Counterstaining improves visualization of the myenteric plexus in immunolabelled whole-mount preparations.

Immunocytochemistry has emerged as a powerful research tool in neurobiology. One of the widely used methods is an indirect fluorescence technique that uses FITC- conjugated IgG to visualise protein expression within tissues, but a major drawback of this technique is the high background fluorescence due to non-specific antibody binding. Gut innervation is complex and best visualized in three-dimensions in whole mount preparations. We describe a simple and easy to use counterstaining procedure in conjunction with an indirect immunofluorescence technique in gut whole mount preparations that largely eliminates background fluorescence and creates a contrasting background against the bright antigen-antibody complexes. Furthermore, this technique allows the detailed qualitative and quantitative study of myenteric plexuses in whole-mount preparations.

Aberrations of the intrinsic innervation of the anorectum in fetal rats with anorectal malformations.

BACKGROUND: Fecal accumulation, constipation, soiling, and incontinence are common sequelae after repair of anorectal malformations (ARMs) in children. It is believed that besides the abnormalities of sacral roots, certain inherent abnormalities of the myenteric plexuses may play an important role in the final outcome after definitive repair. METHODS: This study was conducted to investigate the distribution of neuron-specific enolase (NSE), vasoactive intestinal peptide (VIP), and substance P (SP)-100 neurotransmitters in the rectosigmoid and fistulous tract of the ethylenethiourea-treated rat with ARMs. RESULTS: ARMs were induced by administering 1% ethylenethiourea (125 mg/kg) on gestational day 10, and the litter was harvested on gestational day 21 by cesarean section. Forty-eight controls and 63 with ARMs (46 high-type and 17 low-type) were recovered. Whole-mount preparations of each rectosigmoid and fistulous communication between the rectum and genitourinary tract were stained with fluorescent antibodies against NSE, VIP, and SP-100. The tissues were counterstained with Eriochrome black-T and methyl green dyes to improve the visualization of the myenteric plexuses. CONCLUSIONS: The immunoreactivity of NSE, VIP, and SP-100 was markedly reduced in the rectum and fistulous tract of high-type ARMs and slightly reduced in low-type ARMs compared with controls. Intramural nerves stained by VIP and SP-100 antisera were decreased in both types of ARM, indicating that both inhibitory and excitatory motor neural elements were affected, and this may explain the distal colonic dysmotility seen postoperatively in both high and low ARMs.

Abnormalities of the vertebral column and ribs associated with anorectal malformations.

Lumbosacral vertebral abnormalities are a common association of anorectal malformations (ARMs) and are one of the determinants of the eventual level of fecal continence that can be achieved. This study used a fetal rat model to investigate the spectrum of axial skeletal maldevelopment that may occur with ARMs. Time-mated pregnant rats received 125 mg/kg of 1% ethylenethiourea (ETU) (experimental group) or vehicle only (control). Their fetuses were examined for external malformations and prepared for staining of their skeletons using Alcian blue and Alizarin red S. ARMs developed in 67/68 (98%) of ETU-exposed fetuses, of which 28 (42%) also developed rachischisis, mainly involving the lumbosacral vertebrae. No skeletal abnormality was found in control fetuses. ETU-exposed fetuses with ARMs and rachischisis had abnormal ossification of the vertebral centrum, abnormal fusion between the neural arches of vertebrae, localized narrow or interrupted thoracic vertebral canal, a widely open vertebral canal in the lumbosacral area (rachischisis), and absence of the lower two sacral and coccygeal vertebrae. Rib abnormalities included absence of two to three floating ribs, abnormal fusion of adjacent proximal segments, and abnormal ramification, irregularity, and angulation of their distal segments. The vertebral and rib abnormalities found in ETU-exposed fetuses with ARMs but no rachischisis were much less severe. In addition to the lumbosacral anomalies that are common with ARMs, severe abnormalities of the thoracic vertebrae and their corresponding ribs may occur also. Fetuses with both ARM and rachischisis tend to have more extensive and severe vertebral and rib anomalies. These observations imply a possible common aetiology for ARMs and vertebral anomalies and are consistent with our understanding of the perceived role of the notochord in axial development.

Evidence that the notochord may be pivotal in the development of sacral and anorectal malformations.

BACKGROUND/PURPOSE: The notochord is known to organize normal development of central axial structures, such as the spinal cord, vertebral column, and anorectum, but its role in abnormal development of these organs has not been well documented. The current study has used Ethylenethiourea to induce anorectal malformations in fetal rats, allowing investigation of abnormalities of the notochord and their relationship to the axial structural abnormalities that occur. METHODS: Timed-mated pregnant rats were fed Ethylenethiourea by gavage on gestational day 10. Their embryos were harvested on gestational days 13 to 16 and sectioned in either the transverse or sagittal plane. Sections were stained with H and E and examined serially. RESULTS: Anorectal malformations were identified in 29 of 34 embryos and neural tube defects in 24, ranging from an accessory neural tube to lumbo-sacral rachischisis. There was no tail or only a rudimentary tail in the majority of embryos. Abnormalities of the notochord in the lumbo-sacral area included ventro-dorsal branching, ventral deviation, and ectopic notochordal tissue. Most abnormal notochord branches and ectopic notochordal tissue were abnormally close to or in contact with the wall of the cloaca or neural tube. CONCLUSIONS: Given the known role of the notochord in controlling normal development, this study would suggest that abnormal notochord development may be pivotal in producing neural tube defects and anorectal malformations, possibly by altering sonic hedgehog signalling.

Clarification of the processes that lead to anorectal malformations in the ETU-induced rat model of imperforate anus.

BACKGROUND: Ethylenethiourea (ETU) administered to timed-pregnant rats can induce anorectal malformations (ARMs) in about 80% of rat fetuses, thus providing an ideal animal model to study the embryogenesis of ARMs. The current study was undertaken to investigate the embryogenetic events that may be responsible for the development of ARMs in rats. METHODS: Time-mated pregnant rats were divided randomly into control and experimental groups. The experimental rats received 1% ETU (125 mg/kg) by gavage on gestational day 10, and control rats received only the vehicle. Their embryos were harvested by cesarean section on gestational days 13, 14, 15, and 16. They were fixed and embedded in paraffin and serially sectioned in either the sagittal or transverse plane. The sections were stained with H&E, examined, and photographed. The comparative morphogenesis of the hindgut, cloaca, and tailgut of age-matched embryos was studied. RESULTS: The key abnormalities in the experimental embryos were: (1) maldevelopment of cloaca and urorectal septum with no sign of the fusion between the urorectal septum and the cloacal membrane, (2) delay of tailgut regression, (3) abnormal and massive apoptotic cell death involving the posterior cloacal wall, and (4) underdevelopment of the dorsal aspect of the cloaca and its membrane. The type of ARM that was developing was discernible by gestational day 15 and 16. CONCLUSIONS: ARMs induced by ETU in rat embryos seem to be caused by the cumulative effect of aberrations in the development of several components of the hindgut and cloaca. Variation in the extent of maldevelopment of these structures may result in a spectrum of ARMs.

Patterns of apoptosis during degeneration of the pronephros and mesonephros.

PURPOSE: The adult mammalian kidney is preceded developmentally by 2 primitive kidneys. We determine whether apoptosis is involved in the regression of these primitive organs and document its temporospatial characteristics. MATERIALS AND METHODS: Timed pregnant rats were sacrificed at 11 to 16 days of gestation inclusively. The fetuses were histologically examined to determine the timing and pattern of apoptosis in the primitive kidneys, including pronephros and mesonephros. RESULTS: Apoptosis of the pronephros occurred primarily at 12 days of gestation. Apoptosis of the distal mesonephros occurred primarily at 13 days and was completed by 14, which left the proximal mesonephros a functioning kidney during the early period of development. The location and timing of apoptosis were consistent and specific. CONCLUSIONS: Apoptosis appears to be an important mechanism of the normal regression of the primitive kidney and follows a strict temporospatial pattern.