Changes in the Cerebral Vascular Bed in Experimental Hydrocephalus: An Angioarchitectural and Histological Study.

Changes in the Cerebral Vascular Bed in Experimental Hydrocephalus: An Angioarchitectural and Histological Study

Summary. The angioarchitectural and histological changes of small cerebral blood vessels in experimental hydrocephalus were studied in order to assess changes of the vascular bed in the cerebral mantle.

Changes of the microvasculature assessed from microcorrosion casts by scanning electron microscopy (SEM) and histological changes shown by light and electron microscopy were compared before and after shunting for hydrocephalus. Changes of the regional cerebral blood flow (rCBF) were also evaluated by the hydrogen clearance method.

In hydrocephalus, a reduction in the number and caliber of the capillaries was noted in both the white and gray matter in the SEM study, but the capillaries were preserved and changes were mild and nonspecific in the elec­ tron microscopic examination. Shunting resulted in the reversal of all these changes to normal, along with recovery of the rCBF, which had decreased in hydrocephalus.

These observations suggest that changes of the vascular bed participate in the alteration of cerebral mantle width in the hydrocephalic process, and that changes of the microvasculature result not only from damage to the capillaries themselves, but also from changes of the perivascular structures.

Keywords. Hydrocephalus - Ventriculo-peritoneal shunting - Scanning electron microscopy -Electron microscopy -Cerebral blood flow

Introduction

We have previously reported changes of the microvasculature in hydrocephalic rats, as shown by scanning electron microscopy (SEM), in an investigation of the effects of hydrocephalus on the vascular bed in the cerebral mantle (Oka et al. 1986). In order to study changes of the microvasculature after cerebrospinal fluid (CSF) shunting, rabbits were used in the present investigation.

Histological changes were assessed using light and electron microscopy and changes of the rCBF were determined by the hydrogen clearance method. All parameters were evaluated before and after shunting to determine changes of the vasculature produced by the relief of hydrocephalus.

Materials and Methods

Hydrocephalus Model and Ventriculo-Peritoneal Shunting

Rabbits weighing 1.5-2.0kg were used for this experiment. Under intravenous pentobarbital sodium anesthesia (20-25 mg/kg), a 2-cm sterile midline skin incision was made at the occipital region. After exposing and incising the atlanto-occipital membrane, about 1.0-1.5ml CSF was removed. Then 0.6- 0.8 ml of a kaolin suspension (250 mg/ml) was slowly injected into the cisterna magna. To prevent reflux of the kaolin suspension, a piece of muscle with Aron-a was used to cover the incision.

Ventricular size was measured on coronal computed tomography (CT) scans one week, one month, one and a half months, and two months after the kaolin injection. The degree of ventricular dilatation was divided into three categories (mild, moderate, and severe) in accordance with the ratio of the maximum distance between the bilateral anterior horns and the inside diameter of the skull in the same slice (Fig. 1).

In 11 of the 22 rabbits with severe hydrocephalus, ventriculo-peritoneal shunting was performed two months after the kaolin injection.

Scanning Electron Microscopic (SEM) Study

Seven rabbits. with hydrocephalus (three moderate and four severe), four rabbits after shunting , and nine untreated control rabbits were used for this experiment. Two months after the injection of kaolin, thoracotomy was per­ formed under pentobarbital sodium anesthesia (25 mg/kg). After ligation of the innominate artery and the aortic arch, a 19-gauge elastic needle was used to cannulate the ascending aorta from the left ventricle. The rabbits were then perfused with physiological saline solution, followed by the slow injection of cooled polyester resin (Mercox), at 120mmHg pressure, into the ascending aorta until the resin emerged bilaterally from the cut jugular veins.

The heads were decapitated at the neck and the skin and mandibula were removed. Heads were then embedded in 6% carboxylmethyl cellulose and frozen at -70°C with hexan and dry ice. Each head was cut into serial sagittal sections to nearly the midline with a microtome (LKB 2250, PMV450MP). The rest of the hemisphere was soaked at about 40°C in 2% Triton X-100 solution with 25mM NarEDTA and 2N sodium hydroxide, for about three weeks, to digest the muscle, cranium, and brain tissue. The microvasculature of the gray and white matter in the parietal and frontal areas was then examined by SEM (Hitachi X-650).

Light and Electron Microscopic Study

Eight rabbits with hydrocephalus (four moderate and four severe), four rab­ bits after shunting, and five untreated control rabbits were used for this study. After thoracotomy under intravenous pentobarbital sodium anesthesia (25 mg/kg), they were perfused via the left ventricular route with 0.5% glutaraldehyde/4% paraformaldehyde in 0.01 M phosphate buffer. The brains were then sectioned coronally into slices of about 3mm in thickness. For the electron microscopic study, specimens of gray and white matter in the parietal region were soaked in the same fixing fluid for one night, postfixed in buffered 1% osmium tetroxide for 1 hour, dehydrated in ethyl alcohol, and embedded in Epon 812. Ultrathin sections were double-stained with uranyl acetate and lead citrate and examined under an electron microscope (Hitachi H-300). For the light microscopic study, the sections were stained with hematoxylin and eosin (H & E) and periodic acid-methenamine (PAM).

Regional Cerebral Blood Flow (rCBF)

Regional cerebral blood flow was measured in the parietal gray and white matter by the inhalation method in eight rabbits with hydrocephalus (four moderate and four severe), four rabbits after shunting, and five untreated control rabbits.

Results

Scanning Electron Microscopy

Corrosion casts of the rabbits with hydrocephalus showed that the vasculature was sparse; the main trunks of the vessels could be seen more clearly compared with the controls (Fig. 2). In the SEM study, there was an obvious reduction in both the number and the caliber of the capillaries in hydrocephalus, and this change tended to increase in proportion to the severity of the hydrocephalus. The capillaries were about 8-10 Jlm in diameter in the controls and decreased to a diameter of about 5-8 Jlm in severe hydrocephalus. The number of capillaries returned to normal and their caliber also recovered to about 6- 11Jlm after shunting (Figs. 3 and 4, Table 1). These changes were seen almost equally in the gray and white matter.

Cortical straight and parallel vessels showing a typical "palisade" pattern were distorted in hydrocephalus and returned to normal after shunting (Fig. 5).

Light and Electron Microscopic Study

The light microscopic study showed that the spongiomatous change which was found in the periventricular white matter in hydrocephalus disappeared after shunting. Pathological vessels could not be found in either the hydrocephalic or the post-shunting groups.

In the electron microscopic study, swelling of astrocytes in the perivascular area was observed in hydrocephalus (Fig. 6). Vacuoles, microvilli, and webs were noted more frequently in the endothelial cells of the capillaries in hydro­ cf'phalus as compared to controls (Fig. 7), and these changes were found more frequently in the white matter than in the gray matter. Following shunting, these perivascular and endothelial changes disappeared (Fig. 8). Opening of tight junctions, degeneration and reactive proliferation of endothelial cells, and abnormal vessels indicating neovascularization were not observed in this study.

Regional Cerebral Blood Flow (rCBF) The rCBF of the gray and the white matter in five normal controls was 34.7 ± 4.5ml/100g per min and 17.8 ± 3.3ml/100g per min, respectively. Eight

hydrocephalic rabbits had a rCBF of 24.2 ± 6.7 ml/100 g per min in the gray matter and 14.8 ± 3.7ml/100g per min in the white matter. A decrease in rCBF was thus noted in both the gray and the white matter, and this change became more marked in severe hydrocephalus when compared to moderate hydrocephalus . The rCBF of the gray matter was significantly (P < 0.05) reduced in hydrocephalus compared with that in the normal controls . After shunting, the rCBF recovered to 37.1 ± 9.7 ml/100 g per min in the gray matter and to 21.3 ± 4.2 ml/100 g per min in the white matter. These were significant increases (P < 0.05) from the values seen in hydrocephalus (Tables 2 and 3).

Discussion

An early report on the cerebral vasculature in hydrocephalus was made by Penfield (1929) who performed a macroscopic necropsy study. For the inves­ tigation of the cerebral angioarchitecture several methods have been reported,

including microradiography (Hassler 1964; Sato et al. 1984; Wozniak et al. 1975), microangiography (Okuyama et al. 1987; Plets 1986; Sato et al. 1984), histological microscopic examination (De 1950; Del Bigio and Bruni 1988), and the use of vascular corrosion casting (Oka et al. 1986).

Microradiography involves exposing sections of the brain to X-ray films. Although this method produces excellent stereoscopic pictures of the spatial distribution of the vessels, it has several disadvantages: (1) artifacts may be produced during the injection procedure, (2) obstructed vessels cannot be demonstrated, and (3) most of the capillaries and veins do not fill with X-ray contrast medium and are not visualized.

The microangiographic method, which involves observation, under the microscope, of the injection of various dyes into the cerebral vessels, allows the

study of smaller vessels than the microradiographic method, but is not so good at showing the spatial distribution of the vessels, because of the thin sections used.

Several histological studies of the angioarchitecture in hydrocephalus have been reported. De (1950) observed the vascular pattern of the smaller vessels using Pickworth's stain. Del Bigio et al. (1988) examined periventricular blood vessels by light microscopy and assessed them by a quantitative method.

Using microvascular corrosion casting, the cerebral vasculature can be observed more stereoscopically and with a better understanding of the suc­ cessive levels of the vascular tree than is possible by the microradiographic method. Also, the caliber of vessels can be measured more exactly by this method than by microangiography and histological studies, which reveal only sectioned vessels. In addition, this method can demonstrate the capillaries and veins by the injection of polyester resin into the venous system.

Reports on the cerebral angioarchitecture in hydrocephalus have been some­ what varied, probably owing to differences in the methods used and differences in the caliber of the vessels which were observed. Hassler (1964) used micro­ radiography to observe vessels above the precapillary level in experimental hydrocephalus, and described an increase in the number of arteries and veins in the periventricular white matter and in the cortex. Plets (1986) also stated that the arterioles in the subependymal region showed a relative hyper­ vascularization. On the other hand, Wozniak et al. (1975) found a decrease in the number and caliber of the microvessels in advanced hydrocephalus in the congenitally hydrocephalic Hy-3 mouse. There are several other reports that indicate poor visualization of the microvessels in the periventricular region.

As for the capillaries, only a few studies have been published. Hassler (1964) observed the capillaries in the hydrocephalic necropsied brain and in experi­ mental hydrocephalus by the peroxidase staining method, and reported them to seem normal in the atrophic white matter. In contrast, Del Bigio and Bruni (1988) described a significant decrease in the number of capillaries with lamina of 10 11m or less in the paraventricular area one week after silicon injection. De (1950) also reported a decrease of the capillaries in hydrocephalus.

After shunting, most studies reported that the microvasculature was restored to normal, although in several studies (Del Bigio and Bruni 1988; Plets 1986) in which CSF shunting was performed in the late stages of hydrocephalus, changes of the microvasculature were irreversible.

The most marked change noted in our study was the reduction in the number and caliber of the capillaries in the white and gray matter in chronic hydro­ cephalus. This change reversed completely after shunting. However, shunting was performed only two months after the induction of hydrocephalus, so a longer period of observation would be necessary to better assess the reversi­ bility of changes of the cerebral angioarchitecture in hydrocephalus.

Regarding morphological studies of the capillaries using microscopic methods, changes of the capillaries in the hydrocephalic brain seemed to be mild. Nakagawa et al. (1984) found clefts and vacuoles between the tight junctions of the capillaries and postcapillary venules in kaolin-induced hydro-56 N. Oka et a/.

cephalus, and suggested that the tight junction acted as a shunt pathway for interstitial edema fluid and cerebrospinal fluid to enter the microvessels. Regarding changes of the tight junctions themselves and changes of the endo­ thelial cells in hydrocephalus, Okuyama et al. (1987), from the finding of stenotic or occluded capillaries in the late stage of hydrocephalus, suggested the possible disruption of the blood-brain barrier. In our study, vacuoles, microvilli, and webs, which are thought to be an early and nonspecific response to various stresses, were found in the endothelial cells of the capillaries in hydrocephalus. All these changes reversed after shunting.

The reduction in rCBF in hydrocephalus improved after shunting, which was a similar result to that cited in previous reports (Higashi et al. 1986; Hochwald et al. 1975; Nakamura and Hochwald 1983). The marked decrease in rCBF in the gray matter found in our study, which Murata et al. (1980) also described, contrasted with another report (Sato et al. 1984) of reduced rCBF in the white matter. Aseptic meningitis induced by the injection of kaolin suspension might have been related to the reduction in cortical blood flow.

In hydrocephalus, the vascular bed seemed to decrease with ventricular dilatation and to increase again after shunting. These changes are suggested to participate, not in a small way, in the restoration of cerebral mantle width.

Regarding our results using microcorrosion casting, some questions are raised with regard to the capillaries: (1) Was the sparsity of the vascular cast in hydrocephalus caused by the disappearance of capillaries which actually changed irreversibly? (2) Was this changes caused by the lack of flow of the polyester resin into narrowed capillaries which actually still existed? (3) Was the increase in capillaries after shunting due to the same vessels that were present previously or due to neocapillaries? We also investigated histological changes in order to answer these questions. Our histological studies showed that capillaries were found even in the thinned-out cerebral mantle and that neocapillarization did not occur in the re-expanded mantle after shunting. Therefore, the apparent changes of the capillaries which we observed in hydro­ cephalus may not have been caused by changes of the vessels themselves, but may have been secondary to the influence of changes in the periventricular structures. Enlargement of the extracellular space due to interstitial edema and the hypertrophy and accumulation of astrocytes may have caused the capillaries to collapse in hydrocephalus, and these changes were then nor­ malized after shunting, leading to capillary re-expansion.

Conclusions

1. A reduction in the number and caliber of the cerebral capillaries was the most marked change seen in hydrocephalus. This change was found in both the gray and the white matter.

2. The number and caliber of the capillaries returned to normal with re­ expansion of the cerebral mantle after shunting.

3. Capillary endothelial cells in hydrocephalus showed an increase of vacuoles, microvilli, and webs. These changes were reversed by shunting, and neo­ vascularization was not seen.

4. The rCBF was reduced in hydrocephalus and improved after shunting.

5. These findings suggest that, in the hydrocephalic process, changes of the cerebral vascular bed may participate in the alterations of the cerebral mantle. In addition, the reversal of the changes in the capillaries was apparently brought about not by the vessels themselves, but by changes of the perivascular elements.

References

De SN (1950) A study of the changes in the brain in experimental internal hydro­ cephalus. J Pathol 112: 197-209

Del Bigio MR, Bruni JE (1988) Changes in periventricular vasculature of rabbit brain following induction of hydrocephalus and after shunting. J Neurosurg 69: 115-120 Hassler 0 (1964) Angioarchitecture in hydrocephalus. An autopsy and experimental

study with the aid of microangiography. Acta Neuropathol (Berl) 4: 65-74

Higashi K, Asahisa H, Ueda N, Kobayashi K, Hara K, Noda Y (1986) Cerebral blood flow and metabolism in experimental hydrocephalus. Neurol Res 8: 169-176

Hochwald GM, Boa! RD, Martin AE, Kumer AJ (1975) Changes in regional blood flow and water content of brain and spinal cord in acute and chronic experimental hydrocephalus. Dev Med Child Neural [Suppl] 17: 42-50

Murata T, Yamagata S, Mori K, Handa H, Nakano Y (1980) Computed tomography in experimental canine hydrocephalus. Part 4: Periventricular lucency (PVL) and regional blood flow in the chronic stage of hydrocephalus (in Japanese). No To Shinkei 32: 219-227

Nakagawa Y, Cervos-Navarro J, Artigas J (1984) A possible paracellular route for the resolution of hydrocephalic edema. Acta Neuropathol (Berl) 64: 122-128

Nakamura S, Hochwald GM (1983) Effect of arterial PC02 and cerebrospinal fluid volume flow rate changes on choroidal plexus and cerebral blood flow in normal and experimental hydrocephalic cats. J Cereb Blood Flow Metab 3: 369-375

Oka N, Nakada J, Endo S, Takaku A (1986) Angioarchitecture in experimental hydrocephalus. Pediatr Neurosci 12: 294-299

Okuyama T, Hashi K, Sasaki S, Sudo K, Kurokawa Y (1987) Changes in cerebral microvasculature in congenital hydrocephalus of the inbred rat LEW/Jms: light and electron microscopic examination. Surg Neural 27: 338-342

Penfield W (1929) Cerebral pressure atrophy. In: Penfield W (ed) Proceedings, Asso­ ciation for Research in Nervous and Mental Disease, Vol. 6, Internal pressure in health and disease. Williams and Wilkins, Baltimore, pp 346-361

Plets C (1986) Influence of experimental hydrocephalus on cerebral vascularization. In: Baethmann A, Go KG, Unterberg A (eds) Mechanism of secondary brain damage. Plenum, New York, pp 169-178

Sato 0, Ohya M, Nojiri K, Tsugane R (1984) Microcirculatory changes in experimental hydrocephalus: morphological and physiological studies. In: Shapiro K, Marmarou A, Portnoy H (eds) Hydrocephalus. Raven, New York, pp 215-230

Wozniak M, MeLone DG, Raimondi AG (1975) Micro- and macrovascular changes as the direct cause of parenchymal destruction in congenital murine hydrocephalus. J Neurosurg 43: 535-545

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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.

References

Berry RJ (1961) The inheritance and pathogenesis of hydrocephalus-3 in the mouse. J Pathol Bacteriol 81: 157-167

Bicker DS, Adams RD (1949) Hereditary stenosis of the aqueduct of Sylvius as a cause of congenital hydrocephalus. Brain 72: 246-262

Borit A, Sidman RL (1972) New mutant mouse with communicating hydrocephalus and secondary aqueductal stenosis. Acta Neuropathol (Berl) 21: 316-331

Chamberlain JG (1970) Early neurovascular abnormalities underlining 6- aminonicotinamide (6-AN)-induced congenital hydrocephalus in rats. Teratology 3: 377-388

Chamberlain JG, Nelson MM (1963) Multiple congenital anomalies in the rat resulting from acute maternal niacin deficiency during pregnancy. Proc Soc Exp Bioi Med 112: 836-840

D'Amato CJ, O'Shea KS, Hicks SP, Glover RA, Annesley TM (1986) Genetic prenatal aqueductal stenosis with hydrocephalus in rat. J Neuropathol Exp Neurol 45: 665-682

Dandy WE, Blackfen KD (1914) Internal hydrocephalus: An experimental, clinical and pathological study. Am J Dis Child 8: 406-482

Edwards JH, Norman RM, Roberts JM (1961) Sex-linked hydrocephalus. Report of a family with 15 affected members. Arch Dis Child 36: 481-485

Green MC (1970) The developmental effects of congenital hydrocephalus (ch) in the mouse. Dev Bioi 23: 585-608

Hart MN, Malamud N, Ellis WG (1972) The Dandy-Walker syndrome. A clinico­ pathological study based on 28 cases. Neurology 22: 771-780

Herken H, Lange K, Kolbe H, Keller K (1974) Antimetabolic action on the pentose phosphate pathway in the central nervous system induced by 6-aminonicotinamide. In: (Genazzani E, Herken H eds) Central nervous system studies on metabolic regulation and function. Spring, New York, pp 41-54

Higashi K, Asahisa H, Ueda N, Noda Y, Tashiro M (1984) An experimental model of congenital hydrocephalus in the rat (in Japanese). Shoni no Noshinkei 9: 257-264 Holmes LB, Nash A, ZuRhein GM, Levin M, Opitz M (1963) X-linked aqueductal stenosis: Clinical and neuropathological findings in two families. Pediatrics 63: 1104-1110

Horita N, Oyanagi S, Ishii T, Lzumiyama Y (1978) Ultrastructure of 6- aminonicotinamide (6-AN)-induced lesions in the central nervous system of rats. 1. Chromatolysis and other lesions in the cervical cord. Acta Neuropathol (Berl) 44: 111-119

Kohn DF, Chinookoswong N, Chou SM (1981) A new model of congenital hydro­ cephalus in the rat. Acta Neuropathol (Berl) 54: 211-218

Koto M, Miwa M, Shimizu A, Tsuji K, Okamato M, Adachi J (1987) Inherited hydrocephalus in Csk Wistar-Imamichi rats Hyd strain: A new disease model for hydrocephalus. Exp Anim 36: 157-162

Raimondi AJ, Clark SJ, MeLone DG (1976) Pathogenesis of aqueductal occlusion in congenital murine hydrocephalus. J Neurosurg 5: 66-77

Russel DS (1949) Observations on the pathology of hydrocephalus. Medical Res Coun­ cil Spec. Rep. Series No 265. HMSO, London

Sasaki S (1982) Brain edema and gliopathy induced by 6-aminonicotinamide intoxica­ tion in the central nervous system of rats. Am J Vet Res 143: 1691-1695

Sasaki S, Goto H, Nagano H, Furuya K, Ornata Y, Kanazawa K, Suzuki K, Sudo K (1983) Congenital hydrocephalus revealed in the inbred rat LEW/Jms. Neurosurgery 13: 548-554

Schreiber MS, Reye RDK (1954) Posterior fossa cysts due to congenital atresia of the foramen of Luschka and Magendie. Med J Aust 2: 743-748

Taggart JK, Walker AE (1942) Congenital atresia of the foramen of Luschka and Magendie. Arch Neurol Psychiat 48: 583-612 ,

Woollam DHM, Millen JW (1953) Anatomical considerations in the pathology of stenosis of the cerebral aqueduct. Brain 76: 104-112

Yamada H, Oi S, Tamaki N, Matsumoto S, Sudo K (1991) Prenatal aqueductal stenosis as a cause of congenital hydrocephalus in the inbred rat LEW/JMS. Child's Nerv Syst in press Yamada H, Oi S, Tamaki N, Matsumoto S, Taomoto K (1991) Congenital hydro­ cephalus mimicking Dandy-Walker syndrome induced by 6-aminonicotinamide injec­ tion in pregnant rat. Neurol Med Chir (Tokyo) in press

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Postnatal Changes of HRP-Labeled Corticospinal Neurons in Congenital Hydrocephalic Rats (HTX).

Postnatal Changes of HRP-Labeled Corticospinal Neurons in Congenital Hydrocephalic Rats (HTX)

Summary. In the present study we examined the influence of hydrocephalic changes on the growth of corticospinal neurons and the formation of their neuronal connection with the spinal cord in the congenital hydrocephalic rat (HTX) during the postnatal period, using the horseradish peroxidase (HRP)­ labeling method.

We injected 0.1-2.0J..Ll of 20%-50% HRP solution into the cervical cords of HTX rats which had either high or slight dilatation of the lateral ventricles, at postnatal days 1, 7, 14, and 21, and observed the HRP-labeled corticospinal neurons in the cerebral cortex and the structure of the brain stem each day.

At postnatal day 1, HRP-labeled neurons were found in layer V, which was in the dorsal and lateral areas of the cerebral cortex in both types of hydrocephalic rats. From this finding these neurons could be identified as corticospinal neurons and their axons seemed to reach into the spinal cord. By postnatal day 21, HRP-labeled corticospinal neurons were found in the area from the dorsal to the lateral part of the cerebral cortex in the hydrocephalic rats with slight dilatation of the lateral ventricles. However, in the hydro­ cephalic rats with high dilatation of the lateral ventricles, HRP-labeled cortico­ spinal neurons gradually decreased and a few neurons were found in the lateral part of the cerebral cortex. At postnatal day 21, also, degenerative changes appeared in the corticospinal tract through the pyramis of the medulla oblongata.

These findings indicate that the neuronal connection between the cerebral cortex and the spinal cord, made by the corticospinal axons, gradually dis­ appears when cortical neurons, including corticospinal neurons are destroyed, such destruction being caused by the progresive dilatation of the lateral ven­ tricles and subsequently by the secondary degeneration of the corticospinal axons.

Kohn et al. (1981) reported that, in the congenital hydrocephalic rat (HTX), dilatation of the lateral ventricles appeared at birth and that the thickness of the cerebral cortex gradually decreased, depending on the advancement of lateral ventricular dilatation. These changes seem to influence the differentia­ tion and growth of neurons and neuroglial cells in the cerebral cortex.

Some of the descending projection neurons in the cerebral cortex are corticospinal neurons, which connect the cerebral cortex and the spinal cord. In rats the corticospinal axons are already distributed in the spinal cord at postnatal day 1 (Joosten et al. 1987; Ohtani and Shirai 1988). Moreover, the synaptic formation of the corticospinal neurons advances on their axon terminals in the spinal cord and on their somas through postnatal day 14 (Ohtani et al. 1987; Miyabayashi and Shirai 1988).

In the present study the postnatal development of the corticospinal neurons in the congenital hydrocephalic rat (HTX) was investigated, using the HRP­ Iabeling method, in order to define the effect of hydrocephalic changes on the formation of neuronal connections by cerebral cortical neurons .

Keywords. Congenital hydrocephalic rat - HRP-Labeling method Corticospinal neurons- Cerebral cortex- Postnatal development.

Materials and Methods

HTX rats with slight and high dilatations of the lateral ventricles at postnatal days 1, 7, 14, and 21 were used. We anesthetized all rats with Nembutal, and immediately injected 0.1-2.0 J.d of 20%-50% solution of horseradish perox­ idase (HRP) into their cervical cords. One or two days after HRP injection, we perfused them with a fixative consisting of a 1% solution of paraformaldehyde

Fig. 1. HRP-injection site in the cervical cord of HTX rat at postnatal day 14. Most area of the spinal cord is stained dark brown with HRP. X 24

Fig. 2. A A frontal section of the cerebrum with slight dilatation of the lateral ventricles at postnatal day 1. HRP-labeled corticospinal neurons( ...) are found in layer V of the cerebral cortex. x 24. B A high magnification of the dorsal cerebral cortex in Fig. 2.

The corticospinal neurons (...) are labeled with HRP. x 380

and a 1.25% solution of glutaraldehyde and immersed their brains and spinal cords into a 20% solution of sucrose. We made serial sections of their brains and spinal cords in thicknesses of 60 or 100Jlm, reacted these using the tetra­ methylbenzidine (TMB) method (Mesulam 1978), and observed them with a light microscope.

Results

Injection Site of HRP in the Cervical Cord

HRP was injected into almost the whole area of the cervical cord at postnatal days 1, 7, 14, and 21 (Fig. 1).

Distribution of HRP-Labeled corticospinal Neurons in the Cerebral Cortex At postnatal day 1, the cerebral cortex consisted of the molecular layer, the cortical plate, including many small cells, and layer V, containing large cells with slight and high dilatations of the lateral ventricles. HRP-Iabeled corticospinal neurons were found in layer V of the dorsal cortex with both slight and high dilatations of the lateral ventricles (Figs. 2a and 3a). In higher magnification the somas and proximal dendrites of the corticospinal neurons were found to be labeled with HRP (Figs. 2b and 3b).

At postnatal day 7, the dorsal area of the cerebral cortex with the high dilatation of the lateral ventricles appeared to have a thinner cerebral wall than the area with the slight dilatation; HRP-labeled neurons were localized in layer V of both cortices.

Fig. 3. A A frontal section of the cerebrum with high dilatation of the lateral ventricles at postnatal day 1. HRP-labeled corticospinal neurons ( ) are found in layer V of the dorsal cerebral cortex. x 24. 8 A high magnification of the dorsal cerebral cortex in Fig. 3A. The corticospinal neurons ( ) are labeled with HRP. x 380

At postnatal day 14, the cerebral cortex with the high dilatation (Fig. 4a) became thinner than that with the slight dilatation (Fig. Sa). The thinnest area appeared in the dorsal area of the cortex with the high dilatation. Many rows of HRP-labeled neurons were found in layer V of the cortex with the slight dilatation (Fig. Sa); however, several rows of these neurons were found in the same layer of the dorso-lateral part of the cortex with the high dilatation (Fig. 4a). The HRP-labeled neurons formed pyramidal cells in the cortex with the slight dilatation and formed such cells in the dorso-lateral part of the cortex with the high dilatation [Figs. Sb and 4b). The same neurons became smaller and flattened in the dorsal part of the cortex with the high dilatation (Fig. 4c).

At postnatal day 21, HRP-labeled neurons were stratified in layer V of the cortex with the slight dilatation, and the growth of their somas increased (Fig. 6a). Only a few HRP-labeled neurons were found in the dorsal part and a few HRP-labeled small neurons were found in the lateral part (Fig. 6b).

Degenerative Changes Appearing in the Pyramis of the Medulla Oblongata of HTX Rats with High Dilatation of the Lateral Ventricles At postnatal day 21, many macrophages appeared in the pyramis of the lower medulla oblongata and, simultaneously, degeneration of the corticospinal axons was found in this area (Fig. 7).

Discussion

In the normal rat, the corticospinal tract distributed in the posterior horn and the intermediate zone made immature synapses with the developing neurons in these zones at postnatal day 1, and formed a compound synaptic organization, similar to a glomerulus, through postnatal day 14 (Miyabayashi and Shirai 1988).

Also, retrograde labeling studies with HRP have shown that corticospinal neurons were localized in layer V of the cerebral cortex and that their axons reached the cervical cord. By postnatal day 14, their distribution formed an adult pattern and synaptogensis on their somas advanced (Ohtani et al. 1987; Ohtani and Shirai 1988).

In this study we examined the effect of hydrocephalic changes on the postnatal development of corticospinal neurons in the cerebral cortex of the

Fig. 5. A A frontal section of the cerebrum with slight dilatation of the lateral ventricles at postnatal day 14. Several rows of HRP-labeled corticospinal neurons (..,.) are found in layer V of the dorsal cerebral cortex. X 24. B A high magnification of the dorsal cerebral cortex in Fig. 5A. The somas and the proximal dendrites of the corticospinal neurons (..,.) are labeled with HRP. x 380

congenital hydrocephalic rat (HTX), compared with corticospinal neurons in the normal rat. At postnatal day 1, HRP-labeled neurons were found in layer V of the cerebral cortex. Therefore, this pattern was the same as that found in the normal rat. Also, it was confirmed that axons of the corticospinal neurons reached the spinal cord not only in the hydrocephalic rat with slight dilatation of the lateral ventricles, but also in the hydrocephalic rat with high dilatation of these ventricles. These findings seemed to show that the outgrowth of axons from the corticospinal neuronal somas to the spinal cord advanced without

Fig. 6. A HRP-Iabeled corticospinal neurons( ) are found in the dorsal cerebral cortex with slight dilatation of the lateral ventricles at postnatal day 21. x 380. B A few HRP­ labeled corticospinal neurons ( ) are found in the lateral cerebral cortex with high dilatation of the lateral ventricles at postnatal day 21. x 380 being influenced by the progress of lateral ventricular dilatation caused by the birth.

However, as Kohn et al. (1981) have described, the dilatation of the lateral ventricles became remarkable, and subsequently the thickness of the cerebrum reduced during postnatal development. In this process HRP-labeled cor­ ticospinal neurons gradually decreased in number. In particular, a few HRP­ labeled neurons were scattered in layer V of the lateral part of the cerebral cortex at postnatal day 21. At the same time, axons of the corticospinal neurons appeared to undergo degenerative changes through the pyramis of the

Fig. 7. A transverse section of the lower medulla oblongata with high dilatation of the lateral ventricles in the same hydrocyphalic rat as that shown in Fig. 6B at postnatal day 21. The degenerative changes of the corticospinal tract appear in the pyramis of the ventral part. X 24 medulla oblongata. This degeneration in the corticospinal axons seemed to be caused by changes in the corticospinal neurons, depending on the advancement of lateral ventricular dilatation and immediately the anterograde degeneration of the corticospinal axons. The neuronal connection between the cerebral cortex and the spinal cord, through the corticospinal axons, was gradually lost by because of the disappearance of the corticospinal neurons and the degenera­ tion of their axons.

References

Joosten EA, Gribnau AAM, Dederen JWC (1987) An anterograde tracer study of the developing corticospinal tract in the rat: three components. Dev Brain Res 36: 121-130

Kohn DF, Chinookoswong N, Chou SM (1981) A new model of congenital hydro­ cephalus in the rat. Acta Neuropathol (Berl) 54: 211-218

Mesulam M-M (1978) Tetramethylbenzidine for horseradish peroxidase neurochemistry: a noncarcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents. J Histochem Cytochem 26 : 106-117

Miyabayashi T, Shirai T (1988) Synaptic formations of the corticospinal tract in the rat spinal cord . Okajimas Folia Anat Jpn 65:117-140

Ohtani R, Shirai T (1988) The development of corticospinal tract (CST) neurons. Proceedings of the 6th M Singer Symposium pp 473-483

Ohtani R, Shirai T, Kato H (1987) The postnatal distributional changes and synaptogenesis of corticospinal tract neurons in the cerebral cortex of the rat with HRP-labeling method (abstract). Soc Neurosci 13: 1430

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Early Vs Delayed Ventriculoperitoneal Shunt-Effects on the Impairment of the Developing Brain in Congenitally Hydrocephalic HTX-Rats

Early Vs Delayed Ventriculoperitoneal Shunt-Effects on the Impairment of the Developing Brain in Congenitally Hydrocephalic HTX-Rats

Kikuo Suda, Kiyoshi Sato, Nobuaki Takeda, Mitsuru Wada, Takahito Miyazawa, Hajime Arai, Masanori Ito, and Makoto Miyaoka

Summary. In the present investigation, we report on the effects of the early placement of a ventriculoperitoneal shunt (V-P shunt) on the development of cerebral synapses by counting spine density of the cortical pyramidal neurons (stained by rapid Golgi method) and measuring one of the synaptic vesicle proteins, SVP-38. The techniques used were quantitative histochemical and immunoblot analysis. The learning ability of congenitally hydrocephalic HTX­ rats whose hydrocephalus had been arrested by insertion of a V-P shunt 7-9 days after birth (Early Shunt) was assessed by the light-darkness discrimina­ tion test. When a V-P shunt was inserted into the hydrocephalic animals approximately 4 weeks after birth (Delayed Shunt), not only was there no reduction in the size of the abnormally enlarged ventricles, but also there was no increase in cortical mantle thickness. Furthermore, spine density in the cerebral cortex in such animals was found to be decreased. Learning disability could not be corrected by the delayed shunt procedure. Contrary to these observations, early shunt placement was found to result in normalization of the abnormally enlarged ventricles, concomitant with simultaneous cortical mantle thickening and prevention of both decreased spine density and decay of SVP- 38 in the affected cerebral cortex. The learning disability of such animals was not found to be disturbed, compared with that of the sham-operation group. From these observations, it is concluded that early shunt placement may have a beneficial role not only in repairing, but also in preventing the impairment of synaptogenesis caused by the progression of congenital hydrocephalus.

Keywords. Congenital hydrocephalus - Rat - Ventriculoperitoneal shunt synaptogenesis -Learning ability

Introduction

In previous experiments using congenitally hydrocephalic HTX-rats (Kohn et a!. 1981, 1984), we reported that congenital hydrocephalus impaired the development of dendrites and spines of neurons in the affected cerebral cortex (Miyazawa et a!. 1988). Such impairment of neuronal development in the hydrocephalic brain of HTX-rats may not be completely corrected by insertion of a ventriculoperitoneal shunt approximately 4 weeks after birth (Delayed Shunt). We also suggested that the learning disability found in mature HTX­ rats whose hydrocephalus had been arrested by delayed shunt insertion could be related to the aforementioned disturbance of neuronal development, especially synaptogenesis of the brain (Miyazawa and Sato 1991).

In the present study, we report on the beneficial effects of early placement of a ventriculoperitoneal shunt (Early Shunt) in HTX-rats whose hydrocephalus was arrested by insertion of a ventriculoperitoneal shunt 7-9 days after birth. The beneficial effects on the development of cerebral synapses, and also on the learning ability of these animals, were examined.

Materials and Methods

Congenitally hydrocephalic male HTX-rats (HTX) were used, and non­ hydrocephalic male HTX-rats served as controls with and without sham operations.

Preparation of HTX with Early and Delayed Shunts

HTX manifesting hydrocephalus were divided into two groups in accordance with the time of ventriculoperitoneal (V-P) shunt insertion, that is, the Early Shunt Group in which a V-P shunt was intially inserted 7-9 days after birth, and the Delayed Shunt Group in which the V-P shunt was inserted approxi­ mately 4 weeks after birth. Non-hydrocephalic HTX were also divided into two groups, in one of which sham operations were carried out at the same times as the shunt operations in the experimental groups.

The HTX were anesthetized in the prone position by inhalation of 1.0% halothane. The scalp was incised to expose the parietal bone on the left side, and a hole approximately 2 mm in diameter was bored in the skull in an area 2 mm anterior to the left lambdoid suture and 4-5 mm to the left of the sagittal suture. After the dura matter was exposed and coagulated, a laparatomy was performed at the right dorsal flank to expose the peritoneal space. A shunt passer was inserted into the subcutaneous tissue, and then a V-P shunt tube without pressure regulation valve (Dow Corning Co., silastic catheter, inside diameter 0.025 in, length 12em) was passed from the left parietal area to the right flank. The tip of the ventricular catheter was inserted into the left lateral ventricle 4-5 mm from the inner table of the calvaria. After the flow of the cerebrospinal fluid (CSF) from the abdominal catheter was confirmed, the ventricular catheter was fixed to the skull using Aron Alpha. The abdominal catheter was then inserted into the peritoneal space, and the skin incision was closed.

Ventriculography and Magnetic Resonance Imaging (MRI)

The chronological change in ventricular size and thickness of the cortical mantle of the brains of animals subjected to a V-P shunt was assessed at random times after the surgery by ventriculography and by MRI (installed at the National Institute for Physiological Science in Okazaki). During ven­ triculography under Halothane general anesthesia, 0.2-0.3 ml of Iotroran was slowly injected into the lateral ventricle via V-P shunt abdominal catheter or via 27 G scalp needle inserted directly into the lateral ventricle. Softex X-ray films (Softex Corp., Tokyo) were used to obtain ventriculograms, and the lateral ventricular size visualized on the Softex film was measured by planimetry using a computer-assisted image analyzer (!BASS 2000, Zeiss). The MRI used in this study was Hitachi 2.114 T, and T1 weighted images were obtained.

Light-Darkness Discrimination using Y-Maze Test (Takiguchi et al. 1988)

When the experimental and sham-operated animals subjected either to an early or a delayed V-P shunt became sexually mature, their learning ability was assessed by the Y-maze test, as described elsewhere (Miyazawa and Sato 1991). A mean correct response rate and a mean response latency time were calculated for each set of 10 trials for each respective animal. Different condi­ tions in the animals were compared statistically, using three-way analysis of variance (ANOVA) and the Wilcoxon t-test.

Measurement of Locomotor Activity

All animals subjected to the Y-maze test were individually placed on an Automex-II in order to count their motions in a 12-hour period from 19:00 to 07:00. The statistical differences in motion counts among the different experi­ mental animal groups were evaluated using the Wilcoxon t-test.

Neuropathological Study

Animals which had completed the Y-maze test and assessment of locomotor activity were anesthetized by ethyl inhalation. They were sacrificed by trans­ cardial perfusion with a 4% paraformaldehyde solution for conventional light microscope and histochemical examinations, and with a perfusate containing 3% potassium dichromate and 0.2% osmium tetroxide for quantitative Golgi study (Millhouse 1981). In the latter study, the spines of the apical and basal dendrites of the pyramidal neurons in layers II and III of the fronto-parietal cortex (Zilles and Wree 1985) were quantitatively measured, as described elsewhere (Sholl1953; Miyazawa et al. 1988). In brief, the number of spines on 20 J.tm segments of, apical and basal dendrites respectively, at a distance of 100 J.tm from the cell body of a neuron were counted. The number of dendritic spines obtained from 30 pyramidal neurons of one aminal was considered to represent spine density. Student's t-test was used for the statistical comparison of spine densities among different experimental groups (Miyazawa and Sato 1991).

Paraffin-embedded sections of the brain were stained by an indirect immuno­ histochemical technique, with mouse monoclonal antibody (MAb) against synaptic vesicle protein (SVP-38) purified from guinea-pig cerebrum (Obata et al. 1986, 1987); histochemical quantification of SVP-38 was then carried out. In brief, the sections were incubated with MAb and FITC-conjugated sheep anti-mouse IgG, after which the fluorescence intensity of the immunoreactive products in a 5-J.tm spot in the molecular layer of the parieto-occipital cortex was measured with a microphotometer. The fluorescence intensity values were expressed as the mean standard error of the 60 measurements of four rats in each experimental group, and the results were statistically compared by Student's t-test (T. Miyazawa et al., unpublished data).

Immunoblot Analysis of Synaptic Vesicle Protein (SVP-38)

For electrophoresis, animals in each experimental group were sacrificed by decapitation at 4 weeks of age. The isolated brain tissues were homogenized in 10 volumes of electrophoresis-sample buffer and subjected to immunoblot analysis. Sodium dodecy1 sulfate polyacrylamide gel electrophoresis (SDS­ PAGE) was performed by Laemmli's method (1970). The gels were then electroblotted onto nitrocellulose sheets and the blots were reacted successively with MAb, HRP-conjugated goat anti-mouse IgG, and 3,3'-diaminobenzidine (DAB).

Results

Sequelae of Early Ventriculoperitoneal Shunt

The survival rates of 4-week and 8-week-old hydrocephalic HTX treated with early shunts were approximately 80% and 40%, respectively. Because of animal death due to shunt malfunction, shunt infection, and subdural hematoma, only 32% of the animals thus treated grew to a sexually mature age and could be subjected to the Y-maze test. The survival rate of 8-week-olds in the delayed shunt group was approximately 20%.

Fig. 1. Ventriculographic follow-up of HTX-rat. Left, non-hydrocephalic HTX-rat with sham-operation (control); Center, hydrocephalic HTX-rat with early shunt insertion at 9 days after birth; Right, hydrocephalic HTX-rat with delayed shunt insertion at 4 weeks after birth

Ventriculography and MRI

The early shunt and delayed shunt groups of hydrocephalic animals displayed distinct differences in the chronological changes in size of their lateral ven­ tricles. In the former group, the size of the lateral ventricle, as assessed by venticulography early during shunt procedure, was abnormally enlarged. However, follow-up ventriculography approximately 4 and 8 weeks after birth revealed that early shunt resulted in normalization not only of enlarged ven­ tricles, but also of cranial vault bulging (Figs. 1 and 2). Normalization of abnormally enlarged ventricles concomitant with increase in cortical mantle thickness following early shunt placement was clearly demonstrated by MRI (Fig. 3). Contrary to these findings, little reduction in the size of abnormally enlarged ventricles was observed in the delayed shunt group (Figs. 1 and 2).

Y-Maze Test: Early Shunt Group

The learning ability of sexually mature HTX whose congenital hydrocephalus had been arrested by an early shunt was assessed by the Y-Maze test and compared with that of sham-operated animals. In both groups, the correct

response rate increased progressively with advancing sessions, as demonstrated by the significant session effect of analysis of variance [F(23,552) = 23.38, P < 0.01 ANOVA]. Although a statistically significant difference of mean correct response could be found in 2 out of 24 sessions, the group effect of analysis of variance [F(1,23) = 1.43 N.S. ANOVA] did not show any statistically significant difference between the two groups. In addition, there was inter­ action between the two factors of group and session [F(23,552) = 1.77, P < 0.05 ANOVA] (Fig. 4a). The response latency time of both groups was pro­ gressively shortened with advancing sessions, as demonstrated by the significant session effect of analysis of variance [F(23,552) = 6.75, P < 0.01 ANOVA].

Comparisons of mean response latency time in each session with Wilcoxon t-test did not demonstrate significant differences between the two groups in all sessions. And then, there was no significant difference between the two groups as demonstrated by group effect of analysis of variance [F(1,23) = 0.06 N.S. ANOVA]. In addition, no interaction between the two factors of group and session was found [F(1,23) = 0.55, N.S. ANOVA] (Fig. 4b).

Fig. 4. Light-darkness discrimination test (Y-maze test): a Comparison of correct response rate between early shunt and sham-operated groups, and b comparison of response latency time between early shunt and sham-operated groups. Vertical bars indicate standard errors of the mean

These findings, obtained from animals in the early shunt group, were found to be entirely different from those of animals in the delayed shunt group, where a significantly lower mean correct response rate and prolonged mean response latency time were observed [F(1,23) = 10.16, P < 0.01, F(1,23) = 10.35, P < 0.01 ANOVA] (Miyazawa and Sato 1991).

Locomotor Activity in Early and Delayed Shunt Groups

When nocturnal locomotor activity of animals in the early shunt group was compared with that in the sham-operated group, there was no remarkable difference in any one hour from 19:00 to 07:00 or in the mean 12-hour cumulative activity counts (Fig. 5). These findings differed from the observation of another study that nocturnal locomotor activity of animals in the delayed shunt group was remarkably greater than that in the sham-operated group (Miyazawa and Sato 1991).

Qualitative and Quantitative Golgi Studies (Takashima et al. 1978)

Histological features such as thickness, length, and degree of bifurcation of the apical and basal dendrites of the pyramidal neurons in layers II and III of the fronto-parietal cortex stained by rapid Golgi technique were found not to be differ among the early and delayed shunt groups and the sham-operated group. However, the spine density of both apical and basal dendrites was remarkably decreased in animals of the delayed shunt group as compared with the density in animals of the respective sham-operated groups (Miyazawa and Sato 1991). On the other hand, the spine density in animals of the early shunt group was not significantly different from that of sham-operated animals (Fig. 6).

Quantitative Histochemical Measurement of SVP-38

Chronological changes in fluorescence intensity in layer I of the parieto­ occipital cortex of hydrocephalic HTX were measured at different times after birth and compared with changes in sham-operated HTX. Fluorescence inten­ sity of the cortex in both hydrocephalic and sham-operated HTX increased linearly up to 3 weeks of age, but after 4 weeks of age, the values for the hydrocephalic rats were significantly decreased in comparison with those of sham-operated animals (Fig. 7). In contrast, the values obtained from early shunt animals were found to be not significantly different from those of sham­ operated animals (Fig. 8).

Immunoblot Analysis of SVP-38

In the cerebrum of 4-week-old non-hydrocephalic HTX, SVP-38 was detected by immunoblot analysis as an intense band with a molecular weight of 38 kilodalton. In contrast, in the cerebrum of 4-week-old hydrocephalic HTX, SVP-38 decayed in parallel with the progression of hydrocephalus. Such decay

Fig. 5. Locomotor activity assessed with Automex II: A Comparison of nocturnal activity between early shunt and sham-operated groups, and B comparison of mean 12- hour cumulative activity between early shunt and sham-operated groups

of SVP-38 was not observed when progression of hydrocephalus had been arrested by early shunt insertion (Fig. 9)

Discussion

In earlier studies, large animals such as cats and rabbits, in which aquired hydrocephalus was induced by such techniques as intracisternal injection of kaolin, were used as hydrocephalic animal models for experimental shunt oper-

Fig. 6. Comparison of the spine density of apical and basal dendrites of pyramidal neurons between early and sham-operated groups in layers II and III of the fronto­ parietal cortex. Horizontal bars indicate standard errors of the mean

Fig. 9. Immunoblot analysis of SVP-38 in the cerebral cortex of 4-week-old HTX-rats. Lanes 1 and 4, non-hydrocephalic HTX-rats with sham-operation; Lanes 2 and 5, hydroceph alic HTX-rats without ventriculoperitoneal shunt; Lanes 3 and 6, hydro­ cephalic HTX-rats with early shunt insertion ation (Granholm L 1966; Hochwald and Epstain 1973; Rubin and Hochwald 1976; Del Bigio and Bruni 1980; Fried et al. 1987). It has, however, become increasingly difficult in recent years to use such large animals in experiments. Such being the case, the use of congenitally hydrocephalic HTX-rats and development of the ventriculoperitoneal shunt technique in small animals appear to be significant in hydrocephalus research. As was reported in the present study, morbidity and mortality of HTX to which the V-P shunt was applied were still very high. Consequently, we felt that techical improvement of the V-P shunt would have to be established before the universal validity of this experimental model could be generally accepted. Nevertheless, various important observations were made in the present investigation.

When chronological changes in size of the lateral ventricles after shunt insertion, assessed by means of ventriculography, were compared in the early and delayed shunt groups, the abnormally enlarged ventricles in the early shunt group were found to be promptly normalized after V-P shunt placement, but this did not occur in the delayed shunt group. That reduction in ventricular size after early shunt placement was accompanied by thickening of the cortical mantle was clearly demonstrated by MRI. These observations may indicate that selection in timing of V-P shunt placement is one crucial factor in regard to prevention of functional and morphological disturbances of the developing brain caused by congenital hydrocephalus in HTX-rats.

The brain weight of a rat reportedly increases rapidly until the 25th day after birth, by which time it may be 85% of the weight of an adult brain. Migration of nerve cells is considered to be a major factor in the brain weight increase during the first 10 days of life. These migrating nerve cells differentiate to produce the cortical cell laminae. During the next 10 days, the axons and dendrites develop and myelin forms. Completion of the neuropil is thought to increase the weight of the cerebral mantle (Haas et al. 1970; Sugita 1971; Wada 1988).

According to quantitative analysis of the morphological development of synapses in the cerebral cortex of rats, the number of synapses increases in linear fashion during the period between 1 and 3 weeks of life, and reachesa plateau thereafter (Aghajanian and Bloom 1967; Armstrong-James and Johnson 1970; Miller et al. 1988; Adams and Jones 1982; Blue and Parnavelas 1983; Markus and Petit 1987). In congenitally hydrocephalic HTX-rats, progressive enlargement of the lateral ventricles, concomitant with thinning of the cortical mantle and periventricular CSF edema takes place in the white matter during a 3-week period after birth (Wada: 1988). Miyazawa et al. (1988), when they investigated cortical pyramidal neurons of such hydrocephalic brains by quali­ tative and quantitative Golgi technique at 2 weeks after birth, found that the neuronal soma remained morphologically intact, although such drastic changes as winding, tortuosity, and varicosity were noted in the apical and basal dendrites, in association with marked reduction in the spine density (Borit and Sidmann 19-72; Marin-Padilla 1972; Purpura et al. 1982; McAllister et al. 1985). Since dendritic spines are now recognized as representing "specific postsynaptic receptive structures" on the dendrites, it was assumed by us that presynaptic structures also may be affected by hydrocephalic pathology. Consequently Miyazawa et al. (unpublished data) demonstrated, by using quantitative histo­ chemical measurement of the synaptic vesicle protein, SVP-38, that in the cerebral cortex of hydrocephalic HTX-rats, a progressive increase in the amount of SVP-38 occurs during a 3-week period after birth, followed by a marked drop in its amount at 4 weeks after birth. The age-related in­ crease of SVP-38 observed in both hydrocephalic and control HTX-rats up to 3 weeks after birth seemed to be reflected by a progressive increase of the synaptic vesicle protein occurring in association with synaptogenesis. In this regard, we felt that the age-related changes of synaptic density, assessed by quantitative electronmicroscopic analysis of synaptogenesis of the rat cerebral cortex, and reported by several investigators such as Aghajanian et a!. (1967), Blue and Parnavelas (1983), and Markus and Petit (1987), lent support to our observation. The sudden decay of SVP-38 4 weeks after birth is considered to be one of the direct effects of the progression of hydrocephalus. This observa­ tion, obtained by quantitative histochemical methods, was also confirmed by immunoblot analysis in the present investigation. Since reduced spine density was noted in the hydrocephalic animals as early as 2 weeks after birth, we speculated that a dissociation of pre- and post-synaptic structures may be present in the disturbance of synaptogenesis.

According to the observations of Jones and Bucknall (1987, 1988), the resting pressure of cerebrospinal fluid in the lateral ventricles of hydrocephalic HTX-rats was not elevated above normal for up to 10 days after birth, but by 21 days the pressure was nearly twice that of normal rats. Miyaoka eta!. (1988) reported that local cerebral glucose utilization (LCGU) of severely affected hydrocephalic HTX-rats was decreased throughout the brain and that applica­ tion of a delayed shunt to such animals at 4 weeks of age could not completely correct the impairment of LCGU. Therefore, the significance of early shunt insertion in these HTX is believed to lie in eradicating the pathological pro­ cesses, which occur secondary to increased intracranial pressure, that affect cerebral blood flow and metabolism in the developing brain. As was demon­ strated in the present investigation, when a ventriculoperitoneal shunt was inserted into animals with progressive hydrocephalus at approximately 4 weeks after birth, not only was there no reduction in size of the abnormally enlarged ventricles, but there was also no increase in cortical mantle thickness. Further­ more, spine density in the cerebral cortex in such animals also was found to be decreased when compared with that in sham-operated animals, and learning disability could not be corrected which differed from results in with sham operated animals. Contrary to these observations, in the present investiga­ tion in HTX-rats we found that insertion of a V-P shunt as early as 7-9 days after birth prevented most of the unfavorable effects inflicted by hydro­ cephalus upon the morphological and biochemical development of the brain. In fact, early shunt placement was found to result in normalization of the abnormally enlarged ventricles, in association with simultaneous thickening of the cerebral cortical mantle and preservation of normal spine density in the dendrites of cortical pyramidal neurons. The learning ability of such animals 24 K. Suda et at.

was not found to be disturbed compared with that of the sham-operated group.

We assume, based on these observations, that early shunt placement may have a beneficial role both in repairing and in preventing the impairment of synaptogenesis which occurs in association with the progression of congenital hydrocephalus. Whether or not there is a close correlation between learning disability and impairment of synaptogenesis, as observed in the experiment with the delayed shunt, is still not clear. However, such a correlation seems to plausible, since the prevention of disturbance of synaptogenesis and lack of learning disability in the early shunt experiment were confirmed in the present investigation.

During human fetal brain development, differentiation of the neocortex extends from the beginning of the 3rd month to the end of the 6th month at gestational age (Tuchmann-Duplessis et al., 1980). Synaptogenesis of the cere­ bral cortex begins from the 7th month of gestation and reaches a maximum at the age of one year (Huttenlocher 1979). Although direct correlation in cerebral development between human and rat is difficult to define, the early and delayed shunts applied to HTX-rats by ourselves may be regarded as a model for treat­ ment of human congenital hydrocephalus in utero and in late infancy. Many investigators have reported that learning disability was found in children whose congenital hydrocephalus was treated during the postnatal period (Milhorat 1972; Dennis et al. 1981; Pretorius et al. 1985). In regard to this, treatment of congenital hydrocephalus in utero, namely, a ventriculoamniotic shunt, was thought to be most promising (Clewell et al. 1982); however the evolution of this new method has been became slowed by the high morbidity and mortality of the fetuses and/or children thus treated (Michejda et al. 1986).

At present, pediatric neurosurgeons generally consider that when congenital hydrocephalus is diagnosed in utero by ultrasound, the affected fetus should be delivered after 33 weeks of gestation and treated by prompt cerebrospinal fluid diversion, when the respiratory distress syndrome can be overcome by medical treatment (Edwards et al. 1986). However, the sequelae of prompt cerebrospinal fluid diversion in premature infants cannot be ignored. Con­ sidering our experimental observations presented here, the development of new treatments for congenital hydrocephalus in utero seems to be one of the tasks required of pediatric neurosurgeons.

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