Exp Brain Res (2000) 135:41Ð52 DOI 10.1007/s002210000474
RESEARCH ARTICLE
K. Vio · S. Rodríguez · E.H. Navarrete J.M. Pérez-Fígares · A.J. Jiménez · E.M. Rodríguez Hydrocephalus induced by immunological blockage of the subcommissural organ–Reissner’s fiber (RF) complex by maternal transfer of anti-RF antibodies
Received: 14 February 2000 / Accepted: 15 May 2000 / Published online: 9 September 2000 © Springer-Verlag 2000
Abstract Stenosis of the cerebral aqueduct seems to Introduction be a key event for the development of congenital hydrocephalus. The causes of such a stenosis are not Congenital hydrocephalus has been reported to occur not well known. Overholser et al. in 1954 (Anat Rec only in humans, but also in several laboratory mammals. 120:917Ð933) proposed the hypothesis that a dysfunction The latter have been used to investigate certain aspects of the subcommissural organ (SCO) leads to aqueductal of this disease (cf. Pérez-Fígares et al. 1998). One of the stenosis and congenital hydrocephalus. The SCO is a unsolved issues is how much the stenosis and/or the brain gland, located at the entrance of the cerebral aque- complete obliteration of the cerebral aqueduct is in- duct, that secretes glycoproteins into the cerebrospinal volved in the pathogeneses of congenital hydrocephalus. fluid that, upon release, assemble into a fibrous structure Indeed, the disease may course either with a normal, or a known as Reissner’s fiber (RF). By the permanent addi- stenosed, or an obliterated aqueduct. It has even been tion of new molecules to its rostral end, RF grows and postulated that when aqueductal stenosis does occur it is extends along the aqueduct, fourth ventricle, and central due to a secondary effect that results from hydrocepha- canal of the spinal cord. The immunological blockage of lus, and is not the cause of it (Borit 1976; Williams the SCO-RF complex has been used to test Overholser’s 1973) However, most reports support the view that ste- hypothesis. The following was the sequence of events nosis of the aqueduct is a key event for the development occurring in pregnant rats that had been immunized with of congenital hydrocephalus (Borit 1976; Bruni et al. RF glycoproteins: the mother produced anti-RF antibod- 1988; Jones and Bucknall 1988; Jones et al. 1987). ies and transferred them to the fetus through the placenta The causes of such a stenosis, however, are not well and to the pup through the milk, and the antibodies known. Viral infections occurring in several species are reached the brain of the fetus and pup and blocked the known to sequentially trigger ependymal damage, an SCO-RF complex. This resulted in a permanent absence aqueductal stenosis, and a non-communicating hydro- of RF that was followed by stenosis of the cerebral aque- cephalus (Margolis and Kilham 1969; Nielsen and duct, and then by the appearance of hydrocephalus. The Baringer 1972). Wong et al. (1995) have suggested that latter was patent until the end of the 6-month observation N-CAM L1, a molecule inducing cell adhesion, is in- period. The chronic hydrocephalic state appeared, in volved in the differentiation of the ependymal lining; the turn, to induce new alterations of the SCO. It is conclud- same authors have shown that in humans a mutation in ed that a selective immunological knock out of the L1 leads to a congenital hydrocephalus with stenosis of SCO-RF complex leads to hydrocephalus. the cerebral aqueduct (Wong et al. 1995). Recently, a transgenic mouse model with a mutation in the L1 gene Key words Aqueduct stenosis á Hydrocephalus á has been obtained (Dahme et al. 1997). These mice had a Immunoneutralization á Subcommissural organ á Rat hydrocephalus characterized by a dilatation of the lateral and fourth ventricles; the cerebral aqueduct was not ste- K. Vio · S. Rodríguez · E.H. Navarrete · E.M. Rodríguez (✉) nosed but it was irregularly shaped (Dahme et al. 1997; Instituto de Histlogía y Patología, Facultad de Medicina, Fransen et al. 1998). This latter abnormality was consid- Universidad Austral de Chile, Valdivia, Chile ered as a probable cause of the dilatation of the lateral e-mail: [email protected] Tel.: +56-63-221207, Fax: +56-63-221604 ventricles (Fransen et al. 1998). An old hypothesis proposed by Overholser et al. J.M. Pérez-Fígares · A.J. Jiménez Departamento de Biología Celular y Genética, (1954), still valid as a working hypothesis, involved the Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, subcommissural organ (SCO) in the pathogeneses of Spain aqueductal stenosis (see below). The SCO is a brain 42 gland located in a key position, the roof of the third may be impaired when antibodies against the SCO secre- ventricle, at the entrance of the cerebral aqueduct tion reach the CSF. (Rodríguez et al. 1992, 1998). The SCO secretes into The immunological blockage of the SCO-RF complex the ventricle glycoproteins of high molecular weight during the whole of fetal life and the early postnatal (Nualart et al. 1991), that upon release condense into a weeks has been obtained by the transplacental delivery fibrous structure known as Reissner’s fiber (RF). As new to the fetuses, and through the milk to the pups, of spe- molecules are added to its rostral end, RF grows at a cific antibodies against the SCO secretory proteins. constant rate, and extends along the aqueduct, fourth Some of these animals died during the first 3 postnatal ventricle, and central canal of the spinal cord (Rodríguez weeks; those who survived displayed a rise in the CSF et al. 1992, 1998). The SCO differentiates early in ontog- concentration of several amines (Rodríguez et al. 1999). eny (Schöbitz et al. 1986) and remains fully active dur- The aim of the present investigation was to use this ani- ing the fetal life (Oksche 1969). In the human, the SCO mal model in order to test the hypothesis of Overholser differentiates during the 2nd month of pregnancy and et al. (1954) that a dysfunction of the SCO may lead to a reaches a maximal development during the second half congenital hydrocephalus. Indeed, during the first post- of the embryonic life (Oksche 1969; Rodríguez et al. natal weeks, the rats with a permanent blockage of the 1992, 1998). The human SCO appears to secrete glyco- SCO-RF complex by maternal delivery of antibodies de- proteins into the CSF that do not aggregate in the form velop hydrocephalus. of an RF, but remain soluble in the CSF (Rodríguez et al. 1993). Overholser et al. (1954) postulated that: (1) the secre- Materials and methods tion of the SCO released into the rostral end of the aque- duct during fetal life prevents the closure of the cerebral Animals aqueduct, thus facilitating the free circulation of CSF be- Maternal transfer of antibodies tween the third and the fourth ventricles, and (2) a dys- function of the SCO might lead to the aqueductal steno- The model of maternal delivery of antibodies against RF glyco- sis and a congenital hydrocephalus. This hypothesis has proteins, recently developed in our laboratory (Rodríguez et al. 1999), was used in the present investigation. This experimental gained support from recent findings. Maldevelopment of design, that has been explained in detail elsewhere (Rodríguez the SCO induced by X-irradiation during fetal life is fol- et al. 1999), will be briefly described. Twenty female Sprague- lowed by stenosis of the aqueduct and then by congenital Dawley rats were immunized with the constituent glycoproteins of hydrocephalus (Takeuchi and Takeuchi 1986). Accord- RF. The first immunization was with 100 µg RF glycoproteins emulsified with complete Freund’s adjuvant injected subcutane- ing to Takeuchi et al. (1987), the mouse MT/HokIdr ously. The second immunization was with 50 µg RF glycoproteins lacks an SCO and develops a congenital hydrocephalus. emulsified with incomplete Freund’s adjuvant injected into multi- Hydrocephalic SUMS/np mice have been reported to ple subcutaneous sites 17 days after the first immunization. For have aqueductal stenosis and a small SCO (Bruni et al. the third immunization, 17 days after the second immunization 1988; Jones et al. 1987). A drastic reduction in the size 50 µg RF glycoproteins were injected into the peritoneum using phosphate-buffered saline as vehicle. Seven days after the second of the SCO has been reported in rats CWS/Idr (Takeuchi immunization, the immunized rats were mated overnight; fecunda- et al. 1988) and H-Tx (Jones and Bucknall 1988) with tion occurred in all of them. Under this experimental condition, congenital hydrocephalus, and in rats with a postnatally mothers transferred antibodies against RF glycoproteins to the fe- induced hydrocephalus (Irigoin et al. 1990). The SCO of tuses through the placenta, and to the pups through the milk. Thus, during the key period extending from E-12, when the placenta is the mutant mouse hyh, that develops a severe congenital fully developed, to the end of the 1st postnatal month, when the hydrocephalus, shows signs of increased secretory activ- pups stop suckling milk, the animals were permanently provided ity, and releases to the stenosed aqueduct a material that with anti-RF antibodies (Rodríguez et al. 1999). These authors aggregates, but it does not form an RF (Pérez-Fígares et have confirmed that the transferred antibodies actually reached the blood and the CSF of the fetuses and pups, and blocked RF forma- al. 1998). Severe morphological alterations of the SCO tion during the first 2 months of life. The 20 immunized mothers of hydrocephalic human fetuses have been reported delivered 180 pups; 18% died during the first 3 postnatal weeks. (Castañeyra-Perdomo et al. 1994). Thus, in all species One hundred and seven of the 148 surviving pups were divided in- developing congenital hydrocephalus, in which the SCO- to 14 groups (about 10 pups per group), which were processed for RF complex has been investigated, changes in such a light microscopy at birth and at the ages of 1, 2, 3, 4, 5, 6, 7, 8, and 11 weeks and 3, 4, 5, and 6 months. complex have been reported. The important question, however, whether the changes occurring in the SCO pre- cede hydrocephalus or are a consequence of the hydroce- Combined administration of endogenous (maternal source) phalic state has not yet been clarified. and exogenous antibodies Antibodies against the constituent glycoproteins of The titer of anti-RF antibodies in the plasma and CSF of the pups RF have been used to interfere with the function of the of rats immunized with RF glycoproteins starts to decline during SCO. Thus, a single injection of these antibodies into the the 2nd month of life (Rodríguez et al. 1999). In order to keep a CSF of adult rats leads to a transient blockage of RF for- high titer of antibodies in the CSF beyond the 1st month of life, 41 out of the 148 surviving pups, when they reached 1 month of age, mation (Rodríguez et al. 1990), followed by disturbances started to receive a weekly injection into a lateral brain ventricle in the CSF circulation (Cifuentes et al. 1994). These ex- of 10 µg anti-RF IgG, purified by affinity chromatography. The periments demonstrated that the function of the SCO weekly injections continued until the rats were 6, 11, or 12 weeks 43 old. The antibody was dissolved in 20 µl saline and perfused into the ventricle during 15 min, using a perfusing pump. Total CSF Results volume of a 1-month-old rat is about 350 µl. One week after the last injection, they were processed for light microscopy (cf. Rod- Changes occurring during the first 3 months of life due ríguez et al. 1999). to maternal delivery of anti-RF antibodies During the first 3 months of life, the pups of immunized Control rats mothers displayed an SCO that did not differ from the Two groups of control rats were prepared: SCO of control rats with respect to its shape, spatial ori- entation, and immunoreactivity with AFRU (Fig. 1). In 1. Thirty-six pups of control untreated mothers were processed for light microscopy at birth and at the ages of 1, 2, 3, 4, and the normal rat, newly released secretory glycoproteins 8 weeks and 3, 5, and 6 months. undergo a certain degree of aggregation and form a film 2. Fifteen pups of mothers immunized with rabbit IgG (Sigma, on the ventricular surface of the SCO. This film of secre- USA) following the same immunization and pregnancy proto- tory material has been designated as pre-RF (Rodríguez col described above (100, 50, and 50 µg IgG for the three im- munization sessions) were processed for light microscopy at et al. 1992). About 1 h after being released, the glyco- birth and at the ages of 1, 2, 3, 4, and 8 weeks. proteins become further aggregated to form the RF prop- er (Rodriguez et al. 1992). In the experimental rats a nor- The care and experimental manipulation of animals were accord- ing to the regulations of the Universidad Austral de Chile, which mal pre-RF was not visualized. Irregularly aggregated are in accordance with International Regulations and Policies on AFRU-immunoreactive material, forming fibrils and the use of animals in research. masses, was seen in the rostral end of the cerebral aque- duct (Fig. 1). Immunoreactive fibrils were also found in Microscopy locations where RF material has never been seen in nor- mal animals, such as the rostral (Fig. 1 inset B) and ven- Animals from the experimental and control groups were anesthe- tral (infundibular recess) regions of the third ventricle. tized with ether and their central nervous system fixed by vascular In the rat, the first RF is formed during the 1st postna- perfusion with Bouin’s fluid, dissected out, and immersed in the tal week (Schöbitz et al. 1993; Fig. 2). The pups of same fixative for 48 h. Dehydration was in increasing concentra- tions of alcohol, and embedding was in paraffin. The region of the mothers immunized with RF glycoproteins lacked the lateral ventricles was oriented for transversal sectioning; the rest capacity to form an RF, indicating that the SCO-RF com- of the brain and the brain stem were orientated for sagittal section- plex had been immunologically blocked since the 1st ing; the spinal cord was divided into several fragments all of postnatal week. During the first 2 months of life, abnor- which were embedded to obtain transversal sections. Parallel se- ries of sections obtained by mounting one every ten sections were mal aggregates of RF material were seen along the aque- stained with hematoxylin-eosin or processed for immunocyto- duct and rostral parts of the fourth ventricle. These ag- chemistry. gregates appeared as an irregularly shaped fiber with bound masses of AFRU-immunoreactive material or as Immunocytochemistry small spheres (Fig. 3). These aggregates immunoreacted with AFRU and with anti-rat IgG. A film of AFRU- Series of paraffin sections of the brain and spinal cord were pro- immunoreactive material was seen on the walls of the cessed for the immunoperoxidase method of Sternberger et al. aqueduct and fourth ventricle. In control rats, AFRU im- (1970). The sections were sequentially incubated in: munoreactivity was circumscribed to the normally struc- 1. The primary antiserum AFRU (A=antibody, FR=fiber of tured RF (Fig. 2). Reissner, U=urea). This antibody was raised in rabbits against After the 3Ð4 postnatal weeks, the experimental rats the constitutive glycoproteins of the bovine RF and extracted in a medium containing urea (Rodríguez et al. 1984). AFRU presented a marked stenosis of the distal end of the cere- specifically reacts with high molecular weight glycoproteins bral aqueduct; some of these rats also presented a steno- secreted by the SCO into the CSF, where they aggregate to sis of the cephalic end of the aqueduct (Fig. 1). At this form RF (Nualart et al. 1991). AFRU was used at a dilution level, some of the ventral ependymal cells facing the 1:1000 and incubation was for 18 h. SCO displayed AFRU immunoreactivity (Fig. 1 inset A). 2. The secondary antibody (anti-rabbit IgG), diluted 1:15, for 30 min. These rats presented a dilatation of the lateral and third 3. Rabbit PAP (Sigma), 1:75 dilution, for 30 min. ventricles, compatible with a moderate hydrocephalus. 3,3′-Diaminobenzidine tetrahydrochloride (DAB; Sigma) was Some of the changes described above for the pups of used as electron donor. All incubations were performed in a moist immunized mothers became more pronounced when lit- chamber at room temperature. The antisera and the PAP complex ter pups, 1 month old, received a weekly injection into a were diluted in TRIS buffer, pH 7.8, containing 0.7% non-gelling lateral brain ventricle of an anti-RF antibody (Figs. 4, 5, seaweed lambda-carrageenan (Sigma) as saturating agent, and 6, 7, 8, 9). The dilatation of the third and lateral ventri- 0.5% Triton X-100 (Sigma). Some of the series were further stained with hematoxylin. Omission of incubation in the primary cles was patent (Figs. 4, 8). The lateral ventricles and a antiserum and use of preimmune serum in the immunostaining discrete region of the third ventricle close to the rostro- procedure were used as control tests. dorsal aspect of the thalamus, presented ependymal de- nudation (Figs. 4, 7). The denuded areas presented a smooth surface; the subependymal tissue layer, normally present under the ependyma, was also present in the de- nuded areas (Figs. 4, 7). The SCO showed modifications 44
Fig. 1 Sagittal section through the epithalamus of an 8-week-old Detailed magnification of the fibrils of RF material located in the pup with a blockage of the subcommissural organÐReissner’s fiber third ventricle. ×500 (SCO-RF) complex by maternal transfer of antibodies against RF glycoproteins. Immunostaining using AFRU as primary antibody. Fig. 2 Eight-week-old control untreated rat, immunostained using AFRU as primary antibody. Distal end of the cerebral aqueduct An abnormal pre-RF (asterisk, arrowhead), fibrils of RF material × located out of place, such as the third ventricle (arrow), and steno- (ca) containing a normal RF (RF). C Cerebellum. 500 sis of the rostral end of the cerebral aqueduct (CA) are shown. At Fig. 3 Sagittal section through the distal end of the cerebral aque- the site of stenosis, aqueductal ependymal cells facing the SCO duct (ca) of an 8-week-old pup of a mother immunized with RF become AFRU immunoreactive (square). CP Choroid plexus, glycoproteins. Immunoperoxidase staining using AFRU as prima- III V third ventricle. ×125. Inset A Higher magnification of area ry antibody. A row of immunoreactive spheres is found in the ce- framed in square showing immunoreactive ventral ependymal rebral aqueduct (arrows) and an RF is missing. ×500 cells (arrow) facing the stenosed cerebral aqueduct. ×500. Inset B 45
Fig. 4Ð7 Legend see page 46 46 similar to those described in hydrocephalic rats with a have had anti-RF antibodies in their plasma and CSF postnatally induced hydrocephalus (Irigoin et al. 1990), during the first 2 months of life (cf. Rodríguez et al. such as a change in the shape of the organ and a reduc- 1999), presented alterations in the SCO-RF complex and tion in the height of its secretory cells (Fig. 8). These rats in the ventricular system, most of which corresponded to showed a marked stenosis of the distal end of the cere- the same alterations found during the first 3 months of bral aqueduct (Figs. 4, 5, 6). At the site of stenosis, the life (see above). Thus, the lateral and third ventricles ependyma of the ventral and dorsal walls of the aqueduct continued dilated (compare Figs. 10, 11 with Figs. 12, appeared to establish contact, with a virtual absence of 13, 14, 15, 16), the collicular recess appeared enlarged aqueductal lumen (Figs. 5, 6). Abnormal fragments of (Figs. 12, 14, 16), and in some animals the volume ex- RF and masses of AFRU-immunoreactive material accu- pansion of the ventricles reached a degree not found dur- mulated in the aqueduct in the vicinity of the stenosed ing the first 3 months of life (Fig. 16). In these latter region (Fig. 9 and inset A). The walls of the aqueduct, cases, an atrophy of the choroid plexus was evident but not those of the third or fourth ventricle, had a film (Fig. 16). The degree of stenosis of the distal end of the of AFRU-immunoreactive material (Figs. 8 inset, 9). The aqueduct varied among animals. In some rats the steno- ependymal cells of the collicular recess had abundant sis was marked and extended along the whole post- AFRU-immunoreactive material associated with the api- collicular region of the aqueduct; in others it was cir- cal plasma membrane and as small masses lying on top cumscribed to a small stretch at the distal end of the aq- of the cilia (Fig. 8 inset). AFRU-immunoreactivity was ueduct; in a few rats the aqueduct was opened. also seen in the neuropile of the brain stem (Fig. 9). In those rats with a chronic hydrocephalus the brain Clusters of macrophages containing AFRU-immunoreac- appeared flattened with a patent reduction of its cross- tive material in their cytoplasm and cells most likely cor- sectional area (Fig. 12). The way in which the brain was responding to lymphocytes were seen in the third ventri- dissected out did not allow to establish whether or not cle and aqueduct (Figs. 4, 9 inset B). there was a dilatation of the subarachnoid space. The None of the changes described above were found in SCO of these rats became flattened, with an evident re- the pups of mothers immunized with rabbit IgG. duction in the height of the secretory ependymal cells. There was a change in the spatial relationship between the SCO, deep pineal, and habenular commissure, with Changes occurring during months 4Ð6 of life due the latter two structures changing from a dorsal to a ros- to maternal delivery of anti-RF antibodies tral position with respect to the SCO (Figs. 11, 13). In many rats, the AFRU-immunoreactive material secreted Adult rats, 4Ð6 month old, born from mothers immu- into the ventricle aggregated abnormally, resulting in ir- nized with RF glycoproteins and which, consequently, regular fibrous structures that projected toward the aque-