和生物力学的影响

目的:探讨咪达普利对维甲酸所致的实验性骨质疏松的大鼠骨代谢和生物力学的影响.方法:将60只SD雌性大鼠随机分为3组:对照组(Ⅰ组)、维甲酸诱导骨质疏松模型组(Ⅱ组)、咪达普利组(Ⅲ组),每组20只.对Ⅱ组和Ⅲ组的大鼠用维甲酸80mg/(kg.d)连续灌胃21d建立实验性骨质疏松模型.造模后,Ⅲ组给予按大鼠体重10 mg/(kg.d)咪达普利灌胃,Ⅰ组和Ⅱ组采用生理盐水对照,持续7d后处死大鼠,分别检测各组大鼠的体重、股骨骨密度和干湿重、骨代谢指标和骨生物力学指标.结果:与Ⅰ组相比,Ⅱ组在大鼠体重、股骨骨密度、股骨干湿重、骨代谢指标上差异均有统计学意义(P＜0.05).咪达普利干预后,与Ⅱ组相比,Ⅲ组大鼠的体重增加,股骨干湿重增加,差异有统计学意义(p＜0.05);股骨中钙和磷的含量明显增加,差异有统计学意义(p＜0.05),血清中骨钙素(bone gamma-carboxyglutamic-acid-containing proteins,BGP)浓度降低,差异有统计学意义(P＜0.05);抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)浓度下降,差异有统计学意义(P＜0.05);肱骨生物力学最大载荷(Pmax)、最大挠度(Lmax)、最大弯矩(Mmax)、骨应力(σ)和骨应变增加,差异有统计学意义(P＜0.05).结论:咪达普利能增加维甲酸所致的实验性骨质疏松大鼠骨密度,增加股骨干湿重,减少骨质流失,促进骨形成和矿物质的沉积,增加骨的生物力学强度,减少骨折的发生,对骨质疏松具有保护作用.     

Dual excitatory actions of acetylcholine at the neuromuscular junction in the guinea-pig vas deferens

The action of acetylcholine (ACh) on the smooth muscle of guinea-pig vas deferens was studied using the sucrose-gap method. ACh, when applied at a concentration of 10^–6 M, evoked a depolarization of the smooth muscle membrane which was slow in time course (slow depolarization). When ACh was applied at higher concentrations, another depolarization which was fast in time course (fast depolarization) occurred, overlapping the early part of the slow depolarization. The magnitudes of both depolarizations were concentration-dependent on ACh. TTX and adrenergic receptor antagonists had little effect on either depolarizations, while guanethidine and nicotinic receptor antagonists mainly suppressed the fast depolarization. In contrast, atropine suppressed the slow depolarization. The membrane conductance observed by current application, was reduced during the slow depolarization, and the reversal potential of the depolarization was 18.3 mV negative to the resting membrane potential. Whereas, the reversal potential of the fast depolarization was 27.6 mV positive to the resting membrane potential. This reversal potential was quite similar to that of the adenosine triphosphate (ATP)-induced depolarization, previously observed in the same tissue. From these observations, it is suggested that in the guinea-pig vas deferens, ACh acts on nicotinic receptors at the sympathetic postganglionic nerve terminal, causing the release mostly of a non-adrenergic transmitter, probably ATP. In addition, ACh also acts on muscarinic receptors on the smooth muscle membrane, inducing membrane depolarization resulting from a reduction of the membrane conductance to potassium ions.     

内皮细胞Jagged1下调促进PDGF诱导的大鼠平滑肌细胞增殖迁移

目的： 探讨内皮细胞Jagged1表达对PDGF诱导的大鼠血管平滑肌细胞增殖迁移的调节作用。方法: 分离培养大鼠主动脉内皮和平滑肌细胞,将内皮接种于下室、平滑肌接种于上室建立细胞共培养体系,根据内皮是否行Jagged1小RNA干扰分为对照组、空载体组和Jagged1小RNA干扰组。用Western blotting检测内皮细胞Jagged1的干扰效率。于下室加入PDGF（10 μg/L）干预24 h后分别用［3H］-TdR 掺入和平滑肌迁移计数检测平滑肌细胞增殖迁移能力,用Western blotting检测平滑肌细胞α-SM-actin蛋白表达。结果: 与对照组相比空载体组内皮细胞Jagged1蛋白表达无明显差异,Jagged1小RNA干扰组内皮细胞Jagged1蛋白吸光度相对值明显降低（0.26±0.02 vs 0.67±0.02, P<0.05）;PDGF+空载体组平滑肌［3H］-TdR 掺入量和迁移数与PDGF组相比无明显差异,PDGF+Jagged1小RNA干扰组平滑肌［3H］-TdR 掺入量和迁移数高于PDGF组{［3H］-TdR 掺入(23 074±2 702)counts·min-1·well-1 vs (16 442±1 803)counts·min-1·well-1,n=5,P<0.05;迁移(27±4)cells/field vs (15±3) cells/field, n=5,P<0.05};PDGF+空载体组平滑肌α-SM-actin蛋白表达与PDGF组相比无明显差异,PDGF+Jagged1小RNA干扰组平滑肌α-SM-actin吸光度相对值低于PDGF组（0.25±0.06 vs 0.49±0.04, n=3,P<0.05）。结论: 内皮细胞Jagged1下调促进PDGF诱导的平滑肌细胞增殖迁移,提示血管内皮细胞Jagged1表达在维持平滑肌收缩表型、抑制平滑肌过度增殖迁移中起一定调控作用。     

Insulin enhances angiotensin II induced DNA synthesis in vascular smooth muscle cells of the rat

Summary Hypertension has a high prevalence among subjects with decreased insulin sensitivity and/or hyperinsulinemia. Furthermore, angiotensin II plays a pivotal role in the regulation of vascular tone and is known to induce hypertrophy and/or hyperplasia in vascular smooth muscle cells. In the present study, the effect of insulin on angiotensin II induced smooth muscle cell growth (Wistar-Kyoto rat) was investigated. Cell growth was assessed by the measurement of [³H]thymidine incorporation into cell DNA. Insulin in a concentration range of 1.7 × 10^–10–1.7 × 10^–6 M lacked any effect on cell DNA synthesis. However, insulin enhanced the angiotensin 11 induced DNA synthesis in a concentration-dependent manner. This effect was similar in cells with a weak and in cells with a marked response in DNA synthesis to stimulation with 100 nM angiotensin 11. In conclusion, insulin is able to enhance angiotensin 11 induced DNA synthesis and may therefore function as a growth cofactor in vascular smooth muscle cells.Abbreviations AngII angiotensin II - EGF epidermal growth factor - bFGF basic fibroblast growth factor - PDGF-BB platelet-derived growth factor-BB - VSMC vascular smooth muscle cells Dedicated to Prof. Dr. N. Zöllner on the occasion of his 70th birthday     

Qualitative and quantitative analysis of changes in caudate nucleus neurons during postnatal development of rats

A qualitative and quantitative analysis was made of changes in caudate nucleus neurons of newborn rats and rats aged 7, 14, and 30 days. An increase in complexity of structure of components of the nerve cell was found during early postnatal ontogeny. Maturation of neurons takes place most intensively during the first 2 weeks of life and is virtually complete by the 30th day of the postnatal period. The results suggest that the caudate nucleus becomes involved in the integrative activity of the brain in this period of development.Laboratory of Functional Morphology and Pathohistology of the Brain and Laboratory of Architectonics of the Brain, Brain Institute, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kupriyanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 7, pp. 94–96, July, 1978.     

结缔组织生长因子在单侧输尿管梗阻大鼠肾组织中的表达

目的：检测大鼠单侧输尿管梗阻（UUO）模型不同时期，肾组织中结缔组织生长因子（CTGF）、转化生长因子β1（TGF-β1）和α-平滑肌肌动蛋白（α-SMA）的表达，观察比较在间质纤维化不同阶段，3者的动态变化及关系。 方法： 采用雄性SD大鼠36只，分为假手术组和模型组，模型组行左侧输尿管结扎术，再分3、7、14、21和28 d共6组，每组6只，于各时点处死大鼠，取肾组织，常规HE、Masson染色，按小管间质损害的特征进行半定量评分。免疫组化检测CTGF、TGF-β1和α-SMA表达。 结果： 随梗阻时间的延长，小管间质纤维化加重，28 d间质已基本被纤维化组织所代替。随间质纤维化程度的加重，CTGF和α-SMA表达逐渐增加，两者与小管间质损害积分呈正相关，CTGF与α-SMA的表达之间也呈正相关。TGF-β1表达在7-14 d达高峰后，逐渐减少，但仍高于对照组。 结论： UUO致CTGF表达增加可能与TGF-β升高有关，CTGF可能通过促进间质中肌成纤维细胞的形成而参与肾间质纤维化。     

Inhibition of medullary reticulospinal neurons by excitation of the dorsolateral parts of the pons which block movement and muscle tone in rats

Analysis of the response of 128 reticulospinal neurons in the magnocellular and ventral reticular nuclei showed that 36.7% of these cells responded with short-latency (2–4 msec) action potentials and increased their tonic activity in response to electrical stimulation of the central parts of the hypothalamus, which evoked increases in hindlimb muscle tone in rats. These cells completely stopped producing action potentials during electrical stimulation and during chemical stimulation of the dorsolateral parts of the pons, which inhibited movement and muscle tone. A total of 23.4% of the cells produced only short-latency (1–4 msec) action potentials in response to stimulation of the inhibitory parts of the pons. A total of 3.9% of reticulospinal neurons increased their activity during stimulation of the hypothalamic zones and pontine areas of the brain. No responses were obtained from 35.9% of neurons. It is suggested that excitation of pontine structures inhibiting movement and muscle tone may prevent conduction in descending activatory systems from the rostral parts of the brain (which increase muscle tone) to the reticulospinal neurons of the medulla oblongata. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 3, pp. 353–359, March, 1999.     

Prenatal effects of retinoic acid on lumbar spinal cord development and liver antioxidants in rats

During embryonic and early postnatal development, retinoic acid (RA) regulates genes that control neuronal differentiation and neurite outgrowth from the neural tube. The effects of high levels of RA on the CNS can be detected via nitric oxide (NO), which plays a crucial role in neural transmission. The aim of the study was to investigate the prenatal influence of high levels of RA on postnatal development of nitrergic structures in lumbar spinal cord and antioxidant status. RA was administered orally at a dose of 10 mg/kg body weight to pregnant female Wistar rats during days 8–10 of gestation. Neuronal nitric oxide synthase (nNOS) of lumbar spinal cord sections was processed for visualization via nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry on postnatal day one, day twenty-one and in adults. The results suggest that prenatal administration of high levels of RA is not associated with postnatal morphological changes in nNOS-positive neurons in the rat lumbar spinal cord. An estimation of the activity of enzymes related to the storage of retinoid in the liver showed possible side effects. Suppression and deepening of superoxide dismutase activity persisted into adulthood, and a concurrent downregulation of glutathione reductase was noted. A decrease in reduced glutathione persisted until adulthood when other compensatory mechanisms were probably active to maintain an appropriate level.