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Lysophosphatidyl choline-induced focal demyelination in the rabbit corpus callosum
Journal of the Neurological Sciences, 1979, 44:45 53
45
© Elsevier/North-Holland Biomedical Press
L Y S O P H O S P H A T I D Y L C H O L I N E - I N D U C E D F O C A L D E M Y E L I N A T I O N IN THE RABBIT C O R P U S C A L L O S U M Light-microscopic Observations
S. G. WAXMAN, J. D. KOCSIS and K. C. NITTA Department of Neurology, Stanford University School of Medicine and Veterans Admhlistration Medical Center, Palo Alto, CA (U.S.A.)
(Received 24 April, 1979) (Accepted 19 June, 1979)
SUMMARY The local application of lysophosphatidyl choline (LPC) by microinjection into the region of the corpus callosum of the rabbit produced demyelinating lesions. The lesions were assessed histologically using the Luxol fast blue myelin stain and the Holmes silver nitrate stain for axis cylinders. Survival times for the animals ranged from 7 to 14 days. The center of the lesion was marked by infiltration of macrophages and necrosis, but the major area of the lesion was characterized by demyelination. By consideration of anatomical factors influencing LPC diffusion and of the appropriate placement of the injection, the entire vertical extent (about 0.5 mm) of the corpus callosum could be demyelinated with minimal amounts of necrosis. Since focal demyelination was possible in the fine caliber axons of the corpus callosum which are anatomically representative of many forebrain fiber systems, and since this fiber system is amenable to chronic physiological investigation, the corpus callosum may serve as an experimental model for morpho-physiological studies of mammalian central demyelinating pathways.
INTRODUCTION Focal demyelinating lesions have been produced in the central nervous system This work was supported by Grant RG 1231 from the National Multiple Sclerosis Society. Facilities used in this study were equipped in part through grants from the Medical Research Service of the Veterans Administration and the Paralyzed Veterans of America. Address for correspondence: Stephan G. Waxman, M.D., Ph. D., Department of Neurology, Veterans Administration Medical Center, 3801 Miranda Avenue, Palo Alto, CA 94304, U.S.A.
46 (CNS) by a number of methods. The injection of diphtheria toxin (McDonald and Sears 1970a) permitted the first early studies on the physiology of conduction in demyelinated spinal cord (dorsal column) axons (McDonald and Sears 1970b). More recently, lysophosphatidyl choline (LPC) has been used to produce focal demyelinating lesions in spinal cord (Hall 1972; Blakemore et al. 1977). Another useful model of focal spinal cord demyelination has been provided through the use of transient compression (Gledhill et al. 1973). In contrast, there have been few studies of anatomically controlled focal demyelination in the mammalian cerebrum. One such study used diphtheria toxin to produce demyelination of deep cortical white matter (Wisniewski and Raine 1971). Jacobs (1967) has shown that there may be significant differences in the response of large and small myelinated fibers to demyelination with diphtheria toxin. Most previous studies on focal demyelination have been carried out in large diameter fiber systems, which may not be representative of the axons in cerebral white matter. In the present study we have applied LPC to the fine caliber fibers of the corpus callosum in the rabbit. These axons are especially well suited for study, since they are unbranched (and therefore do not exhibit the complex conduction characteristics of branched central axons (see, e.g., Chung et al. 1970)), and they present the opportunity for monitoring conduction in CNS axons for periods of several hours (Swadlow 1974; Swadlow and Waxman 1976; Waxman and Swadlow 1976a, 1977). This method, which also produces few clinical deficits and therefore obviates problems of animal survival, and has implications in terms of providing a model for studies on the characteristics of focally demyelinated CNS axons within the mammalian cerebrum. MATERIALS AND METHODS Ten adult female Dutch rabbits were used in the present study. The animals were anesthetized with sodium pentobarbital (35 mg/kg) which was injected into the marginal vein of the ear. Their heads were fixed rigidly in a rabbit head holder (David K o p f Instruments) and a small hole was drilled in the bone overlying the corpus callosum (4-6 mm posterior to bregma and 1.5 mm lateral to the midline). A small incision was made in the exposed dura to allow passage of a glass micropipette. The pipette (tip diameter of 30-50 #m) was pulled on a microelectrode puller, and was fixed to the needle of a Hamilton microsyringe. The syringe was mounted to a micromanipulator to allow for accurate placement of the pipette into the brain. A 1 ~ solution of lysophosphatidyl choline (Sigma Chemical Co.) in normal saline was injected into or near the corpus callosum (3-4 mm ventral from the cortical surface), in amounts ranging from 1/A to 4/~1, delivered over a period of 20-40 min. Upon completion of the injection the needle was left in place for 10-20 min prior to withdrawal. After removal of the pipette from the brain the dura was closed, the bone opening was sealed with dental acrylic, and the scalp incision was sutured. Two animals were injected with normal saline to serve as controls. The animals were allowed to survive from 7 to 14 days, and subsequently perfused through the left ventricle of the heart with 10 ~ buffered formalin. Paraffin
47 s e c t i o n s ( 1 0 / ~ m ) w e r e p r e p a r e d and stained with a c o m b i n a t i o n o f L u x o l fast b l u e for m y e l i n a n d the H o l m e s silver n i t r a t e m e t h o d for a x o n staining. S o m e sections were stained with h e m a t o x y l i n and eosin.
Fig. 1. A : lesion site 14 days after LPC injection (left). Normal saline was injected on the right side. Scale bar -- 0.5 mm. B: Higher magnification showing demyelinated fibers in the corpus callosum. The histological sections in this and subsequent figures stained with Luxol fast blue and Holmes silver nitrate method. Scale bar -- 0.2 mm.
48 RESULTS The spatial extent and configuration of the lesions produced from LPC injections were directly related to the volume of injected LPC and to the geometric organization of the fiber tracts near the injection site. In general, larger volumes of injected LPC induced more extensive lesions, and the spread of the lesion was also more extensive along the longitudinal axis of the fibers. The light region within the corpus callosum and fimbria shown in Fig. 1A, delineates an LPC injection site. The lighter tone of the lesion area is the result of myelin loss (absence of Luxol fast blue staining), macrophage infiltration, and the loss of some fibers. The presence of silver-stained axons within the lesion area can be seen in the higher power photomicrograph of Fig. 1B. The nuclei of macrophages can also be seen dispersed amongst the demyelinated fibers. Deep to the corpus callosum, within the dorsal aspect of the fimbria, an area of heavy macrophage infiltration with nearly complete loss of axons is present. Part of this region of necrosis can be seen in Fig. 1A, and is more completely identified in the shaded area of the camera lucida drawing of Fig. 2. Necrosis in this animal was most extensive at the section shown in Fig. 2. This area corresponds to the center of the lesion (notice the demyelinating track in the subcortical white matter marking the course of the micropipette penetration). In all of our lesions, necrosis was most prominent at the center of the lesion closest to the injection site. To minimize damage to the axons of the corpus callosum, but to maximize demyelination of these fibers, we positioned the tip of the micropipette deep to the corpus callosum into the dorsal aspect of the fimbria. At this level, 2/~1 of LPC were injected over a period of 30 min. The pipette was then moved 0.5 m m dorsally and an additional 1/~1 of LPC was injected. A third injection of 1 #l was also
$CW !' \ " " - ~ '. . . .". J~ ~.... .,, ~
......
.....
--
,.~ ~ ~ii~i:~~.../''Fig. 2. Camera lucida drawing showing central region of lesion (inside of dashed lines) and the area of necrosis (shaded). SCW, subcortical white matter; CC, corpus callosum; Fim, fimbria. Scale bar -0.5 mm.
49 applied at 0.5 mm dorsal to the second injection. A banding in the lesion indicative of the 3 injection levels is apparent in Fig. 1A. The slow application of LPC in time, and the vertical distribution of the injections (over 1 mm) maximized the demyelinating effects, but minimized necrosis within the corpus callosum. When the injections were centered dorsal to the corpus callosum, i.e., within the subcortical white matter, even
Fig. 3. A: transition zone to demyelination in the corpus callosum. Scale bar : 40/;m. B: demyelinated fibres cut in cross-section (upper part of field) in the subcortical white matter. Scale bar 16/~m.
50
Fig. 4. A: demyelinated fiber fascicles in the corpus callosum. B: a fascicle of fine caliber fibers in the corpus callosum after focal demyelination (between arrows). Scale bar for A and B = 6.0/~m.
5l large volumes of LPC ( 4 / d ) did not appreciab,y spread into the corpus callosum. Rather, the longitudinal extent of the lesion within the subcortical white matter was greater than with smaller volume injections. The transition zone between myelinated and demyelinated axons was relatively abrupt. This can be seen in the low power photomicrographs of Figs. 1A and I B, and in the higher power photomicrographs of Figs. 3A and 3B. Fascicles of longitudinally oriented myelinated fibers of the corpus callosum are present on the left of Fig. 3A. As one follows these fascicles from left to right, the dark staining myelin is lost and the increased presence of macrophage nuclei can be seen. The cross-cut fibers of the subcortical white matter also illustrates the abrupt transition to demyelination (Fig. 3B). The fibers in the upper half of Fig. 3B have maintained their general anatomical organization, i.e., their grouping into small bundles ef fibers separated by longitudinally directed axons, but they are conspicuously devoid of myelin. The integrity of the fiber fascicles after demyelination can also be seen in Fig. 4A. Discrete fiber bundles composed of axons with a variety of diameters are present, and macrophage nuclei are distributed between these fiber bundles. A grouping of especially fine caliber fibers is shown in Fig. 4B. Many of these fibers were di~cult to photograph in a single plane because of their extremely fine caliber and their tortuous course through the corpus callosum. Injections of normal saline delivered in the same manner as the LPC solution injections, did not produce noticeable damage in our experiments. The corpus callosal region contralateral to the LPC injection site in Fig. 1A was injected with 4/~l of normal saline, but virtually no evidence of tissue damage could be found in the Luxol fast blue-Holmes silver nitrate sections. The visibility of the electrode track on the side of the LPC injection was due primarily to the demyelinating effects of LPC leakage (Fig. 1A). DISCUSSION The present studies show that it is possible to produce, by microinjection of small amounts of LPC through glass micropipettes, focal demyelinating lesions in the corpus callosum of the rabbit. A variable amount of necrosis was found nearest the center of the lesion as has been reported following microinjections of diphtheria toxin in spinal cord (McDonald and Sears 1970a) and of LPC in peripheral nerve and spinal cord (Hall and Gregson 1971; Hall 1972; Blakemore et al. 1977). Since the dorsoventral extent of the corpus callosum is only about 0.6 ram, direct injections into this fiber bundle are not desirable because of the necrosis that would occur at the lesion center. In order to minimize necrosis and to maximize demyelination in the corpus callosum the injections were centered deep to the corpus callosum in the dorsal aspect of the fimbria. After an injection at this site the pipette was moved dorsally and smaller volume injections were also made. The center of necrosis was established, by this injection regimen, in the fimbria and the entire dorsal-ventral extent of the corpus callosum was demyelinated.
52 The diffusion of the LPC appeared very dependent upon the anatomical orientation of the fibers, with spread of the lesion being more pronounced along the longitudinal axis of the fibers. Furthermore, at regions where fibers change direction, such as between the orthogonally oriented fibers of the corpus callosum and the subcortical white matter, there is an apparent barrier or at least impediment to diffusion. This is demonstrated by the difficulty with which LPC located in the corpus callosum will spread dorsally into the subcortical white matter (e.g., notice the relatively sharp border of the demyelinated corpus callosum fibers with the overlying white matter in Fig. 1A). As we have shown previously in the rabbit (Waxman and Swadlow 1976b) and primate (Swadlow et al. 1979), the splenium of the corpus callosum contains relatively small diameter myelinated fibers, with more than 50 ~o of fibers smaller than 0.85/zm and more than 90~o of fibers smaller than 1.5/~m in the rabbit. We were initially concerned that the production of focal demyelinating lesions in rabbit corpus callosum might not be possible in view of Jacobs' (1967) observation that in the peripheral nervous system smaller fibers are more severely damaged by diphtheria toxin than larger fibers. Yet, our data indicate that a wide range of fiber sizes are present in the corpus callosum after LPC application. Of course, with light microscopy precise axon diameters cannot be determined, and future electron-microscopic studies of the demyelinated corpus callosum will be useful in this regard. The present study indicates that it is possible to produce focal demyelinating lesions along the unbranched axons of a fine caliber white matter tract in the mammalian cerebrum, at a site in which clinical deficits are minimal and animal survival therefore optimized. Furthermore, we would note that the rabbit corpus callosum has already been studied in detail in terms of quantitative morphology (Waxman and Swadlow 1976b) and the electrophysiology of action potential conduction (Swadlow 1974; Swadlow and Waxman 1976; Waxman and Swadlow 1976a; Kocsis et al. 1979). Moreover, in contrast to central axonal systems (Hall 1972; Blakemore 1976, 1978; Blakemore et al. 1977), and peripheral nerve (Hall and Gregson 1971; Hall 1973) previously demyelinated with LPC, the rabbit corpus callosum contains many small diameter myelinated fibers (Waxman and Swadlow 1976b) and is therefore similar to many other mammalian white matter tracts (Waxman and Swadlow 1977). The production of LPC-induced focal demyelination in the rabbit corpus callosum may therefore provide a useful model system for the examination, over protracted periods of time, of the physiological, morphological, and biochemical properties of focally demyelinated cerebral axons. ACKNOWLEDGEMENTS We thank Mary E. Smith for excellent assistance with the histological preparations and Friederike Boost for her assistance in photography.
53 REFERENCES Blakemore, W. F. (1976) Invasion of Schwann cells into the spinal cord of the rat following local injection of lysolecithin, J. Neuropath. appl. Neurobiol., 2:21 39. Blakemore, W. F. (1978) Observation on remyelination in the rat spinal cord following demyelination induced by lysolecithin, J. Neuropath. appl. NeurobioL, 4: 47-59. Blakemore, W. F., R. A. Eames, K. J. Smith and W. I. McDonald (1977) Remyelination in the spinal cord of the cat following intraspinal injections of lysolecithin, J. Neurol. Sci., 33: 31-43. Chung, S. H., S. A. Raymond and J. Y. Lettvin (1970) Multiple meaning in single visual units, Brain Behav. Evol. 3 : 7 2 101. Gledhill, R. G., B. M. Harrison and W. I. McDonald (1973) Pattern of remyelination in the CNS, Nature (Lond.), 244: 443-444. Hall, S. M. (1972) The effects of injections of lysophosphatidylcholine into white matter of the adult mouse spinal cord, J. Cell Sci., 10: 535-546. Hall, S. M. (1973) Some aspects of remyelination after demyelination produced by the intraneural injection of lysophosphatidylcholine, J. Cell. Sci., 13: 461-477. Hall, S. M. and N. A. Gregson (1971) The in vivo and ultrastructure effects of injection of lysophosphatidylcholine into myelinated peripheral nerve fibers of the adult mouse, J. CellSci., 9 : 769-789. Jacobs, J. M. (1967) Experimental diphtheritic neuropathy in the rat, Brit. J. exp. Path., 48:204 216. Kocsis, J. D., H. A. Swadlow, S. G. Waxman and M. H. Brill (1979) Variation in conduction velocity during the relative refractory and supernormal periods A mechanism for impulse entrainment in central axons, Exp. Neurol., In press. McDonald, W. 1. and T. A. Sears (1970a) Focal ex!cerimental demyelination in the central nervous system, Brain, 93:575 582. McDonald, W. 1. and T. A. Sears (1970b) The effects of exrerimental demyelination on cenducticn in the central nervous system, Brain, 93 : 583 598. Swadlow, H. A. (1974) Properties of antidromically activated callosal neurons and neurons responsive to callosal input in rabbit binocular cortex, Exp. Neurol., 43:424 444. Swadlow, H. A. and S. G. Waxman (1976) Variations in conduction velocity and excitability following single and multiple impulses of visual callosal axon in rabbit, Exp. Neurol., 53:128-150. Swadlow, H. A., S. G. Waxman and N. Geschwind (1979) Small diameter non-myelinated axons in the primate corpus callosum, Arch. Neurol. (Chic'.), In press. Waxman, S. G. and H. A. Swadlow (1976a) Morphology and physiology of visual callosal axons - Evidence for supernormal period in central myelinated axons, Brain Res., 113: 179-187. Waxman, S. G. and H. A. Swadlow (1976b) Ultrastructure of visual callosal axons in the rabbit, E.vp. Neurol., 53:115-128. Waxman, S. G. and H. A. Swadlow (1977) The conduction properties of axons in central white matter, Progr. Neurol., 8: 297-324. Wisniewski, H. and C. S. Raine (1971) An ultrastructure study of experimental demyelination and remyelination, Part 5 (Central and peripheral nervous system lesions caused by diphtheria toxin), Lab. Invest., 25: 73-80.
45
© Elsevier/North-Holland Biomedical Press
L Y S O P H O S P H A T I D Y L C H O L I N E - I N D U C E D F O C A L D E M Y E L I N A T I O N IN THE RABBIT C O R P U S C A L L O S U M Light-microscopic Observations
S. G. WAXMAN, J. D. KOCSIS and K. C. NITTA Department of Neurology, Stanford University School of Medicine and Veterans Admhlistration Medical Center, Palo Alto, CA (U.S.A.)
(Received 24 April, 1979) (Accepted 19 June, 1979)
SUMMARY The local application of lysophosphatidyl choline (LPC) by microinjection into the region of the corpus callosum of the rabbit produced demyelinating lesions. The lesions were assessed histologically using the Luxol fast blue myelin stain and the Holmes silver nitrate stain for axis cylinders. Survival times for the animals ranged from 7 to 14 days. The center of the lesion was marked by infiltration of macrophages and necrosis, but the major area of the lesion was characterized by demyelination. By consideration of anatomical factors influencing LPC diffusion and of the appropriate placement of the injection, the entire vertical extent (about 0.5 mm) of the corpus callosum could be demyelinated with minimal amounts of necrosis. Since focal demyelination was possible in the fine caliber axons of the corpus callosum which are anatomically representative of many forebrain fiber systems, and since this fiber system is amenable to chronic physiological investigation, the corpus callosum may serve as an experimental model for morpho-physiological studies of mammalian central demyelinating pathways.
INTRODUCTION Focal demyelinating lesions have been produced in the central nervous system This work was supported by Grant RG 1231 from the National Multiple Sclerosis Society. Facilities used in this study were equipped in part through grants from the Medical Research Service of the Veterans Administration and the Paralyzed Veterans of America. Address for correspondence: Stephan G. Waxman, M.D., Ph. D., Department of Neurology, Veterans Administration Medical Center, 3801 Miranda Avenue, Palo Alto, CA 94304, U.S.A.
46 (CNS) by a number of methods. The injection of diphtheria toxin (McDonald and Sears 1970a) permitted the first early studies on the physiology of conduction in demyelinated spinal cord (dorsal column) axons (McDonald and Sears 1970b). More recently, lysophosphatidyl choline (LPC) has been used to produce focal demyelinating lesions in spinal cord (Hall 1972; Blakemore et al. 1977). Another useful model of focal spinal cord demyelination has been provided through the use of transient compression (Gledhill et al. 1973). In contrast, there have been few studies of anatomically controlled focal demyelination in the mammalian cerebrum. One such study used diphtheria toxin to produce demyelination of deep cortical white matter (Wisniewski and Raine 1971). Jacobs (1967) has shown that there may be significant differences in the response of large and small myelinated fibers to demyelination with diphtheria toxin. Most previous studies on focal demyelination have been carried out in large diameter fiber systems, which may not be representative of the axons in cerebral white matter. In the present study we have applied LPC to the fine caliber fibers of the corpus callosum in the rabbit. These axons are especially well suited for study, since they are unbranched (and therefore do not exhibit the complex conduction characteristics of branched central axons (see, e.g., Chung et al. 1970)), and they present the opportunity for monitoring conduction in CNS axons for periods of several hours (Swadlow 1974; Swadlow and Waxman 1976; Waxman and Swadlow 1976a, 1977). This method, which also produces few clinical deficits and therefore obviates problems of animal survival, and has implications in terms of providing a model for studies on the characteristics of focally demyelinated CNS axons within the mammalian cerebrum. MATERIALS AND METHODS Ten adult female Dutch rabbits were used in the present study. The animals were anesthetized with sodium pentobarbital (35 mg/kg) which was injected into the marginal vein of the ear. Their heads were fixed rigidly in a rabbit head holder (David K o p f Instruments) and a small hole was drilled in the bone overlying the corpus callosum (4-6 mm posterior to bregma and 1.5 mm lateral to the midline). A small incision was made in the exposed dura to allow passage of a glass micropipette. The pipette (tip diameter of 30-50 #m) was pulled on a microelectrode puller, and was fixed to the needle of a Hamilton microsyringe. The syringe was mounted to a micromanipulator to allow for accurate placement of the pipette into the brain. A 1 ~ solution of lysophosphatidyl choline (Sigma Chemical Co.) in normal saline was injected into or near the corpus callosum (3-4 mm ventral from the cortical surface), in amounts ranging from 1/A to 4/~1, delivered over a period of 20-40 min. Upon completion of the injection the needle was left in place for 10-20 min prior to withdrawal. After removal of the pipette from the brain the dura was closed, the bone opening was sealed with dental acrylic, and the scalp incision was sutured. Two animals were injected with normal saline to serve as controls. The animals were allowed to survive from 7 to 14 days, and subsequently perfused through the left ventricle of the heart with 10 ~ buffered formalin. Paraffin
47 s e c t i o n s ( 1 0 / ~ m ) w e r e p r e p a r e d and stained with a c o m b i n a t i o n o f L u x o l fast b l u e for m y e l i n a n d the H o l m e s silver n i t r a t e m e t h o d for a x o n staining. S o m e sections were stained with h e m a t o x y l i n and eosin.
Fig. 1. A : lesion site 14 days after LPC injection (left). Normal saline was injected on the right side. Scale bar -- 0.5 mm. B: Higher magnification showing demyelinated fibers in the corpus callosum. The histological sections in this and subsequent figures stained with Luxol fast blue and Holmes silver nitrate method. Scale bar -- 0.2 mm.
48 RESULTS The spatial extent and configuration of the lesions produced from LPC injections were directly related to the volume of injected LPC and to the geometric organization of the fiber tracts near the injection site. In general, larger volumes of injected LPC induced more extensive lesions, and the spread of the lesion was also more extensive along the longitudinal axis of the fibers. The light region within the corpus callosum and fimbria shown in Fig. 1A, delineates an LPC injection site. The lighter tone of the lesion area is the result of myelin loss (absence of Luxol fast blue staining), macrophage infiltration, and the loss of some fibers. The presence of silver-stained axons within the lesion area can be seen in the higher power photomicrograph of Fig. 1B. The nuclei of macrophages can also be seen dispersed amongst the demyelinated fibers. Deep to the corpus callosum, within the dorsal aspect of the fimbria, an area of heavy macrophage infiltration with nearly complete loss of axons is present. Part of this region of necrosis can be seen in Fig. 1A, and is more completely identified in the shaded area of the camera lucida drawing of Fig. 2. Necrosis in this animal was most extensive at the section shown in Fig. 2. This area corresponds to the center of the lesion (notice the demyelinating track in the subcortical white matter marking the course of the micropipette penetration). In all of our lesions, necrosis was most prominent at the center of the lesion closest to the injection site. To minimize damage to the axons of the corpus callosum, but to maximize demyelination of these fibers, we positioned the tip of the micropipette deep to the corpus callosum into the dorsal aspect of the fimbria. At this level, 2/~1 of LPC were injected over a period of 30 min. The pipette was then moved 0.5 m m dorsally and an additional 1/~1 of LPC was injected. A third injection of 1 #l was also
$CW !' \ " " - ~ '. . . .". J~ ~.... .,, ~
......
.....
--
,.~ ~ ~ii~i:~~.../''Fig. 2. Camera lucida drawing showing central region of lesion (inside of dashed lines) and the area of necrosis (shaded). SCW, subcortical white matter; CC, corpus callosum; Fim, fimbria. Scale bar -0.5 mm.
49 applied at 0.5 mm dorsal to the second injection. A banding in the lesion indicative of the 3 injection levels is apparent in Fig. 1A. The slow application of LPC in time, and the vertical distribution of the injections (over 1 mm) maximized the demyelinating effects, but minimized necrosis within the corpus callosum. When the injections were centered dorsal to the corpus callosum, i.e., within the subcortical white matter, even
Fig. 3. A: transition zone to demyelination in the corpus callosum. Scale bar : 40/;m. B: demyelinated fibres cut in cross-section (upper part of field) in the subcortical white matter. Scale bar 16/~m.
50
Fig. 4. A: demyelinated fiber fascicles in the corpus callosum. B: a fascicle of fine caliber fibers in the corpus callosum after focal demyelination (between arrows). Scale bar for A and B = 6.0/~m.
5l large volumes of LPC ( 4 / d ) did not appreciab,y spread into the corpus callosum. Rather, the longitudinal extent of the lesion within the subcortical white matter was greater than with smaller volume injections. The transition zone between myelinated and demyelinated axons was relatively abrupt. This can be seen in the low power photomicrographs of Figs. 1A and I B, and in the higher power photomicrographs of Figs. 3A and 3B. Fascicles of longitudinally oriented myelinated fibers of the corpus callosum are present on the left of Fig. 3A. As one follows these fascicles from left to right, the dark staining myelin is lost and the increased presence of macrophage nuclei can be seen. The cross-cut fibers of the subcortical white matter also illustrates the abrupt transition to demyelination (Fig. 3B). The fibers in the upper half of Fig. 3B have maintained their general anatomical organization, i.e., their grouping into small bundles ef fibers separated by longitudinally directed axons, but they are conspicuously devoid of myelin. The integrity of the fiber fascicles after demyelination can also be seen in Fig. 4A. Discrete fiber bundles composed of axons with a variety of diameters are present, and macrophage nuclei are distributed between these fiber bundles. A grouping of especially fine caliber fibers is shown in Fig. 4B. Many of these fibers were di~cult to photograph in a single plane because of their extremely fine caliber and their tortuous course through the corpus callosum. Injections of normal saline delivered in the same manner as the LPC solution injections, did not produce noticeable damage in our experiments. The corpus callosal region contralateral to the LPC injection site in Fig. 1A was injected with 4/~l of normal saline, but virtually no evidence of tissue damage could be found in the Luxol fast blue-Holmes silver nitrate sections. The visibility of the electrode track on the side of the LPC injection was due primarily to the demyelinating effects of LPC leakage (Fig. 1A). DISCUSSION The present studies show that it is possible to produce, by microinjection of small amounts of LPC through glass micropipettes, focal demyelinating lesions in the corpus callosum of the rabbit. A variable amount of necrosis was found nearest the center of the lesion as has been reported following microinjections of diphtheria toxin in spinal cord (McDonald and Sears 1970a) and of LPC in peripheral nerve and spinal cord (Hall and Gregson 1971; Hall 1972; Blakemore et al. 1977). Since the dorsoventral extent of the corpus callosum is only about 0.6 ram, direct injections into this fiber bundle are not desirable because of the necrosis that would occur at the lesion center. In order to minimize necrosis and to maximize demyelination in the corpus callosum the injections were centered deep to the corpus callosum in the dorsal aspect of the fimbria. After an injection at this site the pipette was moved dorsally and smaller volume injections were also made. The center of necrosis was established, by this injection regimen, in the fimbria and the entire dorsal-ventral extent of the corpus callosum was demyelinated.
52 The diffusion of the LPC appeared very dependent upon the anatomical orientation of the fibers, with spread of the lesion being more pronounced along the longitudinal axis of the fibers. Furthermore, at regions where fibers change direction, such as between the orthogonally oriented fibers of the corpus callosum and the subcortical white matter, there is an apparent barrier or at least impediment to diffusion. This is demonstrated by the difficulty with which LPC located in the corpus callosum will spread dorsally into the subcortical white matter (e.g., notice the relatively sharp border of the demyelinated corpus callosum fibers with the overlying white matter in Fig. 1A). As we have shown previously in the rabbit (Waxman and Swadlow 1976b) and primate (Swadlow et al. 1979), the splenium of the corpus callosum contains relatively small diameter myelinated fibers, with more than 50 ~o of fibers smaller than 0.85/zm and more than 90~o of fibers smaller than 1.5/~m in the rabbit. We were initially concerned that the production of focal demyelinating lesions in rabbit corpus callosum might not be possible in view of Jacobs' (1967) observation that in the peripheral nervous system smaller fibers are more severely damaged by diphtheria toxin than larger fibers. Yet, our data indicate that a wide range of fiber sizes are present in the corpus callosum after LPC application. Of course, with light microscopy precise axon diameters cannot be determined, and future electron-microscopic studies of the demyelinated corpus callosum will be useful in this regard. The present study indicates that it is possible to produce focal demyelinating lesions along the unbranched axons of a fine caliber white matter tract in the mammalian cerebrum, at a site in which clinical deficits are minimal and animal survival therefore optimized. Furthermore, we would note that the rabbit corpus callosum has already been studied in detail in terms of quantitative morphology (Waxman and Swadlow 1976b) and the electrophysiology of action potential conduction (Swadlow 1974; Swadlow and Waxman 1976; Waxman and Swadlow 1976a; Kocsis et al. 1979). Moreover, in contrast to central axonal systems (Hall 1972; Blakemore 1976, 1978; Blakemore et al. 1977), and peripheral nerve (Hall and Gregson 1971; Hall 1973) previously demyelinated with LPC, the rabbit corpus callosum contains many small diameter myelinated fibers (Waxman and Swadlow 1976b) and is therefore similar to many other mammalian white matter tracts (Waxman and Swadlow 1977). The production of LPC-induced focal demyelination in the rabbit corpus callosum may therefore provide a useful model system for the examination, over protracted periods of time, of the physiological, morphological, and biochemical properties of focally demyelinated cerebral axons. ACKNOWLEDGEMENTS We thank Mary E. Smith for excellent assistance with the histological preparations and Friederike Boost for her assistance in photography.
53 REFERENCES Blakemore, W. F. (1976) Invasion of Schwann cells into the spinal cord of the rat following local injection of lysolecithin, J. Neuropath. appl. Neurobiol., 2:21 39. Blakemore, W. F. (1978) Observation on remyelination in the rat spinal cord following demyelination induced by lysolecithin, J. Neuropath. appl. NeurobioL, 4: 47-59. Blakemore, W. F., R. A. Eames, K. J. Smith and W. I. McDonald (1977) Remyelination in the spinal cord of the cat following intraspinal injections of lysolecithin, J. Neurol. Sci., 33: 31-43. Chung, S. H., S. A. Raymond and J. Y. Lettvin (1970) Multiple meaning in single visual units, Brain Behav. Evol. 3 : 7 2 101. Gledhill, R. G., B. M. Harrison and W. I. McDonald (1973) Pattern of remyelination in the CNS, Nature (Lond.), 244: 443-444. Hall, S. M. (1972) The effects of injections of lysophosphatidylcholine into white matter of the adult mouse spinal cord, J. Cell Sci., 10: 535-546. Hall, S. M. (1973) Some aspects of remyelination after demyelination produced by the intraneural injection of lysophosphatidylcholine, J. Cell. Sci., 13: 461-477. Hall, S. M. and N. A. Gregson (1971) The in vivo and ultrastructure effects of injection of lysophosphatidylcholine into myelinated peripheral nerve fibers of the adult mouse, J. CellSci., 9 : 769-789. Jacobs, J. M. (1967) Experimental diphtheritic neuropathy in the rat, Brit. J. exp. Path., 48:204 216. Kocsis, J. D., H. A. Swadlow, S. G. Waxman and M. H. Brill (1979) Variation in conduction velocity during the relative refractory and supernormal periods A mechanism for impulse entrainment in central axons, Exp. Neurol., In press. McDonald, W. 1. and T. A. Sears (1970a) Focal ex!cerimental demyelination in the central nervous system, Brain, 93:575 582. McDonald, W. 1. and T. A. Sears (1970b) The effects of exrerimental demyelination on cenducticn in the central nervous system, Brain, 93 : 583 598. Swadlow, H. A. (1974) Properties of antidromically activated callosal neurons and neurons responsive to callosal input in rabbit binocular cortex, Exp. Neurol., 43:424 444. Swadlow, H. A. and S. G. Waxman (1976) Variations in conduction velocity and excitability following single and multiple impulses of visual callosal axon in rabbit, Exp. Neurol., 53:128-150. Swadlow, H. A., S. G. Waxman and N. Geschwind (1979) Small diameter non-myelinated axons in the primate corpus callosum, Arch. Neurol. (Chic'.), In press. Waxman, S. G. and H. A. Swadlow (1976a) Morphology and physiology of visual callosal axons - Evidence for supernormal period in central myelinated axons, Brain Res., 113: 179-187. Waxman, S. G. and H. A. Swadlow (1976b) Ultrastructure of visual callosal axons in the rabbit, E.vp. Neurol., 53:115-128. Waxman, S. G. and H. A. Swadlow (1977) The conduction properties of axons in central white matter, Progr. Neurol., 8: 297-324. Wisniewski, H. and C. S. Raine (1971) An ultrastructure study of experimental demyelination and remyelination, Part 5 (Central and peripheral nervous system lesions caused by diphtheria toxin), Lab. Invest., 25: 73-80.