Embryopathoetiology of Congenital Hydrocephalus in Experimental Models: A Comparative Morphological Study in Two Different Models.

Embryopathoetiology of Congenital Hydrocephalus in Experimental Models: A Comparative Morphological Study in Two Different Models

Hiroshi Yamada, Shizuo Oi, Norihiko Tamaki, Satoshi Matsumoto 1 ,

and Katsushi Taomoto2

Summary. We studied the morphological aspects of two different kinds of exerimental hydrocephalic model in rats. LEW/Jms rats were used as an inherited congenital hydrocephalic strain and 6-aminonicotinamide (6-AN, a niacinamide antimetabolite )-induced hydrocephalus was studied as an exogenous insult-induced hydrocephalus.

In LEW/Jms rats, aqueduct obstruction was observed on gestational day 17, prior to definite evidence of ventricular enlargement. The form of the aque­ ductal obstruction was found to be simple stenosis, according to Russell's classification. This finding suggested that aqueductal stenosis was the primary cause of hydrocephalus in the LEW/Jms hydrocephalic strain. This strain might be a model of human sex-linked hydrocephalus.

In 6-AN-induced hydrocephalic rats, dilatation of the whole ventricular system was observed. In this model, evidence of cerebral dysgenesis was suggested by bromodeoxyuridine (BUdR) immunostaining. However, the ventricular dilatation was resulted not only from cerebral dysgenesis, but also from increased intracranial pressure. This model was characteristic in that, in addition to the hydrocephalic state, various central nervous system malforma­ tions existed, such as cerebellar dysgenesis, absence of corpus callosum, and so on. These pathological findings suggested that 6-AN-induced hydrocephalus might be a model of human Dandy-Walker syndrome.

Keywords. Congenital hydrocephalus - X-Linked Hydrocephalus - LEW/ Jms strain- 6-Aminonicotinamide- Dandy-Walker syndrome

Introduction

The cause of congenital hydrocephalus is various in both human and ex­ perimental forms of the condition. However, an important problem, the etiopathogenesis of hydrocephalus in many strains, has been left unsolved.

We studied two kinds of experimental congenital hydrocephalus in rats, the LEW/Jms strain as an inherited hydrocephalus and 6-aminonicotinamide (6- AN)-induced hydrocephalus as an exogenous insult-induced hydrocephalus. The purpose of this study was to observe the light microscopic pathological findings of congenital hydrocephalus in both these models during the perinatal period and to elucidate the etiology of hydrocephalus in these rats.

Materials and Methods

Inherited Congenital Hydrocephalus

In the LEW/Jms strain, hydrocephalic anomaly was present in about 20% of the animals, as reported by Sasaki et al. (1983). Normal male and female siblings of hydrocephalic rats were mated. After mating, vaginal smears were inspected each morning for signs of sperm. The day copulation was confirmed was designated as day 0 of gestation. Fetuses were collected by uterotomy on gestational days 17, 18, and 20. Newborn pups were also sacrificed. Materials were put in Bouin's solution and embedded in paraffin. In order to inves­ tigate and elucidate the morphological changes of the entire CSF pathway, serial sagittal sections, 4Jlm thick, were stained with hematoxylin and eosin. Serial coronal sections were also made as the need arose. As it was impossible to. distinguish between hydrocephalic and normal embryos from their physical appearance, specimens from all of the siblings were checked. When hydro­ cephalic rats were included, their morphological changes were compared with those of normal siblings.

Exogenous Insult Hydrocephalus

Male and female Sprague-Dawley (SD-JCL, CLEA Japan) rats were allowed to mate. Vaginal smears were inspected each morning for signs of sperm after mating. The day copulation was confirmed was designated as day 0 of gesta­tion. On the 13th day of gestation, 8mg/kg of 6-AN was given as a single ip injection; this dosage is known to cause a high frequency of hydrocephalus in fetuses near term (Chamberlain and Nelson 1963). Fetuses were collected by uterotomy 1, 2, 4, and 8 days after injection. Materials were put into Bouin's solution, embedded in paraffin, and cut into S1.1m sections. All fetuses were serially sectioned either sagittally or coronally. Untreated fetuses at the same periods of development were used as controls; all materials were stained with hematoxylin and eosin.

On gestational day 17 (4 days after 6-AN injection), bromodeoxyuridine

(BUdR), at a dose of 50mg, was given as a single ip injection to one pregnant rat 1h before uterotomy. The fetuses collected from this rat were then put into 70% ethanol and embedded in paraffin, cut into 5J.1m sections, and deparaffinized. Specimens were denatured for 30 min in 2N HCl and incubated for 30min in ethanol with 0.3% H202 to avoid endogenous peroxidase activity.

Fig. 1. Newborn rats in coronal section . A and C show a hydrocephalic and B and D show a normal rat; C and D are magnifications of the aqueduct. Complete obstruction of the aqueduct with a simple stenosis is observed in hydrocephalus (C), while in the normal rat, the aqueduct is patent with a triangular shaped lumen (D) (A.B From H. Yamada et al (1991) Published with permission)

They were then reacted with a 1:30 dilution of purified anti-BUdR monoclonal antibody in phosphate buffer solution (PBS) for 30 min at room temperature. The specimens were then covered with peroxidase-conjugated anti-mouse im­munoglobulin G antibody for 30 min and reacted with 5 mg of diaminobenzidine tetrahydrochloride and 4Jl 1 of 30% H202 of Tris buffer for 5 min. Myer hematoxylin was used to counterstain the tissue sections. Untreated fetuses at the same period of development were labeled with BUdR by the same method.

Results

LEW/Jms Strain

Figure 1 shows coronal sections of 1-day-old normal and hydrocephalic rats. In normal rats, the smallest diameter was located at the anterior part of the aqueduct, which was triangular in shape with the base facing the dorsal side. The shape of aqueductal sections varied from the cephalic to the caudal level, as in the human aqueduct (Woollam and Millen 1953). In hydrocephalic rats, the smallest part of the aqueduct was completely obstructed by a collection of oval shaped ependymal cells . The number of ependymal cells lining the aqueduct at the level of obstruction was less than that in the smallest area of the aqueduct in normal rats.

On gestational days 20 and 18, the basic appearance was the same as that of the new-born pups. In hydrocephalus, the lateral and third ven+ricles were dilated and the pineal body was compressed and shifted behind by the enlarged third ventricle. A serial sagittal section also showed that the aqueduct was obstructed. The obstructed site was next to the caudal side of the junction between the third ventricle and the aqueduct . Normal rats showed a patent aqueduct. There was no difference between the hydrocephalic and normal fetuses in the posterior part of the aqueduct or in the fourth ventricle.

On gestational day 17, eight rats were examined; their ventricles were the same size. Only one of these eight rats was found to be occluded at the aqueduct; the other seven rats had patent aqueducts (Fig. 2). The entire ven­ tricular system of each rat was the same size, irrespective of aqueductal form.

Throughout the gestational period, the site of occlusion was the anterior part of the aqueduct, that is, the level of the anterior colliculus. No difference was detected between hydrocephalic and normal rats in the size or form of the subarachnoid space, brain stem, and spinal cord.

6-AN Induced Hydrocephalus

All 6-AN treated fetuses near term showed evidence of hydrocephalus. Head enlargement could be detected from their physical appearance and the size of the body was smaller than that of the head, in contrast with the appearance of control rats.

The cerebral mantle facing the ventricle was examined on gestational day 14 in control and 6-AN treated (24h after injection) rats. Many mitotic figures were noted in the cerebral mantle in the control, however, no such figures were seen in the 6-AN treated fetus. Cellular rarefaction was also seen in the 6-AN treated rats. These findings suggested that 6-AN had some toxic effects in the developing brain.

On gestational day 17, the 6-AN treated rat showed more severe hypoplasia in all parts of the brain and cellular rarefaction was seen, particularly in the cerebellum. Mild petechial hemorrhage was seen in the tect m of the midbrain. Macrocephalus became clear and ventriculomegaly was confirmed by histological examination. Enlargement of all ventricles, including the aqueduct , was seen, in contrast with findings in the control (Fig. 3). The finding of a thin cortex suggested cerebral hypoplasia. The fourth ventricle was dilated, with cerebellar hypoplasia. In the control, many BUdR positive cells were found in the cerebral mantle around the ventricle. In the 6-AN treated rat, no BUdR positive cells were found in the central nervou s system.

On gestational day 21, the finding of macrocephalus became clearer from the physical appearance of the 6-AN treated rats . The skull showed marked dis­ tension at the parietal dome. The fact that CSF gushed out when the skull was punctured suggested a high intracranial pressure. The whole ventricular system showed enlargement, including the aqueduct and the fourth ventricle . Agenesis of the corpus callosum was evident on coronal and sagittal sections and normal features characteristic of the cerebellum were not observed (Fig. 4).

Discussion

Many inherited congenital hydrocephalic models have been reported (Berry 1961; Borit and Sidman 1972; D'Amato et al1986; Green 1970; Higashi et al. 1984; Kohn et al. 1981; Koto et al. 1987; Raimondi et al. 1976); however, few examples of primary aqueductal stenosis have been described. The strain we used , LEW /Jms, was first studied by Sasaki et al. (1983), who reported postnatal developmental changes which they examined histologically. They observed the unbalanced dilatation of both the posterior horn of the lateral ventricle and the upper part of the third ventricle in the postnatal period, and speculated that stenosis of the third ventricle and the anterior part of the

lateral ventricle in fetal life was a main cause of hydrocephalus. Our study in the fetal period revealed that aqueductal obstruction preceded hydrocephalus and we concluded that aqueductal obstruction in the rats we studied was a primary change and not a secondary phenomenon due to compression by ventricular dilatation. We find support for this thesis in the observation that there was a decrease in the number of ependymal cells lining the aqueduct . Although the entire aqueduct was lined with ependymal cells, the sit of ob­ struction was the anterior part of the aqueduct.

In humans, there is a form of hydrocephalus with some resemblance to the models presented. In 1949, Bicker and Adams described a family in which all of the sons and four of six brothers of a healthy woman died at birth with hydrocephalus. An autopsy in one case showed evidence of aqueductal stenosis. There have been a number of related reports (Edwards et al. 1961; Holmes et al. 1963) and more information about this sex-linked hydrocephalic disease has been compiled. In the case cited above the entire aqueduct was narrowed; However, there was no septum formation, periaqueductal gliosis, or ependymitis. The narrowest site was reported to be at the rostral portion of the inferior colliculus. As the pathological findings in human hydrocephalus at autopsy are usually those of an advanced stage of hydrocephalus, it is very difficult to conclude whether the aqueductal stenosis described is a primary or a secondary change. In human congenital hydrocephalus, case reports have speculated upon this, but it has yet not been proven that aqueductal stenosis or obstruction are primary changes. In the present study of the hydrocephalic fetal model LEW/Jms rat; we conclude that aqueductal obstruction is a pri­ mary change and not a secondary one. The mechanism of aqueductal obstruc­ tion in this model is still unclear; however, it is our position that morphological studies in this model will be helpful in resolving the cause of hydrocephalus.

The remarkable pharmacological and toxicological properties of the agent 6-AN have been revealed in experimental animals (Horita et al. 1978; Sasaki 1982). This agent acts not only on the spinal cord, but also on the other sites of the central nervous system (CNS) (Henken et al. 1974; Sasaki 1982). The large amount of 6-phosphogluconate which accumulates in neural tissue in adult rats supports the concept that the primary action of this drug is its inhibition of 6- phosphogluconate dehydrogenase in the pentose phosphate pathway(Henken et al. 1974).

Chamberlain (1970) first reported the morphological changes of 6-AN induced congenital hydrocephalus; however, he showed no histological studies of the models he described. We studied the same model, investigating the developing morphological changes by light microscopic study. This model is very characteristic of with cerebral dysgenesis confirmed by the BUdR immunohistochemical method and various anomalies in the central nervous system. The ventricular dilatation in this model was a result not only of cerebral dysgenesis, but also of increased intracranial pressure, shown by the enlarged head size compared with body size.

Representative human forms of hydrocephalus associated with CNS and systemic anomalies include Dandy-Walker syndrome, Arnold-Chiari malfor­ mation, and so on. Several theories relating to the pathogenesis of Dandy­ Walker syndrome have been proposed. The most widely accepted view is that the foramina of Luschka and Magendie fail to open, resulting in cystic enlarge­ ment of the fourth ventricle with consequent failure of the proper development of the cerebellar vermis (Dandy and Blackfen 1914; Schreiber and Reye 1954; Taggart and Walker 1942). Hart et al. (1972) reported a clinicopathological study on 28 cases of the Dandy-Walker syndrome and concluded that this syndrome was likely to be caused by foraminal atresia. However, none of these proposed mechanisms provide explanations for the many and various associated CNS anomalies. We feel that there is still room for further study. The present model showed cystic enlargement of the fourth ventricle and many CNS anomalies, such as absence of the corpus callosum, cerebellar hypoplasia, maldevelopment of the choroid plexus, and so on, which are associated with hydrocephalus. These findings show that there are interesting similarities between this model and the Dandy-Walker syndrome; the findings indicate that the cause of the Dandy-Walker syndrome is not limited to malfunction in the vicinity of the fourth ventricle, but is due to general systemic metabolic errors, such as niacin deficit in fetal life, as described above. The anomalies seen around the fourth ventricle in the Dandy-Walker syndrome may be one of the features of the systemic disease.

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