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Autoradiographic and ultrastructural studies of areas of spinal cord occupied by Schwann cells and Schwann cell myelin
Brain Research, 239 (1982) 365-375
365
Elsevier Biomedical Press
A U T O R A D I O G R A P H I C A N D U L T R A S T R U C T U R A L STUDIES OF AREAS OF S P I N A L C O R D O C C U P I E D BY S C H W A N N CELLS A N D S C H W A N N CELL MYELIN
SHIRLEY A. GILMORE, T E R R Y J. SIMS and JEANNE K. HEARD Departments of Anatomy and Pathology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (U.S.A.)
(Accepted October 15th, 1981) Key words: spinal cord - - intraspinal Schwann cell - - X-radiation - - intraspinal peripheral-type
myelin
SUMMARY Schwann cells, peripheral-type myelin and connective tissue elements develop within the dorsal portion of the X-irradiated spinal cord in immature rats. Factors controlling the distribution of these elements within the irradiated site are not fully understood. In the present study [3H]thymidine autoradiography was used to examine proliferative activities of cells in these areas occupied by peripheral nervous system components, and correlative ultrastructural evaluations were made. At 15 and 20 days post-irradiation (P-I), the Schwann cells occupied the dorsolateral portions of the dorsal funiculi, and heavily labeled cells occurred throughout these areas. By 25 days P-I the Schwann cells extended ventrally into the depths of the dorsal funiculi and into the dorsal gray matter, and labeled cells were concentrated in the deeper portions of these areas. Ultrastructurally, the Schwann cells and peripheral-type myelin were more mature in the superficial portions where proliferative activity was diminished. In contrast, much less mature, peripheral-type myelin occurred in the depths where the labeled cells were concentrated. At 30 and 45 days P-I, labeled cells were much less frequent but usually occurred in the depths when observed. Similarly, a dorsal-ventral gradient in maturity of peripheral-type myelin was evident ultrastructurally. By 60 and 90 days P-I, labeling was rare, and mature Schwann cell myelin was present throughout the areas. Astrocytes and their processes were less numerous in regions invaded by Schwann cells, as compared to controls, and studies are in progress to evaluate the relationships between these glial elements and intraspinal peripheral nervous system components.
0006-8993/82/000(00000/$02.75 © Elsevier Biomedical Press
366 INTRODUCTION Schwann cells and peripheral-type myelin develop within the lumbosacral spinal cord following exposure of that structure to ionizing radiation during the early postnatal period 2,3,8,10,12. Although these intraspinal Schwann cells occupy a foreign environment, they do not spread throughout the spinal cord in an unhindered fashion. In all animals in which these peripheral nervous system elements were observed within the lumbosacral spinal cold, they occupied portions of the dorsal funiculi and the dorsal gray matter. This pattern was usually established by two months post-irradiation 2,1°,12. The regions of spinal cord occupied by Schwann cells are the same at 6 months 11 as at 2 months 12 following exposure to 4000 R, and a similar trend is found in rats receiving smaller amounts of radiation (2000 R and 1000 R) and examined at intervals extending beyond one years. Autoradiographic studies of these regions of intraspinal peripheral nervous tissue demonstrated incorporation of [3H]thymidine into many of the cells during the early stages of their development within the spinal cord (9-15 days post-irradiation) 7. The marked dilution of the label within these cells by 3 days post-injection indicated also that the cells were undergoing division repeatedly. The lack of progressive spread of Schwann cells and other peripheral elements throughout the extent of the irradiated spinal cord, or into adjacent non-irradiated areas, suggests that factors, as yet not understood, limit the development of peripheral nervous tissue within the central nervous system. The present study was undertaken to examine autoradiographically the incorporation of [SH]thymidine into cells in these areas during the first 3 months following irradiation and to correlate these findings with the ultrastructural characteristics of these cells. MATERIALS AND METHODS Litters of Charles River CD rats, each containing a maximum of 6 irradiated and 2 sham-irradiated rats, were used. The rats were irradiated when 3 days of age with the exposed area restricted to a 5-mm length of lumbosacral spinal cord in the manner described previously12. The radiation was administered as a single dose from a Philips Contact Therapy Apparatus operating under the following conditions: 50 KVP; 2 mA; filter added, 0.25 mm AI; HVL, 0.16 mm A1. Each rat received a single total exposure of 4000 R (722 R/rain).
Light microscopy and autoradiography Groups of rats containing at least 3 irradiated and one sham-irradiated animal were killed at the following post-irradiation intervals: 15, 20, 25, 30, 45 and 60 days. Two irradiated rats were killed at 90 days post-irradiation (P-I). One hour prior to autopsy each of these animals received a single injection of [SH]thymidine intraperitoneally (2/~Ci/g body weight; specific activity, 5 Ci/mmol; Amersham). All of these animals were perfused with 10 % phosphate-buffered formalin while under Nembutal anesthesia. The irradiated and immediately adjacent non-irradiated portions and
367 equivalent sham-irradiated portions of spinal cord were removed and placed in fixative for an additional 24 h. Following paraffin embedment, transverse sections were cut (8 /zm) and mounted on glass slides in an interrupted serial fashion. Some of these slides were then coated with Kodak NTB2 emulsion by the dipping technique and exposed for 5 weeks at 4 °C. Following photographic processing, the autoradiographs were stained with hematoxylin and eosin according to the methods described by Baserga and MalamudL The slides not used for autoradiography were stained by the following techniques: hematoxylin and eosin; gallocyanin; gallocyanin-periodic acid Schiff (PAS); Wilder's reticular stain; and Luxol fast blue-PAS for myelin. The slides were examined light microscopically to define the intraspinal areas occupied by Schwann cells and associated peripheral-type myelin and to determine the locations and distribution of labeled cells within these areas at the different post-irradiation intervals.
Ultrastructural studies On each of the post-irradiation intervals of 25, 45, 60 and 90 days, 3 irradiated and one sham-irradiated rats were anesthetized by intraperitoneal injections (0.2 ml/100 g) of a 35 ~,~solution of chloral hydrate. The rats were then perfused via the left ventricle with a fixative containing 2 ~ paraformaldehyde, 2 ~ glutaraldehyde, 0.5 ~.,~, acrolein and 0 . 5 ~ dimethylsulfoxide in a 0.12 M phosphate buffer at pH 7.2. Two hours following perfusion the irradiated or equivalent level of sham-irradiated spinal cord was removed, cut into 1 mm segments and placed in fixative overnight at 4 ~'C. The tissues were then rinsed in buffer and post-fixed in 2 ~ OsO4 (in buffer) for 2 h at 4 "C. After post-fixation the tissues were rinsed in buffer and washed in 30 ~ acetone. They were stained en bloc in 2 ~ uranyl acetate (in 50 ~ alcohol) for 1.5 h and then dehydrated in a graded series of alcohol washes. Following dehydration the spinal cord segments were washed in acetone, infiltrated with Spurr plastic, and embedded in flat molds to facilitate sectioning in the transverse plane. One micron thick sections of the entire spinal cord were stained with toluidine blue and examined light microscopically to locate areas for subsequent thin-sectioning. Thin sections were poststained in uranyl acetate and lead citrate and then examined with a Siemens 101 electron microscope. RESULTS
Fifteen and 20 days post-irradiation At these intervals the areas occupied by Schwann cells and peripheral-type myelin were restricted to the dorsolateral portions of the dorsal funiculi (Fig. 1A). These areas were hypercellular compared to the same region of spinal cord in a control animal (Fig. 1B). The numerous, labeled cells (arrows, Fig. I A) in these areas of peripheral nervous system elements markedly outnumbered those in the control animal (arrows, Fig. 1B).
Twenty-five days post-irradiation By this time the hypercellular areas containing Schwann cells and peripheral-
368
Fig. 1. Dorsolateral portion of the dorsal funiculus and extreme dorsal portion of the gray matter m an animal killed 15 days P-I (A) and in an age-matched (18-day-old) control rat (B). Note the hypercellularity of the area occupied by Schwann cells and peripheral-type myelin in the irradiated ral (A), in contrast to a similar control area (B). Many cells labeled with [3H]thymidine (arrows) are distributed throughout the areas occupied by intraspinal Schwann cells (A), whereas labeled cells (arrows) are rare in the normal, control rat (B). Hematoxylin and eosin stained autoradiographs. 580. type myelin were located throughout sizeable portions of the dorsal funiculi and extended into the dorsal gray matter (Fig. 2A). Areas near the dorsal surface of the spinal cord, such as those indicated by the small asterisk in Fig. 2A, contained few labeled cells at this time (Fig. 2B), in spite of the concentration of cells in these areas. This pattern was characteristic of all the hypercellular areas located in the superficial, dorsal portions of the dorsal funiculi. Ultrastructural examination revealed the presence of Schwann cells and compact myelin in these areas (Fig. 3). These Schwann cells were characterized by an irregularly-shaped nucleus, dense cytoplasm containing numerous free ribosomes, and a distinct basal lamina surrounding each cell (Fig. 3). The myelin sheaths varied in thickness with the caliber of the axons, but generally were thinner than myelin sheaths surrounding comparably sized, peripheral axons in the same animal. Extracellular space was abundant in regions occupied by Schwann cells as compared to the same space in a similar location in control rats. In deeper areas occupied by Schwann cells (large asterisk, Fig. 2A), many cells were labeled (Fig. 2C)
369
Fig. 2. A: dorsal portion of spinal cord from an animal killed 25 days P-I. Note the extensive development of the hypercellular areas containing intraspinal Schwann cells in both the dorsal funiculi (DF) and the dorsal gray matter (DG). For orientation, a vertically oriented line has been placed along the dorsal median septum. The asterisks indicate the sites photographed at a higher power on an autoradiograph of an adjacent section and shown as B and C. Gallocyanin-PAS. ~: 180. B: highpower view of the area indicated by the small asterisk in A. This autoradiograph shows that few cells are labeled by [3H]thymidine (arrows) in this region of intraspinal Schwann cells near the surface of tile dorsal funiculus. Hematoxylin and eosin. × 580. C: high-power view of area indicated by large asterisk in the gray matter in A. Many cells are labeled by [3H]thymidine (arrows) in these more deeply situated, hypercellular areas occupied by Schwann cells. Compare with the labeling in an area of intraspinal Schwann cells near the dorsal surface in the same section (B). Hematoxylin and eosin. • 580.
370
Fig. 3. Superficial portion of the dorsal funiculus covered by basal lamina (arrows) and pia mater (PM) in an animal killed 25 days P-l. At this time Schwann cells (SC) in this region myelinate largecaliber axons (Ax) and are surrounded by a basal lamina (arrowheads). Axons of smaller caliber remain unmyelinated (asterisks). Astroeyte processes (As) terminate in subpial regions, formin 8 a partial 81ia limitans, ~ 16,000,
371
Fig. 4. In the deeper regions of the dorsal funiculus of an animal killed 25 days P-l, the Schwann cells (SC) are in all earlier stage of myelination. This earlier stage of myelination is evidenced by the loose, Schwann cell cytoplasmic spirals around large-caliber axons (Ax) and the lack of major dense lines within the spirals. FloccuIent material (arrows) surrounds Schwann cells and, in some instances, it appears to have formed a basal lamina (arrowheads)., .: 16,000. Inset: axons with mature, compact Schwann cell myelin in a rat killed 90 days P-I. A smaller caliber axon (Ax) remains unmyelinated, but surrounded by Schwann cell cytoplasm (asterisks). Note the presence of collagen fibrils (C) in the extracellular space. • 20,000.
372 when compared to the superficial areas (Fig. 2B). The Schwann cells in these deeper regions were in an earlier stage of myelination than those located more superficially as evidenced by the loose spiralling of their mesaxons around large caliber axons and the lack of major dense lines (Fig. 4). Dense, flocculent material surrounded Schwann cells and appeared to have coalesced to form a thin basal lamina at some sites.
Thirty and 45 day post-irradiation By these intervals, the areas containing Schwann cells and peripheral-type myelin were well-established in both the dorsal funiculi and the dorsal gray matter, where they appeared as the darkly-stained, hypercellular regions in Fig. 5A. Labeled cells were rarely found in the more dorsal or superficial portions of the dorsal funiculi (Fig. 5B), and only a few cells were labeled in similar regions in the dorsal gray matter (Fig. 5C). There was no marked dorsal-ventral gradient in labeling pattern as was noted at the 25-day interval (Fig. 2B, C). Ultrastructural studies of animals killed 45 days P-I revealed that Schwann cells near the surface of the dorsal funiculi continued to form compact myelin by increasing the thickness of the myelin sheaths. Schwann cells situated more deeply in the dorsal funiculi formed compact myelin at this time, but a dorsal-ventral gradient in the maturity of the myelin sheaths was still present. Immature Schwann cells were still observed but were generally located within the dorsal gray matter.
Sixty and 90 day post-irradiation The general pattern of distribution of intraspinal Schwann cells and peripheraltype myelin was the same as that seen at 30 or 45 days post-irradiation. Cells in these areas were rarely labeled, and the few that were labeled were located in the depths of these areas, i.e. in the gray matter. Mature Schwann cell myelin was present in both dorsal funiculi and dorsal gray matter by 60 days P-I, and there was no increase inthe thickness of the myelin sheaths or the number of major dense lines between 60 and 90 days P-I (inset, Fig. 4). Many small-caliber axons remained unmyelinated yet surrounded by Schwann cell processes. At no time during this study were fibroblasts observed within the spinal cord, although collagen fibrils were seen in extracellular spaces between Schwann cells (inset, Fig. 4). DISCUSSION The general pattern of development of Schwann cells and peripheral-type myelin in the spinal cord following exposure to ionizing radiation in this study is the same as that described previously by Heard and GilmorO 2. Briefly, the areas occupied by these peripheral nervous system component are present initially in the dorsolateral portions of the dorsal funiculi (near the spinal cord~lorsal root junction). Thereafter, there is a spread medially and into the depths of the dorsal funiculi, and finally into the dorsal gray matter. The proliferative activity of cells in areas occupied by Schwann cells and peripheral-type myelin was examined in this study by light microscopic autoradio-
373
Fig. 5. A: low-power view of dorsal half of spinal cord from an animal killed 45 days P-L The darklystained, hypercellular areas occupied by Schwann cells and peripheral-type myelin occur throughout substantial portions of the dorsal funiculi and the dorsal gray matter. Hematoxylin and eosin. 7: 53. B : high-power view from an autoradiograpb of a section adjacent to that shown in A showing portions of the dorsal funiculus (DF) occupied by intraspinal Schwann cells and of the dorsal root (DR). The boundary between these two regions is indicated by the line. Only one cell (arrow) is labeled in the area occupied by intraspinal Schwann cells. Hematoxylin and eosin, x 580. C: high-power view from the same autoradiograph as in B showing an area of intraspinal Schwann cells in the gray matter. Only two cells (arrows) are labeled in contrast to the situation noted in a similar area at 25 days P-I (Fig. 2C). Hematoxylin and eosin. ~580. graphy following injection o f [3H]thymidine. M a n y o f the cells were labeled in animals killed 15 and 20 days P-I; this finding is consistent with that reported previously in rats killed at 11 and 15 days P-I 7. The n u m b e r o f labeled cells was m a r k e d l y decreased by 45 days P-l, and by 60 and 90 days P-1 labeled cells were rarely observed.
374 The distribution of the labeled cells within the areas occupied by Schwann cells varied with the post-irradiation interval. The labeled cells were distributed throughout these areas in rats killed 15 and 20 days P-I. At 25 days P-I, however, there was a concentration of labeled cells in the depths of these areas, i.e. in the deeper portions of the dorsal funiculi and in the dorsal gray matter, as compared with the more superficial portions. This observation suggested that there was a 'front' of dividing Schwann cells extending into the substance of the spinal cord. This dorsal-ventral gradient of proliferative activity had essentially disappeared by 45 days P-l, and the presence of only a few labeled cells by 60 days suggested that the mitogenic stimuli were no longer exerting their influence on these cells. Ultrastructurally the Schwann cells observed within the spinal cord in this study had the same characteristics as Schwann cells described in peripheral nervous tissue 13. A dorsal-ventral gradient in the development and maturation of the intraspinal Schwann cell myelin was observed and paralleled the data obtained with [ZH]thymidine autoradiography. This gradient was particularly evident in the 25 day P-! group in which axons in earlier stages of myelination were observed in the depths of the areas where many cells were labeled with [aH]thymidine. In contrast, myelin in a more mature state was observed in the superficial dorsal portions of these areas where cell proliferation was minimal. This pattern of a dorsal-ventral gradient in the state of maturity of the myelin persisted through 45 days P-I. Thereafter, maturation of the Schwann cell myelin and the ensheathment of unmyelinated axons occurred. A known potent mitogenic stimulus for Schwann cells in tissue culture is the presence of naked, growing axons of dorsal root ganglion cells14. Although the stimulus in the present study is not known, it is possible that naked axons in the irradiated portions of the spinal cord serve as a mitogenic stimulus for the Schwann cells within the intraspinal regions. The lumbosacral region of the spinal cord was not yet myelinated by oligodendrocytes at 3 days of age, when the animals were irradiated. Exposure to ionizing radiation results in a marked depletion of the neuroglial population and a subsequent, temporary inhibition of myelinogenesis in the irradiated area, with the adjacent, non-exposed areas appearing to be normal 2,5,~,9,1'~. It is possible then that these irradiated central axons which lack their oligodendroglial components provide a strong mitogenic stimulus for the Schwann cells that have come to reside within the spinal cord. At later post-irradiation intervals, especially beyond 45 days, the neuroglial population increases, and some of the irradiated central axons are myelinated by 01igodendrocytes 2.'~,1°. Perhaps by this time the ensheathment of the axons by both Schwann cells and oligodendrocytes is essentially complete, thereby eliminating that mitogenic stimulus, and additional myelinating cells are no longer produced. The intermingling of axons myelinated by oligodendrocytes and by Schwann cells had been reported previously by Gilmore and Duncan 1° and Blakemore and Patterson 3. An interesting observation was the ensheathment of unmyelinated axons and the myelination of larger axons by Schwann cells in the absence of well-defined collagen. Collagen fibrils were occasionally noted between Schwann cells, especially at the later post-irradiation intervals, but these were certainly less abundant than in peripheral
375 nerves. F u r t h e r m o r e , fibroblasts were not observed in any of this material. This e n s h e a t h m e n t and m y e l i n a t i o n by intraspinal Schwann cells in the absence of an extracellular connective tissue matrix is in marked contrast to tissue culture requirements for Schwann cell myelination4 and will be studied further in this in vivo model. Astrocytes a n d their processes were observed much less frequently than in equivalent areas of n o r m a l control tissue. Blakemore and Patterson 3 also reported a virtual absence of astrocytes from areas occupied of Schwann cells in the irradiated spinal cord a n d attributed this to a toxic or inhibitory effect of Schwann cells on astrocytes. F u r t h e r studies are u n d e r w a y to examine the astrocyte p o p u l a t i o n in more detail in the irradiated, i m m a t u r e rats and to determine a possible relationship between this p o p u l a t i o n a n d the initial occurrence of Schwann cells within the spinal cord. ACKNOWLEDGEMENTS Supported in part by N I H G r a n t NS 04761 from the N a t i o n a l Institute of Neurological and C o m m u n i c a t i v e Disorders and Stroke. The excellent technical assistance of Mr. N a p o l e o n Phillips is gratefully acknowledged.
REFERENCES I Baserga, R. and Malamud, D., Autoradiography: techniques and applications. In Modern Methods in Experimental Pathology, Hoeber Medical Division, Harper and Row, New York, 1969, pp. 21-22. 2 Beal, J. A. and Hall, J. L., A light micrsocopic study of the effects of X-irradiation on the spinal cord of neonatal rats, J. Neuropath. exp. Neurol., 33 (1974) 128-143. 3 B[akemore, W. F. and Patterson, R. C., Observations on the interactions of Schwann cells and astrocytes following X-irradiation of neonatal rat spinal cord, J. Neurocytol., 4 (1975) 573-585. 4 Bunge, R. P. and Bunge, M. B., Evidence that contact with connective tissue matrix is required for normal interaction between Schwann cells and nerve fibers, J. Cell Biol., 78 (1978) 943-950. 5 Gilmore, S. A., The effects of X-irradiation on the spinal cords of neonatal rats. II. Histological observations, J. Neuropath. exp. Neurol., 22 (1963) 294-301. 6 Gilmore, S. A., Delayed myelination of neonatal rat spinal cord induced by X-irradiation, Neurology, 16 (1966) 749-753. 7 Gilmore, S. A., Autoradiographic studies of intramedullary Schwann cells in irradiated spinal cords of immature rats, Anat. Rec., 171 (1971) 517-528. 8 Gilmore, S. A., Long-term effects of ionizing radiation on the rat spinal cord: intramedullary connective tissue formation, Amer. J. Anat., 137 (1973) 1 18. 9 Gilmore, S. A., Patterns of neuroglial proliferation in spinal cord white matter following exposure to ionizing radiation, Experientia, 35 (1979) 1237. 10 Gilmore, S. A. and Duncan, D., On the presence of peripheral-like nervous and connective tissue within irradiated spinal cord, Anat. Ree., 160 (1968) 675-690. 11 Heard, J. K., Changes Induced by X-irradiation of the Mid-thoracic and Lumbosacral Levels of Neonatal Rat Spinal Cord With Special Reference to the Occurrence of lntramedullary Schwann Cells', Dissert. Abstr. Int., Vol. 42, Issue 2B, 1981. 12 Heard, J. K. and Gilmore, S. A., lntramedullary Schwann cell development following X-irradiation of mid-thoracic and lumbosacral spinal cord levels in immature rats, Anat. Rec., 197 (1980) 85-93. 13 Peters, A., Palay, S. L. and Webster, H. de F., The Fine Structure of the Nervous System : The Neurons and Supporting Cells, W.B. Saunders Co., Philadelphia, 1976, pp. 186-200. 14 Wood, P. M. and Bunge, R. P., Evidence that sensory axons are mitogenic for Schwann ceils, Nature (Lond.), 256 (1975) 662-664.
365
Elsevier Biomedical Press
A U T O R A D I O G R A P H I C A N D U L T R A S T R U C T U R A L STUDIES OF AREAS OF S P I N A L C O R D O C C U P I E D BY S C H W A N N CELLS A N D S C H W A N N CELL MYELIN
SHIRLEY A. GILMORE, T E R R Y J. SIMS and JEANNE K. HEARD Departments of Anatomy and Pathology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (U.S.A.)
(Accepted October 15th, 1981) Key words: spinal cord - - intraspinal Schwann cell - - X-radiation - - intraspinal peripheral-type
myelin
SUMMARY Schwann cells, peripheral-type myelin and connective tissue elements develop within the dorsal portion of the X-irradiated spinal cord in immature rats. Factors controlling the distribution of these elements within the irradiated site are not fully understood. In the present study [3H]thymidine autoradiography was used to examine proliferative activities of cells in these areas occupied by peripheral nervous system components, and correlative ultrastructural evaluations were made. At 15 and 20 days post-irradiation (P-I), the Schwann cells occupied the dorsolateral portions of the dorsal funiculi, and heavily labeled cells occurred throughout these areas. By 25 days P-I the Schwann cells extended ventrally into the depths of the dorsal funiculi and into the dorsal gray matter, and labeled cells were concentrated in the deeper portions of these areas. Ultrastructurally, the Schwann cells and peripheral-type myelin were more mature in the superficial portions where proliferative activity was diminished. In contrast, much less mature, peripheral-type myelin occurred in the depths where the labeled cells were concentrated. At 30 and 45 days P-I, labeled cells were much less frequent but usually occurred in the depths when observed. Similarly, a dorsal-ventral gradient in maturity of peripheral-type myelin was evident ultrastructurally. By 60 and 90 days P-I, labeling was rare, and mature Schwann cell myelin was present throughout the areas. Astrocytes and their processes were less numerous in regions invaded by Schwann cells, as compared to controls, and studies are in progress to evaluate the relationships between these glial elements and intraspinal peripheral nervous system components.
0006-8993/82/000(00000/$02.75 © Elsevier Biomedical Press
366 INTRODUCTION Schwann cells and peripheral-type myelin develop within the lumbosacral spinal cord following exposure of that structure to ionizing radiation during the early postnatal period 2,3,8,10,12. Although these intraspinal Schwann cells occupy a foreign environment, they do not spread throughout the spinal cord in an unhindered fashion. In all animals in which these peripheral nervous system elements were observed within the lumbosacral spinal cold, they occupied portions of the dorsal funiculi and the dorsal gray matter. This pattern was usually established by two months post-irradiation 2,1°,12. The regions of spinal cord occupied by Schwann cells are the same at 6 months 11 as at 2 months 12 following exposure to 4000 R, and a similar trend is found in rats receiving smaller amounts of radiation (2000 R and 1000 R) and examined at intervals extending beyond one years. Autoradiographic studies of these regions of intraspinal peripheral nervous tissue demonstrated incorporation of [3H]thymidine into many of the cells during the early stages of their development within the spinal cord (9-15 days post-irradiation) 7. The marked dilution of the label within these cells by 3 days post-injection indicated also that the cells were undergoing division repeatedly. The lack of progressive spread of Schwann cells and other peripheral elements throughout the extent of the irradiated spinal cord, or into adjacent non-irradiated areas, suggests that factors, as yet not understood, limit the development of peripheral nervous tissue within the central nervous system. The present study was undertaken to examine autoradiographically the incorporation of [SH]thymidine into cells in these areas during the first 3 months following irradiation and to correlate these findings with the ultrastructural characteristics of these cells. MATERIALS AND METHODS Litters of Charles River CD rats, each containing a maximum of 6 irradiated and 2 sham-irradiated rats, were used. The rats were irradiated when 3 days of age with the exposed area restricted to a 5-mm length of lumbosacral spinal cord in the manner described previously12. The radiation was administered as a single dose from a Philips Contact Therapy Apparatus operating under the following conditions: 50 KVP; 2 mA; filter added, 0.25 mm AI; HVL, 0.16 mm A1. Each rat received a single total exposure of 4000 R (722 R/rain).
Light microscopy and autoradiography Groups of rats containing at least 3 irradiated and one sham-irradiated animal were killed at the following post-irradiation intervals: 15, 20, 25, 30, 45 and 60 days. Two irradiated rats were killed at 90 days post-irradiation (P-I). One hour prior to autopsy each of these animals received a single injection of [SH]thymidine intraperitoneally (2/~Ci/g body weight; specific activity, 5 Ci/mmol; Amersham). All of these animals were perfused with 10 % phosphate-buffered formalin while under Nembutal anesthesia. The irradiated and immediately adjacent non-irradiated portions and
367 equivalent sham-irradiated portions of spinal cord were removed and placed in fixative for an additional 24 h. Following paraffin embedment, transverse sections were cut (8 /zm) and mounted on glass slides in an interrupted serial fashion. Some of these slides were then coated with Kodak NTB2 emulsion by the dipping technique and exposed for 5 weeks at 4 °C. Following photographic processing, the autoradiographs were stained with hematoxylin and eosin according to the methods described by Baserga and MalamudL The slides not used for autoradiography were stained by the following techniques: hematoxylin and eosin; gallocyanin; gallocyanin-periodic acid Schiff (PAS); Wilder's reticular stain; and Luxol fast blue-PAS for myelin. The slides were examined light microscopically to define the intraspinal areas occupied by Schwann cells and associated peripheral-type myelin and to determine the locations and distribution of labeled cells within these areas at the different post-irradiation intervals.
Ultrastructural studies On each of the post-irradiation intervals of 25, 45, 60 and 90 days, 3 irradiated and one sham-irradiated rats were anesthetized by intraperitoneal injections (0.2 ml/100 g) of a 35 ~,~solution of chloral hydrate. The rats were then perfused via the left ventricle with a fixative containing 2 ~ paraformaldehyde, 2 ~ glutaraldehyde, 0.5 ~.,~, acrolein and 0 . 5 ~ dimethylsulfoxide in a 0.12 M phosphate buffer at pH 7.2. Two hours following perfusion the irradiated or equivalent level of sham-irradiated spinal cord was removed, cut into 1 mm segments and placed in fixative overnight at 4 ~'C. The tissues were then rinsed in buffer and post-fixed in 2 ~ OsO4 (in buffer) for 2 h at 4 "C. After post-fixation the tissues were rinsed in buffer and washed in 30 ~ acetone. They were stained en bloc in 2 ~ uranyl acetate (in 50 ~ alcohol) for 1.5 h and then dehydrated in a graded series of alcohol washes. Following dehydration the spinal cord segments were washed in acetone, infiltrated with Spurr plastic, and embedded in flat molds to facilitate sectioning in the transverse plane. One micron thick sections of the entire spinal cord were stained with toluidine blue and examined light microscopically to locate areas for subsequent thin-sectioning. Thin sections were poststained in uranyl acetate and lead citrate and then examined with a Siemens 101 electron microscope. RESULTS
Fifteen and 20 days post-irradiation At these intervals the areas occupied by Schwann cells and peripheral-type myelin were restricted to the dorsolateral portions of the dorsal funiculi (Fig. 1A). These areas were hypercellular compared to the same region of spinal cord in a control animal (Fig. 1B). The numerous, labeled cells (arrows, Fig. I A) in these areas of peripheral nervous system elements markedly outnumbered those in the control animal (arrows, Fig. 1B).
Twenty-five days post-irradiation By this time the hypercellular areas containing Schwann cells and peripheral-
368
Fig. 1. Dorsolateral portion of the dorsal funiculus and extreme dorsal portion of the gray matter m an animal killed 15 days P-I (A) and in an age-matched (18-day-old) control rat (B). Note the hypercellularity of the area occupied by Schwann cells and peripheral-type myelin in the irradiated ral (A), in contrast to a similar control area (B). Many cells labeled with [3H]thymidine (arrows) are distributed throughout the areas occupied by intraspinal Schwann cells (A), whereas labeled cells (arrows) are rare in the normal, control rat (B). Hematoxylin and eosin stained autoradiographs. 580. type myelin were located throughout sizeable portions of the dorsal funiculi and extended into the dorsal gray matter (Fig. 2A). Areas near the dorsal surface of the spinal cord, such as those indicated by the small asterisk in Fig. 2A, contained few labeled cells at this time (Fig. 2B), in spite of the concentration of cells in these areas. This pattern was characteristic of all the hypercellular areas located in the superficial, dorsal portions of the dorsal funiculi. Ultrastructural examination revealed the presence of Schwann cells and compact myelin in these areas (Fig. 3). These Schwann cells were characterized by an irregularly-shaped nucleus, dense cytoplasm containing numerous free ribosomes, and a distinct basal lamina surrounding each cell (Fig. 3). The myelin sheaths varied in thickness with the caliber of the axons, but generally were thinner than myelin sheaths surrounding comparably sized, peripheral axons in the same animal. Extracellular space was abundant in regions occupied by Schwann cells as compared to the same space in a similar location in control rats. In deeper areas occupied by Schwann cells (large asterisk, Fig. 2A), many cells were labeled (Fig. 2C)
369
Fig. 2. A: dorsal portion of spinal cord from an animal killed 25 days P-I. Note the extensive development of the hypercellular areas containing intraspinal Schwann cells in both the dorsal funiculi (DF) and the dorsal gray matter (DG). For orientation, a vertically oriented line has been placed along the dorsal median septum. The asterisks indicate the sites photographed at a higher power on an autoradiograph of an adjacent section and shown as B and C. Gallocyanin-PAS. ~: 180. B: highpower view of the area indicated by the small asterisk in A. This autoradiograph shows that few cells are labeled by [3H]thymidine (arrows) in this region of intraspinal Schwann cells near the surface of tile dorsal funiculus. Hematoxylin and eosin. × 580. C: high-power view of area indicated by large asterisk in the gray matter in A. Many cells are labeled by [3H]thymidine (arrows) in these more deeply situated, hypercellular areas occupied by Schwann cells. Compare with the labeling in an area of intraspinal Schwann cells near the dorsal surface in the same section (B). Hematoxylin and eosin. • 580.
370
Fig. 3. Superficial portion of the dorsal funiculus covered by basal lamina (arrows) and pia mater (PM) in an animal killed 25 days P-l. At this time Schwann cells (SC) in this region myelinate largecaliber axons (Ax) and are surrounded by a basal lamina (arrowheads). Axons of smaller caliber remain unmyelinated (asterisks). Astroeyte processes (As) terminate in subpial regions, formin 8 a partial 81ia limitans, ~ 16,000,
371
Fig. 4. In the deeper regions of the dorsal funiculus of an animal killed 25 days P-l, the Schwann cells (SC) are in all earlier stage of myelination. This earlier stage of myelination is evidenced by the loose, Schwann cell cytoplasmic spirals around large-caliber axons (Ax) and the lack of major dense lines within the spirals. FloccuIent material (arrows) surrounds Schwann cells and, in some instances, it appears to have formed a basal lamina (arrowheads)., .: 16,000. Inset: axons with mature, compact Schwann cell myelin in a rat killed 90 days P-I. A smaller caliber axon (Ax) remains unmyelinated, but surrounded by Schwann cell cytoplasm (asterisks). Note the presence of collagen fibrils (C) in the extracellular space. • 20,000.
372 when compared to the superficial areas (Fig. 2B). The Schwann cells in these deeper regions were in an earlier stage of myelination than those located more superficially as evidenced by the loose spiralling of their mesaxons around large caliber axons and the lack of major dense lines (Fig. 4). Dense, flocculent material surrounded Schwann cells and appeared to have coalesced to form a thin basal lamina at some sites.
Thirty and 45 day post-irradiation By these intervals, the areas containing Schwann cells and peripheral-type myelin were well-established in both the dorsal funiculi and the dorsal gray matter, where they appeared as the darkly-stained, hypercellular regions in Fig. 5A. Labeled cells were rarely found in the more dorsal or superficial portions of the dorsal funiculi (Fig. 5B), and only a few cells were labeled in similar regions in the dorsal gray matter (Fig. 5C). There was no marked dorsal-ventral gradient in labeling pattern as was noted at the 25-day interval (Fig. 2B, C). Ultrastructural studies of animals killed 45 days P-I revealed that Schwann cells near the surface of the dorsal funiculi continued to form compact myelin by increasing the thickness of the myelin sheaths. Schwann cells situated more deeply in the dorsal funiculi formed compact myelin at this time, but a dorsal-ventral gradient in the maturity of the myelin sheaths was still present. Immature Schwann cells were still observed but were generally located within the dorsal gray matter.
Sixty and 90 day post-irradiation The general pattern of distribution of intraspinal Schwann cells and peripheraltype myelin was the same as that seen at 30 or 45 days post-irradiation. Cells in these areas were rarely labeled, and the few that were labeled were located in the depths of these areas, i.e. in the gray matter. Mature Schwann cell myelin was present in both dorsal funiculi and dorsal gray matter by 60 days P-I, and there was no increase inthe thickness of the myelin sheaths or the number of major dense lines between 60 and 90 days P-I (inset, Fig. 4). Many small-caliber axons remained unmyelinated yet surrounded by Schwann cell processes. At no time during this study were fibroblasts observed within the spinal cord, although collagen fibrils were seen in extracellular spaces between Schwann cells (inset, Fig. 4). DISCUSSION The general pattern of development of Schwann cells and peripheral-type myelin in the spinal cord following exposure to ionizing radiation in this study is the same as that described previously by Heard and GilmorO 2. Briefly, the areas occupied by these peripheral nervous system component are present initially in the dorsolateral portions of the dorsal funiculi (near the spinal cord~lorsal root junction). Thereafter, there is a spread medially and into the depths of the dorsal funiculi, and finally into the dorsal gray matter. The proliferative activity of cells in areas occupied by Schwann cells and peripheral-type myelin was examined in this study by light microscopic autoradio-
373
Fig. 5. A: low-power view of dorsal half of spinal cord from an animal killed 45 days P-L The darklystained, hypercellular areas occupied by Schwann cells and peripheral-type myelin occur throughout substantial portions of the dorsal funiculi and the dorsal gray matter. Hematoxylin and eosin. 7: 53. B : high-power view from an autoradiograpb of a section adjacent to that shown in A showing portions of the dorsal funiculus (DF) occupied by intraspinal Schwann cells and of the dorsal root (DR). The boundary between these two regions is indicated by the line. Only one cell (arrow) is labeled in the area occupied by intraspinal Schwann cells. Hematoxylin and eosin, x 580. C: high-power view from the same autoradiograph as in B showing an area of intraspinal Schwann cells in the gray matter. Only two cells (arrows) are labeled in contrast to the situation noted in a similar area at 25 days P-I (Fig. 2C). Hematoxylin and eosin. ~580. graphy following injection o f [3H]thymidine. M a n y o f the cells were labeled in animals killed 15 and 20 days P-I; this finding is consistent with that reported previously in rats killed at 11 and 15 days P-I 7. The n u m b e r o f labeled cells was m a r k e d l y decreased by 45 days P-l, and by 60 and 90 days P-1 labeled cells were rarely observed.
374 The distribution of the labeled cells within the areas occupied by Schwann cells varied with the post-irradiation interval. The labeled cells were distributed throughout these areas in rats killed 15 and 20 days P-I. At 25 days P-I, however, there was a concentration of labeled cells in the depths of these areas, i.e. in the deeper portions of the dorsal funiculi and in the dorsal gray matter, as compared with the more superficial portions. This observation suggested that there was a 'front' of dividing Schwann cells extending into the substance of the spinal cord. This dorsal-ventral gradient of proliferative activity had essentially disappeared by 45 days P-l, and the presence of only a few labeled cells by 60 days suggested that the mitogenic stimuli were no longer exerting their influence on these cells. Ultrastructurally the Schwann cells observed within the spinal cord in this study had the same characteristics as Schwann cells described in peripheral nervous tissue 13. A dorsal-ventral gradient in the development and maturation of the intraspinal Schwann cell myelin was observed and paralleled the data obtained with [ZH]thymidine autoradiography. This gradient was particularly evident in the 25 day P-! group in which axons in earlier stages of myelination were observed in the depths of the areas where many cells were labeled with [aH]thymidine. In contrast, myelin in a more mature state was observed in the superficial dorsal portions of these areas where cell proliferation was minimal. This pattern of a dorsal-ventral gradient in the state of maturity of the myelin persisted through 45 days P-I. Thereafter, maturation of the Schwann cell myelin and the ensheathment of unmyelinated axons occurred. A known potent mitogenic stimulus for Schwann cells in tissue culture is the presence of naked, growing axons of dorsal root ganglion cells14. Although the stimulus in the present study is not known, it is possible that naked axons in the irradiated portions of the spinal cord serve as a mitogenic stimulus for the Schwann cells within the intraspinal regions. The lumbosacral region of the spinal cord was not yet myelinated by oligodendrocytes at 3 days of age, when the animals were irradiated. Exposure to ionizing radiation results in a marked depletion of the neuroglial population and a subsequent, temporary inhibition of myelinogenesis in the irradiated area, with the adjacent, non-exposed areas appearing to be normal 2,5,~,9,1'~. It is possible then that these irradiated central axons which lack their oligodendroglial components provide a strong mitogenic stimulus for the Schwann cells that have come to reside within the spinal cord. At later post-irradiation intervals, especially beyond 45 days, the neuroglial population increases, and some of the irradiated central axons are myelinated by 01igodendrocytes 2.'~,1°. Perhaps by this time the ensheathment of the axons by both Schwann cells and oligodendrocytes is essentially complete, thereby eliminating that mitogenic stimulus, and additional myelinating cells are no longer produced. The intermingling of axons myelinated by oligodendrocytes and by Schwann cells had been reported previously by Gilmore and Duncan 1° and Blakemore and Patterson 3. An interesting observation was the ensheathment of unmyelinated axons and the myelination of larger axons by Schwann cells in the absence of well-defined collagen. Collagen fibrils were occasionally noted between Schwann cells, especially at the later post-irradiation intervals, but these were certainly less abundant than in peripheral
375 nerves. F u r t h e r m o r e , fibroblasts were not observed in any of this material. This e n s h e a t h m e n t and m y e l i n a t i o n by intraspinal Schwann cells in the absence of an extracellular connective tissue matrix is in marked contrast to tissue culture requirements for Schwann cell myelination4 and will be studied further in this in vivo model. Astrocytes a n d their processes were observed much less frequently than in equivalent areas of n o r m a l control tissue. Blakemore and Patterson 3 also reported a virtual absence of astrocytes from areas occupied of Schwann cells in the irradiated spinal cord a n d attributed this to a toxic or inhibitory effect of Schwann cells on astrocytes. F u r t h e r studies are u n d e r w a y to examine the astrocyte p o p u l a t i o n in more detail in the irradiated, i m m a t u r e rats and to determine a possible relationship between this p o p u l a t i o n a n d the initial occurrence of Schwann cells within the spinal cord. ACKNOWLEDGEMENTS Supported in part by N I H G r a n t NS 04761 from the N a t i o n a l Institute of Neurological and C o m m u n i c a t i v e Disorders and Stroke. The excellent technical assistance of Mr. N a p o l e o n Phillips is gratefully acknowledged.
REFERENCES I Baserga, R. and Malamud, D., Autoradiography: techniques and applications. In Modern Methods in Experimental Pathology, Hoeber Medical Division, Harper and Row, New York, 1969, pp. 21-22. 2 Beal, J. A. and Hall, J. L., A light micrsocopic study of the effects of X-irradiation on the spinal cord of neonatal rats, J. Neuropath. exp. Neurol., 33 (1974) 128-143. 3 B[akemore, W. F. and Patterson, R. C., Observations on the interactions of Schwann cells and astrocytes following X-irradiation of neonatal rat spinal cord, J. Neurocytol., 4 (1975) 573-585. 4 Bunge, R. P. and Bunge, M. B., Evidence that contact with connective tissue matrix is required for normal interaction between Schwann cells and nerve fibers, J. Cell Biol., 78 (1978) 943-950. 5 Gilmore, S. A., The effects of X-irradiation on the spinal cords of neonatal rats. II. Histological observations, J. Neuropath. exp. Neurol., 22 (1963) 294-301. 6 Gilmore, S. A., Delayed myelination of neonatal rat spinal cord induced by X-irradiation, Neurology, 16 (1966) 749-753. 7 Gilmore, S. A., Autoradiographic studies of intramedullary Schwann cells in irradiated spinal cords of immature rats, Anat. Rec., 171 (1971) 517-528. 8 Gilmore, S. A., Long-term effects of ionizing radiation on the rat spinal cord: intramedullary connective tissue formation, Amer. J. Anat., 137 (1973) 1 18. 9 Gilmore, S. A., Patterns of neuroglial proliferation in spinal cord white matter following exposure to ionizing radiation, Experientia, 35 (1979) 1237. 10 Gilmore, S. A. and Duncan, D., On the presence of peripheral-like nervous and connective tissue within irradiated spinal cord, Anat. Ree., 160 (1968) 675-690. 11 Heard, J. K., Changes Induced by X-irradiation of the Mid-thoracic and Lumbosacral Levels of Neonatal Rat Spinal Cord With Special Reference to the Occurrence of lntramedullary Schwann Cells', Dissert. Abstr. Int., Vol. 42, Issue 2B, 1981. 12 Heard, J. K. and Gilmore, S. A., lntramedullary Schwann cell development following X-irradiation of mid-thoracic and lumbosacral spinal cord levels in immature rats, Anat. Rec., 197 (1980) 85-93. 13 Peters, A., Palay, S. L. and Webster, H. de F., The Fine Structure of the Nervous System : The Neurons and Supporting Cells, W.B. Saunders Co., Philadelphia, 1976, pp. 186-200. 14 Wood, P. M. and Bunge, R. P., Evidence that sensory axons are mitogenic for Schwann ceils, Nature (Lond.), 256 (1975) 662-664.