Exp 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 induced by immunological blockage of the –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 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 . tized with ether and their 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 , presented ependymal de- nudation (Figs. 4, 7). The denuded areas presented a smooth surface; the subependymal tissue layer, normally present under the , was also present in the de- nuded areas (Figs. 4, 7). The SCO showed modifications 44

Fig. 1 Sagittal section through the 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 , 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 , 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-

Fig. 4 Sagittal section through the brain of a 7-week-old pup of a Fig. 8 Sagittal section through the brain of a 12-week-old pup of mother immunized with RF glycoproteins. During the 5th and 6th a mother immunized with RF glycoproteins. During the last weeks of life, the pup received an intraventricular injection of the 7 weeks of life, the pup received a weekly intraventricular injec- reactive IgG fraction of AFRU. The brain was serially cut, and the tion of the reactive IgG fraction of AFRU. The brain was serially section shown corresponds to the midsagittal plane. Hematoxylin- cut, and the section shown corresponds to a plane close to the mid- eosin stain. A marked stenosis of the distal region of the cerebral line. Immunostaining using AFRU as primary antibody; back- aqueduct (delimited by two thick arrows) and dilatation of the ground staining with hematoxylin. A dilatation of the third ventri- third ventricle (III V) and rostral end of the aqueduct (ca) are cle (asterisks) is patent. SO Immunoreactive subcommissural or- patent. The ventricular wall close to rostrodorsal region of the gan, Th thalamus. ×32. Inset Collicular recess organ. AFRU- thalamus (th) displays ependymal denudation (area framed in rect- immunoreactive material is seen associated with the apical plasma angle 1). This area is shown at high magnification in Fig. 7. Clus- membrane of the ependymal cells and with the surface of most cil- ters of lymphocytes and macrophages (short arrows) are present in ia, and as masses lying on top of cilia (short arrows). CR Colli- the infundibular recess (ir) of the third ventricle and associated to cular recess, long arrow immunonegative cilium. ×1250 the ependyma of the (area framed in rectan- gle 2). This area is shown at high magnification in inset of Fig. 9. Fig. 9 Sagittal section through the distal end of the cerebral aque- so Subcommissural organ, c cerebellum. ×25 duct (CA) of the same rat as shown in Fig. 8 immunostained with AFRU. The brain was serially cut, and the section shown corre- Fig. 5 Adjacent section to that shown in Fig. 4, showing the area sponds to the midsagittal plane. The distal end of the aqueduct ap- of transition (arrow) between the dilated and stenosed regions of pears stenosed (large arrow). Rostral to this site, fragments of RF the cerebral aqueduct (CA). C Cerebellum. Hematoxylin-eosin (RF) and masses of RF material (arrowheads) accumulate. A coat stain. ×44 of immunoreactive material on the walls of the aqueduct (thin arrows) is seen. Immunoreactive material is also seen in the neuro- Fig. 6 Detailed magnification of stenosed region of the cerebral × aqueduct (CA). The dorsal and ventral ependymal linings establish pile underneath the ventral ependyma (asterisk). 250. Inset A De- a close contact. C Cerebellum. Hematoxylin-eosin stain. ×128 tailed magnification of Fig. 9 showing the abnormal RF (RF) and the associated clusters of immunoreactive material (arrowhead). Fig. 7 Detailed magnification of the area framed in rectangle 1 of ×1250. Inset B Detailed magnification of area framed in rectan- Fig. 4 showing the area of ependymal denudation (bent arrow). gle 2 of Fig. 4 showing a clusters of lymphocytes and macro- The subependymal region localized under the ciliated ependyma is phages in the vicinity of the median eminence. Macrophages con- also present in the area devoid of ependyma (asterisks). Th Thala- tain AFRU-immunoreactive material in their cytoplasm (arrows). mus. Hematoxylin-eosin stain. ×123 ×500 47

duct as well as toward the third ventricle (Fig. 15). In life (see above), were missing in the 4- to 6-month-old some rats, an RF with a normal appearance was seen in rats. the aqueduct. In all 4- to 6-month-old rats, bundles of Rats 4Ð6 months old, born from immunized mothers, immunoreactive fibrils were seen out of place, in rostral which, in addition, received a weekly injection of puri- and ventral regions of the third ventricle. Clustered im- fied anti-RF antibody into the lateral ventricle during the munocompetent cells, as those seen in the ventricles and 2nd and 3rd month of life, presented alterations in the associated with abnormal RF during the first months of SCO-RF complex and in the ventricular system indistin- 48 49 guishable from those displayed by rats of the same age suckling stops by the end of the 1st month of life, the an- and delivered with maternal antibodies only (see above). tibodies continue to circulate in the plasma and CSF of None of the experimental rats, at any of the ages in- the pups for an additional month (Rodríguez et al. 1999), vestigated (1 week to 6 months), had an RF in the central most likely due to the half-life of IgG; in human infants canal of the spinal cord. Control untreated rats of similar the half-life of IgG is 1 month (Turner 1996). On the ages, and the 1- to 8-week-old pups of mothers immu- other hand, it has been established that a single injection nized with rabbit IgG, all presented an RF in the central of anti-RF antibodies into the lateral ventricle of the rat canal of the spinal cord. leads to the fragmentation of RF and to a transient block- age of the formation of a new RF (Cifuentes et al 1994; Rodríguez et al. 1990, 1999). Thus, alterations in the Discussion SCO-RF starting during the 1st postnatal week and con- tinuing during the first 2 months of life of the pups of The immunoneutralization of the subcommissural organ immunized mothers, may be explained on the following during the fetal and early postnatal periods leads grounds: (1) the antibodies reaching the CSF react with to permanent alterations in the SCO-RF complex the glycoproteins being secreted by the SCO, forming Ag-Ab complexes [the existence of these complexes has Pregnant rats immunized with RF glycoproteins produce been proved (Rodríguez et al. 1999)], (2) the binding of antibodies against these proteins and transfer the immu- the antibodies to the RF glycoproteins interferes with the noreactive IgG to the fetuses through the placenta and to normal aggregation of the latter, resulting in abnormal the pups through the milk (Rodríguez et al. 1999). After aggregates, and (3) the presence of Ag-Ab complexes in the aqueduct and fourth ventricle triggers the arrival to this region of immunocompetent cells; macrophages Fig. 10 Sagittal section through the brain of an adult normal rat. would remove the complexes from the CSF. The occur- The brain was serially cut, and the section shown corresponds to a plane close to the midline. It shows the appearance of the third rence of this series of events is further supported by the ventricle, the subcommissural organ (so), a fully open cerebral following observation. During the 3rd month of life, aqueduct (arrows), and the fourth ventricle. Broken vertical line when anti-RF antibodies are no longer detectable in the shows height of brain tissue extending from corpus callosum to pi- plasma and CSF of the pups of immunized mothers, nei- al surface. p , th thalamus. Hematoxylin-eosin stain. ×14 ther the Ag-Ab complexes nor the immunocompetent cells are visualized in the brain cavities; still, RF contin- Fig. 11 High magnification of the area of the subcommissural or- ues to display abnormal characteristics. If at this age, ex- gan (so) shown in Fig. 10. h commissure, p deep pineal gland, ca cerebral aqueduct. ×32 ogenous antibodies are injected into the CSF, the Ag-Ab complexes and the immunocompetent cells reappear in Fig. 12 Sagittal section through the brain of a 6-month-old rat that the ventricles (Rodríguez et al. 1999). during the fetal period and the first 2 months of life was delivered with maternal antibodies against RF glycoproteins. The brain was In brief, the alterations of the SCO-RF complex dur- serially cut, and the section shown corresponds to the midsagittal ing the first 3 months of life of pups delivered with anti- plane. It shows a dilated third ventricle and collicular recess (as- RF antibodies may be explained on an immunological terisk), and stenosis of the cephalic (small arrow) and caudal basis. However, the alterations of the SCO-RF complex (large arrow) ends of the aqueduct. Broken vertical line indicates a reduction in the height of brain tissue extending from corpus cal- in 4- to 6-month-old pups of immunized mothers, a peri- losum to pial surface (compare with vertical line in Fig. 10). so od of life when anti-RF antibodies are absent, is difficult Subcommissural organ, p pineal gland, th thalamus. Hematoxylin- to interpret. We want to speculate on two alternative ex- eosin stain. ×14 planations: Fig. 13 High magnification of the subcommissural organ region shown in Fig. 12. The spatial relationship between the habenular 1. The exposure of the secretory cells of the SCO to an- commissure (h), deep pineal gland (p), and the subcommissural tibodies specific for the proteins secreted by these organ (so) appears altered (compare with Fig. 11). cp Choroid plexus. ×32 cells would lead to a sustained alteration in the secre- tory process and/or in the process of aggregation of Fig. 14 Section adjacent to that of Fig. 12 immunostained using the secreted glycoproteins. The following observa- AFRU as primary antibody. The immunoreactivity of the subcom- missural organ (arrow) is shown. ×14 tions support this possibility. After a single injections of anti-RF antibody into the CSF of adult rats, the ex- Fig. 15 High magnification of the subcommissural organ (so) re- gion shown in Fig. 14. Irregular fibrous aggregates of RF material isting RF undergoes fragmentation a few hours after extend in opposite directions, namely, the aqueduct and the third the injection, and the formation of a new RF is de- ventricle (arrows). ×32 layed for a week. Although the injected antibodies Fig. 16 Sagittal section through the brain of an 8-week-old rat were available to the SCO for 4 h only, a period in that during the fetal period and the first 2 months of life was deliv- which the CSF is completely renewed, the SCO ered with maternal antibodies against RF glycoproteins. The brain was unable to form a new RF for a whole week was serially cut, and the section shown corresponds to a paramedi- (Rodríguez et al. 1990). In vitro experiments have led an plane. Immunoperoxidase staining using AFRU as primary an- tibody. A marked dilatation of the third ventricle (star) and colli- to similar findings. It has been shown that explants of cular recess (asterisk), and atrophy of the choroid plexus (arrow) bovine SCO secrete glycoproteins into the culture me- are patent. so Subcommissural organ. ×16 dium (Estivill-Torrús et al. 1998; Lehman and Sterba 50 1993) that aggregate on the surface of the explants duced by X-irradiation during fetal life precedes stenosis (González et al. 1999). The addition of anti-RF anti- of the aqueduct and leads to congenital hydrocephalus body to the culture medium for 1 h appears to inhibit (Takeuchi and Takeuchi 1986). According to Takeuchi et the release and/or the aggregation of the secretory al. (1987), the mouse MT/HokIdr lacks an SCO and de- glycoproteins for the whole observation period of velops a congenital hydrocephalus. Distinct changes in 15 days (Schöbitz and Rodríguez unpublished obser- the SCO-RF of the hydrocephalic mutant hyh mice, in- vations). cluding the appearance in them of an extra source of RF 2. The alterations of the SCO-RF complex in 4- to material, have been recently reported (Pérez-Fígares 6-month-old pups of immunized mothers could be a et al. 1998). consequence of the hydrocephalus that started to de- The facts that in the human the SCO is especially velop during the postnatal period in which the SCO is secretorily active during fetal life (Oksche 1969; Rodríguez immunologically blocked (see below). Indeed, in et al. 1993) and that the SCO of hydrocephalic human fe- adult rats, the SCO becomes altered and the formation tuses presents severe alterations (Castañeyra-Perdomo et al. of RF blocked, as a consequence of a hydrocephalus 1994) might be an indication that, in humans, a dysfunction induced by a provoked virus Borna encephalitis, or by of the SCO may lead to congenital hydrocephalus. the injection of kaolin into the cisterna magna (Irigoin What is the mechanism by which the immunoneutral- et al. 1990). ization of the SCO-RF complex leads to hydrocephalus? A likely possibility is that the absence of a normal RF re- Whatever the mechanism may be, the permanent altera- sults in disturbances of the CSF circulation along the aq- tion of the SCO-RF complex and the definitive absence ueduct. A single injection of anti-RF antibody into adult of RF from the central canal of the spinal cord induced rats produces a complete breakdown of the existing RF; by the maternal delivery of anti-RF antibodies is a puz- a few weeks later a new RF has grown and reached the zling phenomenon that needs to be further investigated. entrance of the central canal of the spinal cord but it does That antibodies against brain proteins may be transferred not progress along the central canal (Cifuentes et al. from mother to fetus and pup, reach the sites where such 1994). Although the central canal of these RF-deprived proteins occur in the CNS, immunoneutralize them, and rats remains open, the CSF circulation along this canal is produce a permanent functional damage are all facts that drastically reduced; it has been suggested that this might have obvious clinical implications. be due to turbulence of the CSF at the entrance of the central canal, triggered by the absence of RF (Cifuentes et al. 1994). In the present experimental model, the sus- The alterations of the SCO-RF complex induced tained delivery of anti-RF antibodies to the CSF during by maternal transfer of anti-RF antibodies lead the first months of life leads to an absence of RF in the to hydrocephalus aqueduct during this key period of life. This, in turn, could lead to a disturbance of the CSF circulation along The dilatation of the lateral and third ventricles occur- the aqueduct. The presence of aggregates of RF material ring in rats that have been transferred with maternal anti- out of place, such as in the ventral region of the third bodies against RF glycoproteins, is compatible with the ventricle, could be the reflection of an abnormal CSF development in these rats of a hydrocephalus. This pos- circulation. sibility is further supported by the fact that these rats The stenosis of the distal end of the aqueduct dis- also presented a marked stenosis of the cerebral aque- played by the experimental rats would certainly contrib- duct, ependymal denudation, and had RF material locat- ute to the development of hydrocephalus. What is the ed in abnormal sites, such as the ventral region of the mechanism leading to such a stenosis? Worth mentioning third ventricle, which could be the result of disturbances in this respect is the early hypothesis advanced by Over- in the CSF circulation. holser et al. (1954) that the secretion of the SCO released In the pups of immunized mothers, the immunologi- into the rostral end of the aqueduct prevents the closure cal blockage of the SCO is already evident at birth, indi- of the cerebral aqueduct, thus allowing the free circula- cating that such a blockage also occurs during the fetal tion of CSF between the third and fourth ventricles. Re- period. The presence of anti-RF antibodies in the plas- cent findings support this possibility. The major constitu- ma and CSF of the fetuses supports this possibility tive RF glycoprotein, RF-Gly I has repeated sequences (Rodríguez et al. 1999). Since the dilatation of the ven- (Nualart et al. 1998). Within each repeat there is a core tricles starts to be evident by the end of the 1st postnatal sequence of four amino acids (VTCG) which according month, it seems highly probable that the immunological to Gobron et al. (1996) would be responsible for the ag- blockage of the SCO-RF complex during the perinatal gregative and anti-aggregative properties displayed by period leads to a hydrocephalus. The present findings as RF glycoproteins on neurons in culture (Monnerie et al. well as the sequence of events support the hypothesis 1998). Whether or not RF-Gly I has anti-aggregative that a dysfunction of the SCO leads to the aqueductal properties on ependymal cells is a subject under investi- stenosis and a congenital hydrocephalus (Newberne gation. 1962; Overholser et al. 1954). Other findings also sup- Considering that during the first 2 months of life of port this hypothesis. Maldevelopment of the SCO in- pups of immunized mothers, Ag-Ab complexes with as- 51 sociated lymphocytes and macrophages are present along Cifuentes M, Rodríguez S, Pérez J, Grondona JM, Rodríguez EM, the aqueduct, the possibility that the aqueductal stenosis Fernández-Llebrez P (1994) Decreased flow through the central canal of the spinal cord of rats immu- is due to a discrete and circumscribed inflammatory pro- nologically deprived of Reissner’s fibre. Exp Brain Res 98: cess, can not be ruled out. 431Ð440 Many of the rats with an immunoneutralization of the Dahme M, Bartsch U, Martini R, Anliker B, Schachner M, Mantei SCO by maternal transfer of antibodies used in the pres- N (1997) Disruption of the gene coding for the cell adhesion molecule L1 leads to malformations of the nervous system in ent investigation, were also used to investigate the con- mice. Nat Genet 17:346Ð349 centration of monoamines in the cisternal CSF. A rise in Estivill-Torrús G, Cifuentes M, Grondona JM, Miranda E, the CSF concentration of several amines has been found Berm·dez-Silva FJ, Fernández-Llebrez P, Pérez J (1998) Quan- in these rats, supporting the hypothesis that RF partici- tification of the secretory glycoproteins of the subcommissural pates in the clearance of monoamines from the CSF organ by a sensitive sandwich ELISA with a polyclonal anti- body and a set of monoclonal antibodies against the bovine (Rodríguez et al. 1999). The most pronounced rise corre- Reissner’s fiber. Cell Tissue Res 294:407Ð413 sponded to L-3,4-dihydroxyphenylalanine, a precursor of Fransen E, D’Hooge R, Camp GV, Verhoye M, Sijbers J, Reyniers ; a rise in the CSF concentration of epineph- E, Soriano P, Kamiguchi H, Willemsen R, Koekkoek SKE, De rine, norepinephrine, 3,4-dihydroxyphenylacetic acid, an Zeeuw CI, De Deyn PP, Van der Linden A, Lemmon V, Kooy RF, Willems PJ (1998) L1 knockout mice show dilated ventri- acid metabolite of dopamine, , and its metabo- cles, vermis hypoplasia and impaired exploration patterns. lite 5-hydroxyindolaceteic acid (5-HIAA) was also de- Hum Mol Genet 7:999Ð1009 tected in these rats (Rodríguez et al. 1999). The cells of Gobron S, Monnerie H, Meiniel R, Creveaux I, Lehmann W, the choroid plexus have on their apical (ventricular) Lamalle D, Dastugue B, Meiniel A (1996) SCO-spondin: a new member of the thrombospondin family secreted by the plasma membranes receptors for serotonin (Nilsson et al. subcommissural organ is a candidate in the modulation of neu- 1991) and dopamine (Nicklaus et al. 1988). These two ronal aggregation. J Cell Science 109:1053Ð1061 monoamines as well as norepinephrine, when adminis- González CA, Garcés G, Sáez JC, Schöbitz K, Rodríguez EM tered into the CSF, influence the rate of CSF secretion (1999) The ependymocytes of the bovine subcommissural or- (Johanson 1989; Lindwall-Axelson et al. 1998). The rats gan are functionally coupled through gap junctions. Neurosci Lett 262:175Ð178 with an immunoneutralization of the SCO-FR complex, Irigoin C, Rodríguez EM, Heinrichs M, Frese K, Herzog S, undergoing such a disturbance in the CSF concentration Oksche A, Rott R (1990) Immunocytochemical study of the of monoamines should be expected to suffer an alteration subcommissural organ of rats with induced postnatal hydro- in the CSF secretion. Increased CSF levels of 5-HIAA cephalus. Exp Brain Res 82:384Ð392 Johanson CE (1989) Potential for pharmacologic manipulation of and the dopamine metabolite, homovanillic acid, have the bloodÐcerebrospinal fluid barrier. In: Neuwelt EA (ed) Im- been found in rabbits made hydrocephalic neonatally plications of the bloodÐbrain barrier and its manipulation. Ple- (Olmstead et al. 1995). num Press, New York, pp 223Ð260 In brief, the following is the sequence of events oc- Jones HC, Bucknall RM (1988) Inherited prenatal hydrocephalus in the H-Tx rat: a morphological study. Neuropathol Appl curring in the rats with an immunoneutralization of the Neurobiol 14:263Ð274 SCO-RF complex: the mother produces antibodies Jones HC, Dack S, Ellis C (1987) Morphological aspects of against RF glycoproteins and transfers them to the fetus the development of hydrocephalus in a mouse mutant through the placenta and to the pup through the milk; the (SUMS/NP). Acta Neuropathol (Berl) 72:268Ð276 antibodies reach the CNS of the fetus and pup during the Lehman W, Sterba G (1993) The subcommissural organ in vitro. In: Oksche A, Rodríguez EM, Fernández-Llébrez P (eds) The first 2 postnatal months and block the SCO-RF complex subcommissural organ. An ependymal brain gland. Springer, resulting in the absence of RF; this is followed by Berlin Heidelberg New York, pp 133Ð140 stenosis of the cerebral aqueduct that precedes the ap- Lindwall-Axelson M, Mathew C, Nilsson C, Owman C (1998) Ef- pearance of hydrocephalus. The chronic hydrocephalic fect of 5-hydroxytryptamine on the rate of cerebrospinal fluid production in rabbit. Exp Neurol 99:362Ð368 state would, in turn, induce new alterations of the SCO. Margolis G, Kilham L (1969) Hydrocephalus in hamsters, ferrets, rats and mice following inoculations with reovirus type I. II. Acknowledgements We thank Mr. G. Alveal for his most valu- Pathologic studies. Lab Invest 21:189Ð198 able help in the processing of samples for light microscopy. This Monnerie H, Dastugue B, Meiniel A (1998) Effect of synthetic work was supported by Grants from FONDECYT 197Ð0627, and peptides derived from SCO-spondin conserved domains on 1000Ð435, Chile, to E.M.R., FIS 98Ð1508, Spain, to J.M.P.-F., chick cortical and spinal-cord neurons in cell cultures. Cell and Agencia Española de Cooperación Internacional, Spain, to Tissue Res 293:407Ð418 J.M.P.-F. and E.M.R. Newberne PM (1962) The subcommissural organ of the vita- min B12-deficient rat. J Nutr 76:393Ð413 Nicklaus KJ, McGonigle P, Molinoff PB (1988) [3H]SCH 23390 References labels both dopamine-1 and 5-hydroxytryptamine1c receptors in the choroid plexus. J Pharmacol Exp Ther 247:343Ð348 Borit A (1976) Communicating hydrocephalus causing aqueductal Nielsen SL, Baringer JR (1972) Reovirus-induced aqueductal ste- stenosis. Neuropediatrie 7:416Ð422 nosis in hamsters. Phase contrast and electron microscopic Bruni JE, Bigio MR del, Cardoso ER, Persaud TVN (1988) Neu- studies. Lab Invest 27:531Ð537 ropathology of congenital hydrocephalus in the SUMS/NP Nilsson C, Lindvall-Axelsson M, Owman C (1991) Role of the ce- mouse. Acta Neurochir (Wien) 92:118Ð122 rebrospinal fluid in volume transmission involving the choroid Castañeyra-Perdomo A, Meyer G, Carmona-Calero E, Bañuelos- plexus. In: Fuxe K, Agnati E (eds) Transmission in the brain. Pineda J (1994) Alterations of the subcommissural organ in Raven Press, New York, 24:307Ð315 the hydrocephalic human fetal brain. Brain Res Dev Brain Res Nualart F, Hein S, Rodríguez EM, Oksche A (1991) Identification 79:316Ð320 and partial characterization of the secretory glycoproteins of 52 the bovine subcommissural organ-Reissner’s fiber complex. Rodríguez S, Vio K, Wagner C, Barría M, Navarrete EH, Ramírez Evidence for the existence of two precursor forms. Mol Brain VD, Pérez-Fígares JM, Rodríguez EM (1999) Changes in the Res 11:227Ð238 cerebrospinal-fluid monoamines in rats with an immunoneu- Nualart F, Hein S, Yulis RC, Zárraga AM, Araya A, Rodríguez tralization of the subcommissural organ-Reissner’s fiber com- EM (1998) Partial sequencing of Reissner’s fiber glycopro- plex by maternal delivery of antibodies. Exp Brain Res tein I (RF-Gly I). Cell Tissue Res 292:239Ð250 128:278Ð290 Oksche A (1969) The subcommissural organ. J Neuro-Visc Relat Schöbitz K, Garrido O, Heinrichs M, Speer L, Rodríguez EM 9:11Ð139 (1986) Ontogenetical development of the chick and duck sub- Olmstead CE, Lazareff JA, Orlino EN Jr, Fluharty AL, Faull KF, commissural organ. Histochemistry 84:31Ð40 Peacock WJ, Wehby-Grant MC, Gayek RJ, Fisher RS (1995) Schöbitz K, Rodríguez EM, Garrido O, Del Brío-León MA (1993) Neuramine related compounds in the CSF of hydrocephalic Ontogenetic development of the subcommissural organ with rabbits. Neuroreport 6:1769Ð1772 reference to the flexural organ. In: Oksche A, Rodríguez EM, Overholser MD, Whitley JR, O’Dell BL, Hogan AG (1954) The Fernández-Llebrez P (eds) The subcommissural organ. An ventricular system in hydrocephalic rat produced by a ependymal brain gland. Springer, Berlin Heidelberg New deficiency of vitamin B12 or folic acid in the maternal diet. York, pp 41Ð49 Anat Rec 120:917Ð933 Sternberger LA, Hardy PH, Cuculis JJ, Meyer HG (1970) The un- Pérez-Fígares JM, Jiménez AJ, Pérez-Martín M, Fernández- labeled antibody enzyme method of immunohistochemistry: Llebrez P, Cifuentes M, Riera P, Rodríguez S, Rodríguez EM preparation and properties of soluble antigen-antibody com- (1998) Spontaneous congenital hydrocephalus in the mutant plex (horseradish peroxidase-antiperoxidase) and its use in the mouse hyh. Changes in the ventricular system and the sub- identification of spirochetes. J Histochem Cytochem 18:315Ð commissural organ. J Neuropathol Exp Neurol 57:188Ð202 333 Rodríguez EM, Oksche A, Hein S, Rodríguez S, Yulis R (1984) Takeuchi IK, Takeuchi YT (1986) Congenital hydrocephalus fol- Comparative immunocytochemical study of the subcommissu- lowing X-irradiation of pregnant rats on an early gestational ral organ. Cell Tissue Res 237:427Ð441 day. Neurobehav Toxicol Teratol 8:143Ð150 Rodríguez S, Rodríguez EM, Jara P, Peruzzo B, Oksche A (1990) Takeuchi IK, Kimura R, Matsuda M, Shoji R (1987) Absence of Single injection into the cerebrospinal fluid of antibodies subcommissural organ in the cerebral aqueduct of congenital against the secretory material of the subcommissural organ re- hydrocephalus spontaneously occurring in MT/HOK1dr mice. versibly blocks formation of Reissner’s fiber: immunocyto- Acta Neuropathol 73:320Ð322 chemical investigations in the rat. Exp Brain Res 81:113Ð124 Takeuchi IK, Kimura R, Shoji R (1988) Dysplasia of subcommis- Rodríguez EM, Oksche A, Hein S, Yulis CR (1992) Cell biology sural organ in congenital hydrocephalus spontaneously occur- of the subcommissural organ. Int Rev Cytol 135:39Ð121 ring in CWS/Idr rats. Experientia 44:338Ð340 Rodríguez EM, Jara P, Richter H, Montecinos H, Flández B, Turner M (1996) Antibodies and their receptors. In: Roitt I, Wiegand R, Oksche A (1993) Evidence for the release of CSF- Brostoff J, Male D (eds) Immunology. Mosby, London, soluble secretory material from the subcommissural organ, pp 1Ð12 with particular reference to the situation in the human. In: Williams B (1973) Is aqueductal stenosis a result of hydrocepha- Oksche A, Rodríguez EM, Fernández-Llebrez P (eds) The lus? Brain 96:399Ð412 subcommissural organ. An ependymal brain gland. Springer, Wong EV, Kenwrick S, Willems P, Lemmon V (1995) Mutation in Berlin Heidelberg New York, pp 121Ð131 the cell adhesion molecule L1 causes mental retardation. Rodríguez EM, Rodríguez S, Hein S (1998) The subcommissural Trends Neurosci 18:168Ð172 organ. Microsc Res Tech 41:98Ð123