Developmental abnormalities of the otic capsule and inner ear following application of prolyl-hydroxylase inhibitors in chick embryos.

BACKGROUND: Naturally hypoxic conditions in amniote embryos play important roles in normal development. We previously showed that a hypoxic condition is required to produce a sufficient amount of neural crest cells (NCCs) during embryogenesis and that promoting a hypoxic response by prolyl-hydroxylase (PHD) inhibitors increases NCCs. Given that PHD inhibitors are considered as a potential treatment for anemia and ischemic diseases, we investigated the phenotypic effect of PHD inhibitors on embryonic development. METHODS: Chick embryos were administered with PHD inhibitors prior to the induction of NCCs on day 1.5. Three main events relating to hypoxia, NCCs induction, vasculogenesis and chondrogenesis, were examined. RESULTS: PHD inhibitors caused an increase of Sox10-positive NCCs in vivo. Vasculogenesis was promoted temporarily, although rapid vasculogenesis diminished the effect by day 5 in cephalic and pharyngeal regions. Studies on chondrogenesis at day 7 showed advanced development of the otic capsule, a cartilaginous structure encapsulating the inner ear. Analysis by X-ray micro-computed-tomography (µCT) revealed smaller otic capsule, suggesting premature differentiation. This in turn, deformed the developing semicircular canals within it. Other skeletal structures such as the palate and jaw were unaffected. The localized effect on the otic capsule was considered a result of the multiple effects from the hypoxic responses, increased NCCs and promoted chondrogenesis. CONCLUSION: Given the wide range of clinical applications being considered for PHD inhibitors, this study provides crucial information to caution and guide use of PHD inhibitors when treating women of childbearing age.

An unreported variation of the cervical vagus nerve: anatomical and histological observations.

Variations involving the cervical portion of the vagus nerve are seemingly very rare. We report an adult male found to harbour a right cervical vagus nerve that crossed anterior to the right common carotid artery to terminate in the lateral aspect of the thyroid gland. A very small continuation of this nerve was found to continue distally into the thorax. Histologically, this part of the vagus nerve did not contain ganglion or other cell bodies. There were no heterologous inclusions (thyroid, parathyroid, thymus, salivary gland or branchial cleft remnants) present. Although grossly there was a connection into the thyroid gland, this was not observed histologically. No signs of trauma were found to the ipsilateral neck region. We hypothesise that this variation is due to entanglement between the thyroid gland and cervical vagus nerve during development. This rare variation might be considered by the clinician who operates in the cervical region or interprets imaging of the neck. To our knowledge, a vagus nerve with the above described morphology has not been described.

Development of cranial parasympathetic ganglia requires sequential actions of GDNF and neurturin.

The neurotrophic factors that influence the development and function of the parasympathetic branch of the autonomic nervous system are obscure. Recently, neurturin has been found to provide trophic support to neurons of the cranial parasympathetic ganglion. Here we show that GDNF signaling via the RET/GFR(alpha)1 complex is crucial for the development of cranial parasympathetic ganglia including the submandibular, sphenopalatine and otic ganglia. GDNF is required early for proliferation and/or migration of the neuronal precursors for the sphenopalatine and otic ganglia. Neurturin exerts its effect later and is required for further development and maintenance of these neurons. This switch in ligand dependency during development is at least partly governed by the altered expression of GFR(&agr;) receptors, as evidenced by the predominant expression of GFR(&agr;)2 in these neurons after ganglion formation.

Cat eye syndrome associated with aganglionosis of the small and large intestine.

A newborn male infant is presented with the characteristic phenotype of the cat eye syndrome and a small supernumerary chromosome shorter than a 22. He also had complete absence of parasympathetic ganglion cells throughout the small and large intestine.

Deficient neurogenic innervation of the myenteric plexus with normal submucous plexus involving the entire small and large bowel.

A newborn presented with a picture of intestinal obstruction. Multiple biopsies of the small and large bowel showed an unusual neurogenic innervation. The myenteric plexus of Auerbach was severely depleted of ganglion cells and nerve fibers, while the submucous plexus of Meissner was normally innervated. An ileostomy failed to function and extended trial with experimental smooth muscle stimulant (Cisapride) was equally ineffective. The patient was finally treated by a myectomy from the duodenum to the descending colon with a sigmoid colostomy. This procedure, coupled with a Nissen fundoplication, stopped the vomiting and allowed normal defecation through the colostomy. The patient is presently taking increasing increments of oral fluids with a concomitant decrease in the volume of parenteral nutrition. The myectomy initiated marked hypertrophy of the muscularis mucosa. Could this muscular hypertrophy account for the improvement in bowel function? Possible etiology will be discussed. We caution that rectal submucosal suction biopsy alone may be misleading if normal ganglion cells and nerve fibers are found, yet the patient's clinical symptoms fail to improve. A full thickness bowel wall biopsy is then recommended.

Ultrastructural localization of acetylcholinesterase activity in Hirschsprung's disease.

Localization of acetylcholinesterase activity in congenital megacolon was studied by light and electron microscopy. Acetylcholinesterase activity was strongly positive at the light microscopic level in the Auerbach's plexus of the normal segment and in proliferated nerve fibers of the aganglionic segment. The reaction product observed by electron microscopy was deposited in and between the plasma membranes of the ganglion cells, nerve fibers, and their terminals. The product was also observed in the rough endoplasmic reticulum, nuclear envelope, and Golgi apparatus of the ganglion cells. Acetylcholinesterase activity in the aganglionic segment was observed in and between the plasma membranes of nerve fibers and nerve terminals, which terminated in proximity to smooth muscle cells. Reaction deposits were also observed in the interspace between nerve terminals and smooth muscle cells, suggesting direct innervation of smooth muscles by extrinsic nerve fibers.

Diagnosis of congenital neurogenic abnormalities of the bowel with monoclonal anti-neurofilament antibodies.

The diagnostic properties of two monoclonal, antineurofilament antibodies were tested in a retrospective investigation of bowel specimens of 24 cases of Hirschsprung's disease, eight cases of long-segment aganglionosis, eight cases of pseudo-obstruction, and six cases of chronic constipation. Immunohistochemical staining with the antibodies in combination with hematoxylin counterstaining revealed six distinctive and divergent pictures, demonstrating innervation disturbances in all cases. Apart from Hirschsprung's disease and long-segment aganglionosis, characteristic pictures also appeared for pseudo-obstruction and, diversely, for chronic constipation. In addition, anomalies were revealed in the ganglionic, proximal bowel of five patients with Hirschsprung's and two with long-segment aganglionosis, who had all suffered from chronic constipation postoperatively.

Hirschsprung's disease: catecholamine content, alpha-adrenoceptors, and the effect of electrical stimulation in aganglionic colon.

In order to assess abnormalities in the adrenergic mechanism in the intestine of Hirschsprung's disease, catecholamine concentrations, alpha-adrenoceptors, and the effect of electrical field stimulation were examined in aganglionic segments of colon or rectum. The aganglionic segment had a higher concentration of norepinephrine, assayed with high performance liquid chromatography with an electrochemical detector, whereas concentrations of epinephrine or dopamine were similar in normal and pathological segments. In four patients with extensive aganglionosis, the norepinephrine concentration in aganglionic colon segments decreased progressively in descending, transverse, and ascending colon. The tissue content of alpha-adrenoceptors and their affinity assayed from the specific binding of [3H]dihydro-alpha-ergocryptine appeared similar in normal and aganglionic segments of the rectosigmoidal colon. Electrical field stimulation of normal rectosigmoidal colon segments caused relaxation at low frequencies and contraction at a very high frequency. Relaxation was not abolished by blocking concentrations of propranolol or phentolamine. In aganglionic segments, the predominant response to electrical field stimulation was contraction, which was inhibited by either atropine or tetrodotoxin. These results indicate that an alpha-adrenergic system and cholinergic innervation apparently exist in aganglionic colon segments and that dysfunction of the colon appears to result from lack of a nonadrenergic inhibitory system.

Prenatal sonographic diagnosis of Hirschsprung's disease.

The prenatal sonographic presentation of Hirschsprung's disease is described. This report strongly suggests that bowel distention in Hirschsprung's disease may begin in the late third trimester of intrauterine life. The importance of early diagnosis and treatment cannot be overemphasized. Sonography may greatly contribute to the early diagnosis of Hirschsprung's disease.

Fibromuscular dysplasia of arteries in Hirschsprung's disease.

Abnormal arteries were demonstrated in the intestines of 8 of 25 children with Hirschsprung's disease. The histologic findings were similar to those seen in fibromuscular dysplasia with proliferation of smooth muscle cells in the thickened adventitia. We propose a new pathological entity, "adventitial fibromuscular dysplasia." The abnormal arteries were located only in the transitional zone, which includes the proximal aganglionic, the oligoganglionic, and the distal normoganglionic segments. These findings are compatible with the suggestion that the craniocaudal migration of ganglion cells might be interrupted by intestinal ischemia in the presence of abnormal arteries in the embryo.

Histochemical acetylcholinesterase reactions in total colonic aganglionosis.

Reliability of the histochemical diagnosis in total colonic aganglionosis is controversial. We studied histochemical acetylcholinesterase (AChE) reactions in three children with total colonic aganglionosis. In all three, there was an increase in AChE positive nerve fibers in the rectal mucosa, obtained when the patients were over the neonatal period. However, a proximal extension of the increase in AChE positive nerve fibers was noted in the distal end of the resected bowel obtained at the definitive operation in two cases out of three. In one, there was no proximal extension in the resected bowel, and in another, there was an increase in AChE positive nerve fibers in the mucosa of the ganglionic ileum. Our findings indicate that the histochemical study of AChE activity in rectal suction biopsy is useful, even in cases of total colonic aganglionosis.

A new approach to total colonic aganglionosis.

Two babies with total colonic aganglionosis treated by a modified Martin-Duhamel procedure are described. Ileostomy, long side-to-side ileocolic anastomosis and Duhamel's pull-through were the sequence of steps in this staged management. Neonatal ileostomy followed by ileocoloplasty proximal to the ileostomy facilitates home management of the new born patient who then produces well formed stools. This approach avoids neonatal pull-through and obviates the need for parenteral alimentation.

Abnormal differentiation of selected nuclear centers in the brain of a duck embryo associated with partial duplication of the primitive streak.

In a control set of duck embryos, an abnormal duck embryo of 16 days incubation was found which had two beaks as the only outward sign of duplication. The beaks were of equal size, each with upper and lower bills. Bill-clapping movements were absent. The embryo had two normal eyes placed one on either side of the head, and the rest of the body was normal in external appearance. Sections through the head revealed further duplication of the fore-, mid-, and hindbrain divisions. The medial half of each of the embryonic brain divisions, however, was greatly reduced. Two additional optic primordia were also noticed in sections, each of which was reduced to a mass of tissue representing a lens and a much-folded pigment epithelium. The orbital tissues associated with the rudimentary eyes were greatly disorganized. Abnormal differentiation associated with duplication of the brain divisions was determined by cell counts in selected nuclear centers. Cell numbers in each case appeared to be remarkably proportional to the size of the innervation field. Our data, based on cell counts in the nuclear centers chosen for this study in the abnormal embryo and normal control embryos of the same age, are consistent with the hypothesis that cell survival in related parts of the nervous system may be regulated by their peripheral field of innervation.

[Total colic aganglionosis (author's transl)]. / Aganglionismo cólico total.

Eleven cases of total colic aganglionism are presented in a series of 130 patients with Hirschsprung's disease. Five patients survived, four were operated according to the Duhamel-Roviralta technique and the fifth according to the simpre procedure of Duhamel. Two others, also operated using the Duhamel-Roviralta technique died, one in the immediate postoperatory period and the other at three months due to hepatitis. The four remaining cases died, two after having effected the ileostomy and two due to the inability of applying an immediate solution because the results of the biopsies were lacking. Death was due to septic processes in the immediate postoperatory period. Advantages of the Duhamel-Roviralta operation makes it the selected technique for the greater part of cases, due to its' quickness and simplicity.

Modified endorectal procedure for management of long-segment aganglionosis.

During the past five years, 65 patients with Hirschsprung's disease have been treated at our institution. Ten of these patients had long-segment aganglionosis, five with total colonic involvement, four with aganglionosis to the midtransverse colon, and one child with Hirschsprung's disease extending to the distal jejunum. There are six females and four males in the series, which is a reversal of the normal sex ratio for aganglionosis. An extensive literature review of long-segment Hirschsprung's disease has revealed a total of 489 reported cases. The mean frequency of long-segment aganglionosis in the literature is 12% of all cases of aganglionosis. Our series is comparable, with an incidence of 15%. The overall mortality rate in the literature of 54% indicates the poor therapeutic results obtained with this disease in the past. All ten of our patients have been definitively treated with a modified endorectal pull-through procedure. The mortality rate with this procedure has been zero. There has been one major complication (incorrect pathologic diagnosis of the level of aganglionosis) and four minor complications. The average follow-up period is 42.6 months. The functional results have been excellent with an average of three to four soft bowel movements per day. All of the children over 3 years of age are completely toilet trained. The remaining two patients are too young to be toilet trained, but are continent in the sense that they do not soil between bowel movements. Awareness of this entity of long-segment aganglionosis should lead to earlier diagnosis, appropriate operative therapy, and lower mortality rates.

Myenteric plexus of the hind-gut: developmental abnormalities in humans and experimental studies.

Intraluminal pressure studies on patients with congenital aganglionosis showed that the aganglionic rectum contracted in an uncoordinated manner and failed to relax. Histochemical assessment of the innervation helped to explain the variable severity of the symptoms in this condition. It is concluded that (1) absence of ganglia prevents normal coordinated peristalsis and creates an obstructive element; (2) absence of reflex relaxation adds to the obstruction; (3) the degree of uncoordinated motor activity in distal aganglionic bowel probably relates to the number of cholinesterase-positive nerves in the circular muscle and adds another variable obstructive element; and (4) deficient innervation of distal ganglionic bowel probably creates a poor propulsive force and so accentuates more distal obstructive factors. Neurohistochemical and functional studies in the anorectum of cats reveal a somewhat different innervation pattern from that in humans but show that sphincteric tone is mainly due to alpha-adrenergic neural activity. Reflex relaxation of the internal anal sphincter is a complex function in which inhibitory responses override motor responses, and it involves an important non-adrenergic non-cholinergic component. The role of cholinergic nerves in the sphincter remains uncertain. Neurohistochemical assessment of full thickness biopsy specimens of rectal muscle from patients with disabling constipation shows that developmental neuronal dysplasias of the hind-gut may be divided into three main categories: (1) aganglionosis (Hirschsprung's disease), (2) hypoganglionosis and (3) hyperganglionosis, and that the different neuronal elements may be affected to differing degrees in individuals within each group. Resection of the aganglionic bowel is required in congenital aganglionosis but the combined diagnostic-therapeutic procedure of anorectal myotomy has been found beneficial in patients with hypoganglionosis.