Reissner's fiber and the wall of the central canal in the lumbo-sacral region of the bovine spinal...

Cell Tissue Res (1985) 240:649-662

and R e s e a r c h �9 Springer-Verlag 1985

Reissner's fiber and the wall of the central canal in the lumbo-sacral region of the bovine spinal cord Comparative immunocytochemical and ultrastructural study*

Sara Rodriguez, Silvia Hein, Roberto Yulis, Luis Delannoy, In~s Siegmund, and Est~ban Rodriguez** Instituto de Histologia y Patologia, Universidad Austral de Chile, Valdivia, Chile

Summary. Reissner's fiber (RF) of the subcommissural organ (SCO), the central canal and its bordering structures, and the filum terminale were investigated in the bovine spinal cord by use of transmission electron microscopy, histochemical methods and light-microscopic immunocytochemistry. The primary antisera were raised against the bovine RF, or the SCO proper. Comparative immunocytochemical studies were also performed on the lumbo-sacral region of the rat, rabbit, dog and pig.

At all levels of the bovine spinal cord, RF was strongly immunoreactive with both antisera. From cervical to upper sacral levels of the bovine spinal cord there was an increasing number of ependymal cells immunostainable with both antisera. The free surface of the central canal was covered by a layer of immunoreactive material. At sacral levels small subependymal immunoreactive cells were observed. From all these structures sharing the same immunoreactivity, only RF was stained by the paraldehyde-fuchsin and periodic- acid-Schiff methods.

At the ultrastructural level, ependymal cells with numerous protrusions extending into the central canal were seen in the lower lumbar segments, whereas cells displaying signs of secretory activity were principally found in the ependyma of the upper sacral levels. A few cerebrospinal fluid-contacting neurons were observed at all levels of the spinal cord; they were immunostained with an anti-tubulin serum.

The lumbo-sacral segments of the dog, rat and rabbit, either fixed by vascular perfusion or in the same manner as the bovine material, did not show any immunoreactive structure other than RF.

The possibilities that the immunoreactive ependymal cells might play a secretory or an absorptive role, or be the result of post-mortem events, are discussed.

Key words. Reissner's fiber - Subcommissural organ - Ependyma Immunocytochemistry - Electron microscopy - Bovine

Send offprint requests to." Prof. Est6ban M. Rodriguez, Instituto de Histologia y Patologia, Universidad Austral de Chile, Valdivia, Chile

* Supported by Grant 1/38259 from the Stiftung Volkswagen- werk, Federal Republic of Germany, and Grant RS-82-18 from the Direcci6n de Investigaciones, Universidad Austral de Chile

** The authors wish to thank Dr. Enrique Romeny from the Valdi- via abattoir for kindly providing the bovine spinal cords

Reissner's fiber (RF) is a thread-like structure extending from the subcommissural organ (SCO) to the Sylvian aque- duct, fourth ventricle and central canal of the entire spinal cord (see reviews by Olsson 1955, 1958; Ermisch 1973; Leonhardt 1980). A considerable body of evidence derived from histochemical (Naumann 1968; Diederen 1970), auto- radiographic (Sterba et al. 1967; Leatherland and Dodd 1968; Diederen 1972; Ermisch 1973), ultrastructural (Oksche 1969; Rodriguez 1970; Hofer et al. 1980), immunocytochemical (Sterba et al. 1982; Rodriguez et al. 1984), and experimental (Wingstrand 1953; Olsson 1957) studies strongly indicates that RF is a secretory product of the SCO. Additional evidence supports the view (Olsson 1955, 1958) that the newly released material is incorporated at the rostral end of RF, thus causing the latter to grow in a rostro-caudal direction (Sterba 1969; Ermisch 1973). The daily growth rate of RF and the rate of disappearance from RF of labeled components have been estimated for a number of species (Ermisch et al. 1968; Ermisch 1973). All these findings support the view that in the terminal region of the central canal certain events leading to a "disappearance" of the caudal end of RF must be operative. The nature and time course of these events and the fate of the RF material have not as yet been clarified. Although several morphological studies were focussed on the situation in the terminal ventricle, in which classical staining methods for visualization of RF were used (Olsson 1955; Wislocki et al. 1956; Hofer 1964), they did not solve these questions. Re- cently, Hofer et aI. (1984) have provided ultrastructural evidence for a discharge of RF material into capillaries located in the vicinity of the ampulla caudalis of Ammocoetes.

We have been successful in completely dissolving RF isolated from bovine spinal cord and have raised antibodies against this RF extract (Rodriguez et al. 1984). These antibodies were used to perform an immunocytochemical study of several segments of the bovine spinal cord, with special reference to the content of the central canal and its bordering structures. The results of this study stimulated us to extend the analysis to the lumbo-sacral region of other species as well and to perform an electron-microscopic investigation of the wall of the central canal at the lumbo-sacral levels of the bovine spinal cord.

Materials and methods

Light microscopy. Twelve bovine spinal cords (SPC) were obtained at the slaughterhouse and processed in four differ-

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Fig. 1. Sagittal section through the cervical spinal cord (Group IV). Broken arrow fine threads in core of Reissner's fiber (RF). Small arrows immunoreactive spherical structures on surface of RF; large arrow immunoreactive ependymal cell; E ependyma. • 300

Fig. 2. Cross section through the thoracic spinal cord (Group IV). Small arrows layer of IRM on ependymal surface; large arrows immunoreactive ependymal cells; RF Reissner's fiber. • 230

ent ways. Group I: The SPC were dissected out 20 min after death and segments of about 4 cm in length were perfused via the central canal (CC), using an infusion pump, with Bouin 's fixative for 20 min, at a rate of 1 I.tl/min. Then, the perfused segment was transverselly cut to obtain several slices of approximate ly 4 mm thick, which were immersed in Bouin 's fixative for two days. Group H: Same procedure as in G r o u p I but the pos t -mor tem time was approximate ly 1 h. Group IlI: Cervical, thoracic and lumbar segments of the SPC were divided into two blocks for preparing frontal and sagittal sections, respectively. The tissue material used for frontal sections consisted of a 5-mm thick cross-sec- t ional slice of the entire SPC. The other tissue block was approximate ly 15 mm in length and contained the CC encompassed by the gray- and white-matter commissures. Fo r the sacral segments the following procedure was used: At the level of segment S 1 the diameter of the bovine SPC is much smaller than at the more cranial levels. F rom $2 caudally, the SPC proper cannot be recognized and the CC extends along a terminal filum. Therefore, at these levels the blocks of tissues contained alternatively the entire SPC or the terminal filum. All tissue blocks from this group were immersed in the fixative 20 min after death, and fixation lasted for two days. Group IV: Same procedure as in G r o u p III, but the time lapse between death and fixation was approximate ly 1 h. Segments of SPC from lumbar (L2, Ls, L6) and sacral ($I-$5) levels were obtained from each animal. Tissue samples from cervical (C 1 C2) and thoracic (D2, D4) levels were obta ined only from some of the animals studied.

F o r comparat ive purposes, the lumbo-sacral regions of the following species were also processed: Rat." Three animals were fixed by vascular perfusion and one was fixed by immersion 20 min after death. Rabbit: Two animals were fixed by vascular perfusion and one by immersion 1 h after death (same procedure as bovine SPC in G r o u p IV). Dog: One animal fixed by vascular perfusion. Pig: The SPC of two pigs were obta ined at the slaughterhouse and were processed as the bovine SPC of G r o u p III. In all these species the tissues were fixed in Bouin 's fluid.

After careful washes and dehydrat ion in increasing con-

centrations of alcohols, the material was embedded in Para- plast. Each block was serially cut and every fifth section mounted. In some cases (sacral levels) a complete series of sections was mounted. Prior to dehydrat ion the lumbo- sacral samples of one bovine SPC of Group II I were cut into small blocks (1 mm thick) and, after dehydrat ion in alcohols and acetone, embedded in butyl-methyl-methacrylate according to Rodriguez et al. (1984). One-pm serial sections obtained from these blocks were mounted on separate slides.

Immunostaining. The sections were stained by use of the unlabeled peroxidase method of Sternberger et al. (1970). Two different pr imary antisera were employed. One was raised against bovine R F dissolved in a medium containing ethylene diamine tetraacetic acid, DL-di thiothre i to l and urea ( A F R U , see Rodriguez et al. 1984). The other antiserum was against the bovine SCO extracted in a medium with ammonium bicarbonate and phenylmethylsulfonyl flu- oride. This latter procedure mainly extracts the intracellular secretory material of the SCO complex (Hein et al., in preparation). The immunizat ion schedule for the SCO extract was similar to that used when obtaining the an t i -RF serum (Rodriguez et al. 1984). F o r brevity, in the following the anti-SCO serum has been characterized with the acronym ASO (A for antiserum; SO for subcommissural organ). Both, A F R U and ASO were used at dilutions ranging between 1:1000 and 1:6000. Incubat ion time was 18 h. The second ant ibody was applied at a di lut ion of 1:50. PAP (Bioproducts, Brussels, Belgium) was diluted to 1 : 75. Firs t and second antibodies and PAP were diluted in TRIS buffer, pH 7.8, containing 0.7% non-gelling seaweed gela- tin, l ambda carrageenan (Sigma), and 0.5% Tri ton X-100 (Sigma) (Sofroniew et al. 1979). Coplin jars were used for incubation with the first and second ant ibody, whereas PAP incubat ion was carried out in a moist chamber.

Several batches of each pr imary antiserum were used to stain adjacent sections. No differences were found in the pat tern of the immunoreact ion produced by the different batches. On the other hand, in most cases, sections from the different segments of the SPC were simultaneously

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Fig. 3. Sagittal section through the lower lumbar spinal cord (Group IV). Reissner's fiber (RF) surrounded by densely packed spherical structures (asterisks). Smal l arrows globular immunoreactive formations; large arrows immunoreactive ependymal cells; E ependymal layer, x 400

Fig. 4. Higher magnification of Fig. 3 showing distribution of immunoreactive material in the spaces among the spherical structures (arrows). x 510

Fig. 5. Frontal section of lumbar spinal cord (Group IV), Immunoreactive material in Reissner's fiber (RF), on the free surface of the ependymal layer (E), and in a few ependymal cells (arrows) and their basal processes (broken arrows), x 200

Fig. 6. Section adjacent to that shown in Fig. 5. Smal l arrows immunoreactive ependymal cells; large arrow protruding ependymal cell; broken arrows layer of immunoreactive material on the free surface of ependyma. • 800

stained in the same staining session. In order to test the specificity of the immunoreac t ion and to check the p robab le existence o f endogenous peroxidase in the tissues giving a positive immunostaining, sections from the different segments studied, and from all species investigated, were processed in the same way as for immunostaining, but incubation in the pr imary ant iserum was omitted.

Some sections of the bovine SPC were immunos ta ined by use of an ant i- tubulin serum as pr imary ant ibody. Tubu- lin was extracted from pig brains and purif ied according to Maccioni et al. (1981). The immuniza t ion schedule was similar to the one used when obtaining the an t i -RF serum (Rodriguez et al. 1984). Ant i - tubul in serum was used at a di lut ion o f I : I000.

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Fig. 7. Cross section of sacral spinal cord (Group IV), Sl-level, showing numerous immunoreactive ependymal (arrowheads) and subependymal cells (arrows). RF Reissner's fiber. Broken lines A, B planes of section in Figs. 8 and I0, respectively, x 250

Fig. 8. Sagittal section of sacral cord (Group IV), S l-level. Asterisk immunoreactive globules and non-immunoreactive spherical structures. Broken arrows immunoreactive ependymal cells; arrows immunostained subependymal cells, x 150

Fig. 9. Higher magnification of Fig. 8 showing immunoreactive ependymal cells (large arrows) and inamunoreactive material on the free surface of ependyma (arrowheads) and on bundles of fused cilia (small arrows). • 600

Fig. 10. Sagittal section through the ventral subependyma[ region of sacral spinal cord (Group IV), Sl-level, showing a column formed by immunoreactive cells and their processes, x 100

Fig. 11. Enlarged detail of Fig. 10 showing immunoreactive cell bodies (large arrows), processes (small arrows) and their spatial association to a blood vessel (by). x 400

Other staining procedures. Serial sections from the lumbo- sacral region of the bovine SPC, embedded either in Para- plast or methacrylate , were stained according to the following procedures: a) paraldehyde-fuchsin (Gabe 1968); b) pseudoisocyanin (Sterba et al. 1967); c) PAS according to M c M a n u s (Pearse 1980); d) periodic acid-silver methenamine (Rodriguez et al. 1984).

Electron microscopy. Cervical and lumbo-sacral segments of three SPC obtained 20 min after death were perfused

through the CC with a threefold aldehyde mixture containing 4% paraformaldehyde, 2% acrolein and 2.5% glutaraldehyde buffered to pH 7.4 with 0.2 M monosodic-disodic phosphate (Rodriguez 1969). Perfusion was performed by use of an infusion pump. Fixative was perfused at a rate of 1 ~l/min, and perfusion lasted for 20 rain. When perfusion was completed, small blocks of tissues containing the CC and a thin layer of border ing tissue were prepared. These blocks were then immersed in fresh fixative to complete a period of 2 h of aldehyde treatment. After washing

the blocks with 0.1 M phosphate buffer, pH 7.4, they were fixed for 2 h in 1% OsO4 buffered to pH 7.4 with phosphate. After a few washes in distilled water the blocks were dehydrated in increasing concentrations of alcohols and pure acetone. Embedding was in a mixture of Epon and Araldite.

The blocks were oriented to obtain cross sections of the CC. Ultrathin sections were stained with uranyl acetate and lead citrate. A total of 470 electron micrographs were obtained. One-lam thick sections, adjacent to the ultrathin sections, were stained with toluidine blue-borax.

Results

Light microscopy

Immunocytochemistry of the bovine spinal cord by use of AFRU 1 as primary antiserum

Cervical and thoracic levels. In representative cross sections RF measured approximately 35 ~tm in diameter (Fig. 2). The core of the fiber was less immunoreactive than the peripheral portion. In the core, however, fine thread-like structures extending in longitudinal direction were strongly immunostained (Fig. 1). In sagittal sections the surface of the fiber appeared uneven due to the presence of globular immunoreactive structures protruding into the lumen of the central canal (CC) (Fig. 1). In the cervical and thoracic segments individual ependymal cells displayed strong immunoreactivity (Figs. 1, 2). The immunoreactive material (IRM) was mainly concentrated in the apical cytoplasm, although in some cells also the basal processes were immunoreactive (Fig. 1). In the thoracic segments, however, IRM covered the entire surface of the CC. In consequence, the non-immunoreactive ependymal cells, which largely out- numbered the immunoreactive elements, were covered by a layer of IRM on their free surface (Fig. 2).

Lower lumbar levels. The diameter of RF in cross sections was again in the range of 35 ~tm (Fig. 5). In most specimens belonging to the four groups of animals (I-IV) the lumen of the CC was occupied by spherical structures. In spinal cords fixed 1 h after death, but not in those fixed 20 min after death, the spaces between these spherical profiles contained IRM (Figs. 3, 4, 14). Intermingled with the globular formations, or attached either to the surface of RF or to the ependymal wall, spherical or oval (5-10 ~tm in diameter) immunoreactive structures were observed (Figs. 3, 4). These latter elements were more numerous in specimens fixed 1 h after death (Fig. 3) than in those fixed 20 min after death (Fig. 14), irrespective whether the fixation was by immersion or by perfusion of the CC. In the four groups of animals, the number of immunoreactive ependymal cells at the lower lumbar levels exceeded that at cervical and thoracic levels.

Parallel to these variations there was also a change in the shape of the immunoreactive ependymal cells. In the lower lumbar portion they were elongated with a basal nucleus and endowed with a single, long basal process (Figs. 3-6). Some of the cells were seen to protrude far into the CC (Fig. 6). It was not possible to trace the site of projection or termination of the basal processes of these cells.

1 See page 650 for details

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Fig. 12. Cross section through the sacral spinal cord, S4-1evel (Group IV). Arrowheads layer of immunoreactive material on the free surface of the ependymal layer (E); arrows subependymal immunoreactive cells, x 175

Fig. 13. Cross section of the filum terminale, Ss-level (Group IV). Reissner's fiber (RF) is the only immunoreactive structure; E ependyma. x 230

The IRM found in association with the free surface of the CC showed individual variation within one group of animals. It was, however, evident that in the specimens fixed 20 min after death the IRM appeared in form of thin patches (Fig. 14) or as a layer thinner than that observed in spinal cords fixed 1 h after death (Fig. 5).

Upper sacral levels (S 1 , 32). The main differences with respect to the findings described for the lower lumbar levels refer to the number of immunoreactive ependymal cells and to the presence of immunoreactive cells in the subependymal region. In the upper sacral segments the former were much more numerous than in any other region of the spinal cord (Figs. 7-9). Only in a few specimens, but without any correlation to the four schedules of fixation procedure used, the CC displayed the globular structures (reactive and non- immunoreactive) described for the lumbar levels (Fig. 8). The appearance of the layer of IRM located on the surface of the CC varied with the post-mortem time interval in the same manner as described for the lumbar segments.

Lateral and ventral to the central canal subependymal immunoreactive cells were observed (Fig. 7). These cells

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Figs. 14-16. Cross sections (paraffin-embedded material) through the lower lumbar spinal cord (Group I). Sections of Figs. 14 and 15 were immunostained by use of anti-RF and anti-SCO sera, respectively. Section of Fig. 16 was processed as for immunostaining but incubation with the primary antiserum was omitted. Small arrow Reissner's fiber; large arrow immunoreactive ependymal cells. Insert (in Fig. 15): Higher magnification of the cells labeled with large arrow, x 185, Insert: x 650

Figs. 17-19. Serial t-lam methacrylate sections through the sacral spinal cord (Group Ill) immunostained by use of anti-RF serum (Fig. 17), or stained with periodic acid-Schiff (Fig. 18) or paraldehyde-fuchsin (Fig. 19) methods. Double arrows Reissner's fiber; single arrow immunoreactive ependymal cell; broken arrows immunostained subependymal cells, x 185

were particularly numerous in the anterior gray commissure. Sagittal sections through the latter region showed that these cells and their processes form a longitudinal column (Fig. 10). These elements were ovoid in shape and ranged between 12 and 20 gm in diameter. They displayed a few immunoreactive processes running parallel to the central canal and forming a loose bundle (Figs. 10, 11). Some of them established close contacts with blood vessels (Fig. 11).

Lower sacral levels ( S 3 , $4). Immunoreactive ependymal cells were virtually missing. However, the free surface o f the ependymal cells and more clearly the cilia were covered by a layer of I R M (Fig. 12). A few immunoreactive cells were present in the latero-ventral subependymal region (Fig. 12).

Filum terminale ( S s level). The diameter of RF in cross sections was approximately 13 ~tm, i.e., much smaller than that found in the upper levels of the spinal cord. No immunoreactive material was observed either in the lumen of the CC, or in ependymal or subependymal cells (Fig. 13).

Other staining procedures. The use of ASO as primary antiserum for the immunoperoxidase staining gave similar results to those obtained with A F R U , namely, immunostaining o f RF, ependymal cells and the material associated with the surface of the CC (compare Figs. 14, 15). When the primary antiserum was omitted in the immunostaining procedure, the brown reaction product of peroxidase was completely missing (Fig. 16). When adjacent serial methacrylate sections, 1-gm thick, were stained according to the immunoperoxidase method using A F R U as primary antiserum (Fig. 17), silver methenamine (SM) or PAS (Fig. 18), and pseudoisocyanin (Psi) or paraldehyde-fuchsin (AF)

(Fig. 19), it became clear that from all the immunoreactive structures, i.e., RF, globular formations in the CC, ependy- real and subependymal cells, only the RF gave a positive reaction with SM, PAS, Psi, and AF.

Immunocytochemistry o f the lumbo-sacral region o f the rat, rabbit, dog and pig using A F R U as primary antiserum. Al- though the lumbo-sacral region of rats and rabbits was serially cut and all sections were mounted and immunostained, no immunoreactive structures other than RF were found. This was also the case for the specimens fixed, delib- erately, by a late immersion in the fixative. Worth mentioning is the fact that while the CC of the two rabbits fixed by vascular perfusion lacked the globular structures, that of a rabbit fixed by immersion 1 h after death was filled with such structures. None of these structures was, however, immunoreactive. The lumbo-sacral region of the dog also lacked immunoreactive ependymal cells. The sacral segments of the spinal cord of the pig fixed by immersion 20 rain after death displayed immunoreactive ependymal cells similar in shape and distribution to those observed in the upper sacral levels of the bovine spinal cord. No globular structures were seen in the CC of the pig lumbo- sacral cord.

Electron microscopy o f the bovine lumbo-sacral spinal cord

Lower lumbar levels (L 4, Ls ) . The l-p.m thick sections stained with toluidine blue revealed the presence of RF and densely packed spherical structures, which occupied most of the lumen of the CC (Fig. 20). The electron-microscopic analysis of adjacent sections showed that these formations are in fact surface protrusions of the ependymal cells (Fig. 21). These protrusions appeared filled with a flocculent material, polyribosomes, a few smooth-surfaced

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Fig. 20. One-gm thick Epon section through the lower lumbar level of spinal cord, stained with toluidine blue-borax. Numerous ependymal protrusions fill the central canal (arrows) and surround Reissner's fiber (RF). An area similar to that framed by rectangle is shown in Fig. 21. x 500

Fig. 21. Ultrathin section adjacent to the 1-gm thick section shown in Fig. 20. Protrusions emerging from ependymal cells (arrows) or free in the lumen of central canal (asterisk). RF Reissner's fiber, x 1900

Fig. 22. Ependymal protrusions establishing contact with Reissner's fiber (RF). x 10000

]Fig. 23. Enlarged detail of Fig. 21 showing the fine structure of the ependymal cells protruding into the central canal. Asterisks protrusions; arrowheads lysosome-like bodies; G Golgi apparatus, x 7300

cisternae and lysosome-like bodies (Fig. 21). Many o f the protrus ions established close contacts with R F (Figs. 21, 22). The ependymal cells p roper contained several lysosome-like bodies located in the supranuclear region, a well-developed Golgi apparatus , and numerous free ribo-

somes (Fig. 23). The free surface emitted large prot rus ions and microvilli. Only one cilum, but exceptionally two, were seen to arise from each ependymal cell (Fig. 23). In some regions o f the CC, microvilli and cilia were seen to be embedded in a matr ix of loosely arranged fine filaments. Occa-

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Fig. 28. Electron micrograph of an area similar to that framed by small rectangle in Fig. 24A. C capillary lumen; asterisk perivascular space, arrows processes of the external limiting basement membrane; Ep basal processes of clear ependymal cells ending on an extension of the perivascular basal lamina, x 6650

sionally, this type o f material was also seen between the protrusions.

The ependymal cells were sealed by junctional complexes. Judging from their ultrastructural (transmission electron-microscopic) appearance they seemed to correspond to tight and adhaerens-type junctions and desmo- somes (Fig. 23). These ependymal cells displayed a single basal process mainly filled with ribosomes, densely packed filaments and mitochondria. Scattered among these ependymal cells were a few "clear cells" which will be described in the following section.

Upper sacral levels ($1, $2). Fig. 24A is a picture of a 1-1am thick section o f the S2-segment that belonged to the same spinal cord the L5-segment of which is shown in Figs. 20-23. The CC of the S2-segment appears free of ependymal protrusions. In the ependymal lining clear and dark ependymal cells can be observed (Fig. 24A). At the electron-microscopic level clear and dark ependymal cells become readily distinguishable (Fig. 24 B).

Clear ependymal cells. These cells displayed a large and ovoid nucleus with abundant euchromatin (Fig. 24B). In the l-~tm thick sections treated with toluidine blue these nuclei appeared faintly stained (Fig. 24A). The main ultrastructural feature of the cytoplasm was the large number of dilated cisternae of the rough endoplasmic reticulum located in the supranuclear (Fig. 25) and basal (Fig. 27) regions of the cell as well as in the basal process. Free ribo-

somes were scarce and the Golgi apparatus poorly developed. These cells emitted into the CC small protrusions containing lysosome-like bodies (Fig. 25) and circumscribed cisternae of the rough endoplasmic reticulum. They virtually lacked microvilli and displayed only one cilium (Fig. 25).

Dark ependymal cells. The nucleus of this cell type appeared in the l -gm sections elongated, richer in heterochromatin than the nucleus of the clear cells, and strongly basophilic (Fig. 24A, B). The cytoplasm contained abundant free ribosomes, numerous mitochondria and bundles o f filaments, a well-developed Golgi apparatus and a few non-dilated cisternae of rough endoplasmic reticulum. They also displayed a single basal process filled with filaments and free ribosomes. At variance with the ependymal cells of the lumbar levels, they had only few lysosome-like bodies and did not protrude into the CC (Fig. 24B). Instead, these cells presented a large number of microvilli resembling a brush border (Figs. 24B, 26). Dark cells lacking microvilli but provided with numerous cilia were only occasionally seen (Fig. 24). The multiciliated ependymal cells were numerous at the lower sacral levels (S 3, $4) and in the upper lumbar segments.

Synaptoid contacts between nerve fibers and ependymal cells were never observed.

Blood vessels. The capillaries located in the vicinity of the CC (Fig. 24A) were characterized by a thin non-fenestrated

Fig. 24. A One-gm thick Epon section through the sacral spinal cord, St-level, stained with toluidine blue-borax. The ependymal lining is formed by dark and clear (arrows) cells. Double arrows Reissner's fiber; C capillary. Areas similar to those framed by large and small rectangles are shown in Figs. 24 B and 28, respectively, x 280. B Electron micrograph of an area similar to that included in large rectangle of Fig. 24A. Most dark cells display numerous microvilli (arrowheads) and some of them are multiciliated (asterisk). RF Reissner's fiber. The two clear cells framed by the open rectangle are shown in Fig. 25. x 1900

Fig. 25. Higher magnification of the area delimited by the open rectangle in Fig. 24B, showing some details of the ultrastructure of the clear ependymal cells. Arrowheads dilated cisternae of rough endoplasmic reticulum; large arrows lysosome-like bodies; small arrow flattened cisterna of rough endoplasmic reticulum in a dark cell. x 7300

Fig. 26. Enlarged detail of the free surface of the dark ependymal cells shown in Fig. 24 B. x 7000

Fig. 27. Basal region of the cytoplasm of a clear ependymal cell, mostly occupied by mitochondria and dilated cisternae of the rough endoplasmic reticulum, x 10000

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Fig. 29. Sacral spinal cord (Sl-level), intraependymal CSF-contacting neuron; Ep ependymal cell, RF Reissner's fiber. • 8500, Insert: Enlarged detail of Golgi area of a neuron. Electron-dense granules (arrows) in the vicinity of two Golgi complexes (G). x 15000

Fig. 30. Dendritic process of a CSF-contacting neuron containing several mitochondria and a few electron-dense granules (arrows). x 22 000

Fig. 31. Intraependymal dendritic process (arrow) and ending ofa hypendymal CSF-contacting neuron; Ep ependymal cells, x 8000

Fig. 32. Cross section through the lumbar spinal cord immunostained using anti-tubulin as primary antiserum, a Note a CSF-contacting neuron in the wall of the central canal (arrow); gc gray commissure, x 70. b Enlarged detail of the CSF-contacting neuron shown in Fig. 32a. Single arrow axon; double arrow CSF-contacting terminal; ce central canal, x 400

endothelium and a perivascular space (Fig. 28). The external basal lamina extended into long branches which formed a network around the CC. Each ependymal cell, either clear or dark, established several contacts with this labyrinth of basal laminae (Fig. 28).

Rose t t e - l i ke structures. In the subependymal region occa- sional small clusters of cells were sealed together by junctional complexes and encompassed a small, irregular lumen. These cells projected microvilli and a few cilia into the central cavity. At their opposite pole the cells were in contact

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with the network of basal laminae. The rosetteqike structures were formed by dark and clear cells in analogy to those described above for the ependymal lining of the CC.

Cerebrospinal fluid (CSF)-contacting neurons. Scattered along the ependymal lining of the bovine lumbo-sacral cord were one to three CSF-contacting neurons (FCN) per cross section of paraffin-embedded material. These FCN were strongly immunostained by the anti-tubulin serum, in con- trast to the ependymal cells which did not react with this antiserum (Fig. 32). The neuronal perikarya were located either in the vicinity of the CC or at the base of the ependymal lining; in the following the former will be called intraependymal FCN and the latter hypendymal FCN.

The intraependymal FCN (Fig. 29) displayed typical Nissl bodies, several Golgi complexes and electron-dense granules, approximately 100 nm in diameter, mainly located in the Golgi area. The process projecting to the CC was short and thick. Its free end was smooth or had a few microvilli. The hypendymal FCN displayed a round and large perikaryon located in the interphase between the ependyma and the adjacent neuropil. The cell organelles, includ- ing the dense secretory granules, were similar to those of the intraependymal FCN. The main difference was that the hypendymal FCN had a long process traversing the ependymal lining and protruding into the CC. This process mainly contained microtubules and its dilated ending was mostly filled with numerous mitochondria and a few electron-dense granules 100nm in diameter (Figs. 30, 31). These endings lacked the fine, radially oriented processes, as described by Vigh and Vigh-Teichmann (1971) for the CSF-contacting dendrites of other vertebrate species. Both types of neurons were innervated by axon terminals containing electron-dense vesicles.

Discussion

Several morphological studies dealing with the mode of termination of RF in the terminal portion (terminal ventricle) of the central canal (CC) have been performed by applying classical staining methods, such as chromalum hematoxylin, paraldehyde-fuchsin and PAS (Olsson 1955, 1958; Wislocki et al. 1956; Hofer 1964; Palkovits 1965), histochemical tests (Naumann 1968), transmission electron microscopy (Sterba and Naumann 1966; Oksche 1969; Tulsi 1982; Hofer et al. 1984), and scanning electron microscopy (Tulsi 1982). Based on this morphological evidence some authors have speculated about the fate of RF material at the level of the terminal ventricle. Thus, it has been suggested that RF undergoes an enzymatic degradation (Naumann 1968) or that its material escapes via gaps in the wall of the CC to reach the subarachnoid space or the vascular system of the leptomeninges (Wislocki et al. 1956; Palkovits 1965; Oksche 1969; Hofer et al. 1984). However, convincing evidence for any of these possibilities is still missing.

On the other hand, very little attention has been paid to the events that might occur in the preterminal region of the CC. In his extensive study on RF in the caudal portion of the spinal cord of some lower vertebrates, Olsson (1955) described the presence of "perpendicular threads" extending between the surface of the CC and the RF. He also stated that these threads anastomose and, where they fuse, they appear to stain in the same manner as the fiber itself. Olsson clearly distinguished these threads from cilia

of the ependymal cells. Furthermore, he also described PAS-positive "droplets" which appeared to stick to the ependymal cilia and to the perpendicular threads. In certain species these droplets were seen along the CC, but were absent in the terminal ventricle. In a recent, scanning electron-microscopic study of RF in the sacral region of the spinal cord and the ilium terminale of the possum, Tulsi (1982) described strands of presumed secretory material extending from the apex of the ependymal cells to RF and, in addition, flocculent masses associated with RF. These two descriptions agree well with some of the present findings.

Due to the fact that the immunoreactive structures of the CC described in the present report were found in slaughterhouse material (bovine and pig), a relevant point with respect to these observations needs to be discussed, i.e., the question to what extent these findings are associated with post-mortem events 2.

With the information now available, we want to make the following considerations:

Ependymal protrusions. It is clear that the spherical structures filling the CC as seen in the light-microscopic preparations correspond to ependymal protrusions. Judging from the situation in the rabbit spinal cord, in which these protrusions were present in the specimen fixed 1 h after death but absent in those fixed by vascular perfusion, it could be concluded that, at least in this species, they represent a post-mortem artifact. Although the bovine CC and its bordering structures appear adequately fixed after perfusing the CC with aldehydes 20 min after death, the presence of protrusions within the CC could well represent a post- mortem artifact. However, since the protrusions are restricted to a rather small segment of the CC (lower lumbar levels), one still may wonder about the meaning of this "selective" artifact. Could it be related to a particular type of ependymal cell? The fact that such protrusions do not occur in spinal segments (cervical, thoracic, lower sacral levels) where the CC is lined by multiciliated ependymal cells supports this possibility. Furthermore, in the CC of the upper sacral levels the ependymal cells do not protrude into the CC, despite the fact that the vast majority of them are not multiciliated. There are, however, some interesting differences between the non-protruding (upper level) and the protruding (lower level) ependymal cells; the latter are rich in lysosome-like bodies. Thus, it may appear that the protrusions, although being probably an artifact, could be related to a non-artifactual phenomenon occurring in a particular cell type of ependymal cell. The findings by Erhardt et al. (1983) are worth mentioning in this respect. These authors studied the CC of several primates fixed by vascular perfusion with glutaraldehyde. Fig. 2 a-c of their publica- tion shows ependymal protrusions intimately associated with RF, whereas their Fig. 1 a illustrates a portion of the CC free of protrusions and lined by cells rich in microvilli. No reference is made to the level of the spinal cord at which these electron micrographs were taken.

Layer of immunoreactive material (IRM) on the surface of the central canal (CC). The fact that this layer is much thinner or reduced to patch-like precipitates in bovine speci-

2 This point was raised by the reviewers of the original version of this paper. The authors are grateful to these reviewers for their constructive suggestions

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mens fixed 20 min after death, as compared to the thick layer found in animals fixed I h after death, and the absence of such a layer in the CC of all other mammalian species investigated most of which were fixed by vascular perfusion, suggests that the formation of this layer is a post-mortem event. The same consideration can be made with respect to the IRM filling the spaces between the ependymal protrusions of the lumbar region. Nevertheless, the coexistence of a distinct and strongly immunoreactive RF, and of a thick layer of IRM on the surface of the CC (see Fig. 2), deserves closer consideration. If the material on the surface of the CC is derived from RF, it is obvious that only some RF-material might separate from the fiber to become attached to the CC. Is this partial segregation of RF-material dependent on the length of the post-mortem interval prior to fixation, or could it be due to the presence in RF of components that can readily escape from the fiber, either intra vitam or as a post-mortem event, to bind to the free surface of the ependymal cells? If this layer of IRM is a post-mortem product, why is such a layer missing in the upper cervical spinal cord and filum, where a RF is also present? The findings by Ermisch et al. (1968) that, after injection of 3%ystine to the carp, the radioactivity in the "ependymal border" and lumen of the ilium was higher than in the ependymal border and lumen of CC at higher levels of the spinal cord, could be related to the appearance of the layer of IRM described here. The possibility that the layer of IRM is derived from the immunoreactive ependymal cells will be discussed below.

Ultrastructural immunocytochemistry of the bovine lumbo-sacral region fixed by perfusion of the CC 20 min after death, has revealed that the patch-like deposits of IRM seen in light-microscopic preparations correspond spatially to small clusters of multiciliated ceils with IRM completely encompassing the bundle of cilia (unpublished observation by the authors). In a scanning electron-microscopic study of the caudal spinal cord in the cat and rabbit, Castenholz (1984) described the presence over the ependyma of the sinus terminalis of single deposits or a continu- ous layer of an amorphous material. This material also causes the cilia of the ependymal cells to adhere to each other. Castenholz considered this material to be derived from RF.

Immunoreactive ependymal cells. The main argument in fa- vor of the view that the presence of immunoreactive ependymal cells in the bovine lumbo-sacral region of the spinal cord is the consequence of some kind of post-mortem processes, is the fact that the lumbo-sacral region of the rat, rabbit and dog fixed by vascular perfusion lacked immunoreactive ependymal cells. On the other hand, the following findings may support the possibility that the immunoreac- rive ependymal cells of the bovine spinal cord are not a post-mortem artifact but a state occurring in the living animal :

(1) When the lumbo-sacral segments of the bovine, pig, rat and rabbit were fixed under the same conditions (20 and 60 min after death), only the two former showed immunoreactive ependymal cells. Therefore, the absence of these cells in the spinal cords of rats and rabbits, regardless of whether they were fixed supravitally or late (1 h) after death, indicates that in these species post-mortem events do not lead to an artifactual appearance of immunoreactive ependymal cells.

(2) The immunoreactive ependymal cells of the bovine spinal cord were seen under the same conditions (perfusion of CC 20 rain after death) as in the material processed for electron microscopy, which showed an adequate preserva- tion of the ultrastructure of the ependymal cells.

(3) The electron-microscopic study revealed the presence of ependymal cells with clear signs of secretory activity, which- due to their frequency and distribution in the lumbo-sacral region - may correspond to the immunoreactive cells.

(4) An ultrastructural immunocytochemical study using the pre-embedding technique has shown that in the immunoreactive ependymal cells the IRM is located within membrane-bounded structures that may correspond either to vacuoles or dilated cisternae of the rough endoplasmic reticulum (unpublished observations by the authors). This result might indicate that the IRM present in these cells is either secreted or absorbed, but not the result of a massive (and passive) imbibition with RF material as a consequence of a post-mortem artifact. Post-embedding ultrastructural immunocytochemistry is in progress to identify the immunoreactive cells and the organelles containing the IRM.

(5) The presence of immunoreactive hypendymal cells, even in regions where the immunoreactive ependymal cells are missing (lower sacral levels) would be difficult to explain in terms of post-mortem changes of the RF. Instead, they may correspond to the clear cells that have been observed in the rosette-like structures.

(6) The possibility that in the rat, dog and rabbit the immunoreactive cells are confined to a restricted area located distal to the lumbo-sacral region investigated, should also be considered. The sinus terminalis of these species is now being investigated in our laboratory. The fact that in the cat and rabbit Castenholz (1984) described a layer of an amorphous material, probably derived from RF, located on the ependyma of the sinus terminalis but absent in other levels of the CC, might support this possibility.

Even if we consider the immunoreaction of ependymal cells of the bovine spinal cord as a post-mortem artifact, one may wonder why only certain cells, mainly concentrated in the lumbo-sacral region, are capable of incorporation of RF material despite the fact that all ependymal cells lining the CC are exposed to this type of material or even covered by a layer of IRM. Does this indicate that the artifact reveals the presence in the CC of a particular type of ependymal cell? It should be mentioned here, that most of the ependymal cells of the bovine lumbo-sacral spinal cord differ from the cells lining other segments of the CC. Whereas in cervical, thoracic and lower sacral levels most cells correspond to the classical ciliated ependyma, in the lumbo-sacral segments the ependymal elements display abundant microvilli or protrusions and a single basal process ending on blood vessels possessing a perivascular space; thus they resemble the specialized ependymal cells found in certain circumventricular organs (Scott et al. 1982).

The fact that the layer of IRM and the immunoreactive ependymal cells, at variance with RF, are not stainable with the pseudoisocyanine, paraldehyde-fuchsin, silver methenamine and PAS methods may be interpreted in two different ways:

(1) It could be postulated that molecules escaping from the RF, either in the living animal or as a post-mortem event, are subjected to breakdown so that they lose their

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carbohydra te moiety and/or the chemical groups responsible for the stainabili ty with pseudoisocyanine and paraldehyde-fuchsin. These molecular fragments would continue to be immunoreact ive and may adhere to the free surface of the CC or become incorpora ted into certain ependymal cells. The drastic reduction in the thickness of R F in segments caudal to the area where the immunoreact ive cells are more numerous would suppor t : ( i ) the absorpt ive hy- pothesis and (ii) the assumption that this absorpt ion occurs under intravital condit ions.

(2) The alternative postulate would be that the mater ial found in the ependymal cells and that forming the layer on the free surface o f the CC is secreted by the ependymal cells, and that it displays a cross-reactivity with the R F material but lacks the ca rbohydra te component , at least that responsible for a positive PAS reaction, as well as the chemical group(s) responsible for a positive para ldehyde- fuchsin reaction. Since, for the prepara t ion of the an t i -RF serum, R F was isolated by perfusing the CC of the bovine spinal cord with artificial CSF, it could be argued that the ant iserum used might contain antibodies against substances present in the CC that are not components of RF. These addi t ional ant ibodies might then be responsible for the immunostaining of structures other than RF. However, the ant iserum raised against a par t ia l ly purified extract of the SCO proper reacts not only with R F but also with the above-ment ioned ependymal cells of the CC. Hence, it can be postula ted that the immunosta inable mater ial of the ependymal cells and that o f R F share a par t ia l chemical identity. A complete chemical identity is ruled out by the histochemical differences discussed above. Tulsi (1982) suggested that the s t rands observed in scanning electron micrographs, extending from the free surface of the ependymal cells to RF, represent addi t ional secretory products added to R F by ependymal cells in the sacral and possibly also other regions of the spinal cord. Tulsi 's assumption implies the existence of secretory cells in the wall of the CC. How- ever, he was unable to find ul t rastructural evidence for the existence of such secretory elements in the spinal cord o f the possum.

The study of the CC and its border ing structures needs to be extended to a representative series of species, especially those that can be fixed by vascular perfusion, before arriving at definitive conclusions.

CSF-contacting neurons (FCN) . The use of an anti- tubulin serum appears to be a useful tool for the immunocytochemical study of the FCN. The in t raependymal and hypendymal F C N described here appear to correspond to the proximal group of F C N reported by Vigh and Vigh-Teichmann (1971). The fine structure of the per ikarya and the processes of F C N projecting to the CC of the bovine spinal cord agrees well with the descript ion by Vigh and Vigh-Teich- mann (1971, 1979) of the F C N in the spinal cord of several vertebrate species. There is, however, a difference with respect to the fine structure of the dendrit ic terminal of the process. In all species investigated by Vigh and Vigh-Teich- mann (1971, 1979) the CSF-contact ing dendri te displays villous extensions, radial ly oriented and endowed with fine fi laments; the authors interpret these extensions as stereo- cilia. Al though we systematically searched for such stereo- cilia, we were unable to find them in the bovine material. Instead, the ul trastructure o f the bovine CSF-contact ing dendrites is similar to that described by Leonhard t (1968)

and Erhardt et al. (1983) for the CSF-contac t ing terminals in the spinal cord of the rabbi t and primates.

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Accepted January 7, 1985

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