Neurochemical Research, VoL 15, No. 11, 1990, pp. 1051-1053
Lipid Changes in Central Nervous System Membranes in Experimental Allergic Encephalomyelitis (EAE) Serafina Salvati 1,2, Lucilla Attorri 1, Annamaria Confaloni 1, and Antonella Di Biase I (AcceptedAugust 1, 1990)
Lipid composition of myelin fractions isolated from Lewis rats during the early stage of the development of experimental allergic encephalomyelitis (EAE) were determined by high-performance thin layer chromatography (HPTLC). When comparing the myelin fractions of EAE-affected animals with those of controls, the main differences were observed in the light fraction, where a decrease in the percentage of phospholipids (PH) relative to the total lipids was observed. These findings give further support that the light myelin fraction being the most sensitive at the onset of clinical symptoms must play a key role in demyelinating process. KEY WORDS: Myelin; EAE; CNS; rat.
also affect the insertion of basic proteins in the membrane and consequently its structure. In Lewis rats, with experimental allergic encephalomyelitis (EAE), an autoimmune demyelinating disease, we found in the light fraction, isolated at the onset of clinical symptoms, a morphological aspect similar to that observed in the nutritional experiments described above. In opposition to those experiments, the amount of BP in test animals was lower compared to controls (5). These observations strengthen the opinion that BP does not have a structural role (6). In this paper we analyze the lipid composition of myelin fractions isolated from EAE-affected rats in order to study their role in the morphological and biochemical changes observed.
INTRODUCTION Our previous studies (1,2) have shown that manipulation of dietary lipids induces an accelerated behavioral development. The alterations in functional patterns were related to biochemical and morphological changes in the light myelin fraction (3) isolated by density gradient centrifugation according to Agrawal (4). Electron microscopy observations showed that in test animals this fraction is composed of small vesicles, whereas in the controls the light fraction is composed mainly of large multilayered membranes. Biochemical data showed that the amount of basic protein relative to total proteins increases in test animals. Monolayer experiments (3) have suggested that dietary lipids by influencing the chemical composition of myelin lipids can t Istituto Superioredi Sanita, Roma, Italy. To whom to address reprint requests. NeurochemistrySection, Dept. Metabolismand PathoIogicalBiochemistry,IstitutoSuperioredi Sanitfi, Viale Regina Elena, 299,00161, Roma, Italy. Abbreviations used: EAE, experimentalallergicencephalomyelitis; PH, phospholipids;CH, cholesterol;GL, galactolipid; PE, phosphatidylethanolamine; SPM, sphingomyelin;PC, phosphatidylcholine;PS, phosphatidylserine; PI, phosphatidylinositol;CB, cerebroside;CB-OH, hydroxy-cerebroside;SULF, sulfatides; BP, basic proteins
E X P E R I M E N T A L PROCEDURE Animals. Ten adultmale Lewisrats (CharlesRiver)weighing200250 g at the beginningof the experimentwere used. To induct EAE, 5 rats were inoculatedwith injectionsof a) 0.2 ml of emulsionof Sprague-Dawleyrat spinal cord in completeFreund's adjuvant into the footpadsof the hindlimbs, and b) 0.1 ml of Bordetella
1051 0364-3190/90/1100-1051506.00/0 9 1990 Plenum Publishing Corporation
1052 pertussis culture (20• 10 cells/ml; Institut Merieux, Lyon France), also into the hindlimbs. Five control rats concurrently received footpad injections of complete Freund's adjuvant alone and Bordetella pertussis
(7). All rats were killed by decapitation I0 days postinoculation. The brains were quickly removed and weighed. Four independent fractions experiments were conducted. Isolation of Myelin Fractions. Myelin preparations were isolated from fresh tissue essentially by the procedure of Norton and Poduslo (8). Fractions of the whole myelin was done according to Agrawal (4). The myelin pellet was dispersed in 0.32 M sucrose and distributed equally into freshly made discontinuous gradients, consisting of 0.55 M sucrose layered over 0.75 M, 0.85 M and 0.88 M sucrose solution. The myelin was then centrifuged at 75,000g per 60 min, using a SW 27.2 rotor in a Beckman L8-70 ultracentrifuge. Three bands and a pellet were obtained: a light fraction floating on 0.55 M sucrose, a heavy fraction on 0.75 M sucrose, a membrane fraction on 0.85 M sucrose and a pellet. The corresponding fractions from each of the mixture were combined, dispersed in distilled water, and centrifuged at 12,000 g during 20 rain, to remove the sucrose. This last step was repeated three times. Lyophilized subfractions were stored at 20~ Lipid analysis. Lipids were extracted from myelin fractions with 20 vol of chloroform-methanol (2:1 v/v) according to Folch et al. (9). Lipids were separated in classes by one-dimensional HPTLC and quantified by in situ laser densitometry (md.XL-LKB-Sweden) after staining with specific reagents or phospholipids (PH), cholesterol (CH), and galactolipid (GL) as previously described (10). Peak areas were obtained by an internal digital integrator.
RESULTS AND DISCUSSION The majority of the EAE-affected rats (80%) develop clinical signs at 10 days p.i. The neurological signs involve prevalently a distal tail weakness and a mild paraparesis of hindlimbs. There are no body-weight differences between the test animals and controls. Thus, the biochemical changes observed are solely due to EAE process and other side-effects such as starvation are to be excluded. The lipid composition of myelin fractions is shown in Table I. The major changes are present in the light fraction of EAE-affected rats. In this myelin fraction the phospholipids percentage is reduced because of the significant decrease of SPM, PS + PI, PE. These results confirm our previous biochemical study on CR-EAE affected rats, where the PH changes were mainly located in the light fraction of myelin isolated at various time points. But on the contrary, our previous experiments, the PH changes were due only to the decrease of PE. In this present study, we observe that the lower PH is in relation with the decrease of all PH classes. In EAE the alteration in PH composition is probably so marked because of the acute monophasic nature of the disease. In the CR-EAE experimental model, characterized by recovery and relapse periods, the changes are
Salvati, Attorri, Confaloni, and Di Biase less pronounced, since the remyelinating process that occurs during the course of the disease (12) can balance the demyetinating process. The light fraction shows differences also in galactolipids content. In the test group the percentage is higher because of the significant increase of CB-OH. The decrease of sulfatides percentage is not significant. Although monolayer experiments showed high specific interaction between sulfatides and BP (13), our experimental data make it difficult to correlate the low amount of BP with the slight decrease of sulfatides. These data support the paper of Tennekoon et al. (14) in demonstrating that the sulfatides are probably not the acidic lipids with which BP interacts in vivo, since they have different topological distribution in the membrane. Sulfatides are located at the external surface, while the BP is at the cytoplasmatic side (15). Immunochemical studies have provided evidence that also PE, PS, and PI are located at the cytoplasmatic side (16). Since the lipid changes in EAE-affected rats are so marked in PH percentage, we therefore propose that in vivo their charged polar groups may be important for the interaction with BP. Modification of some of this myelin constituents, probably perturbing intermolecular reactions and ionic permeability, may affect the structure and the function of membranes. These findings support the hypothesis that the morphological aspect is not dependent on the single myelin constituents, but rather on an alteration of lipid/protein or lipid/lipid ratio. No differences are present in cholesterol percentage in both fractions. The percentage of PH and GL in EAE-affected rats does not show significant differences in the heavy fraction when compared to controls. In this study it is difficult to suggest if the observed differences in EAE may be the first consequence of specific disruptive responses of cellular or humoral immunological agents originated in response to encephalitogenic mixture. We can only strengthen the opinion that the light fraction being the most sensitive at the early stages of the disease, must play a key role in triggering demyelinating disease.
ACKNOWLEDGMENTS We gratefully acknowledge the excellent technical assistance of L. Malvezzi Campeggi. This work was supported in part by CNR through "Progetto Finalizzato Invecchiamento".
Changes in Light Myelin Fraction in EAE
1053
Table I. Lipid Composition of Myelin Fractions
Light
Heavy
c (3) SPM PC PS + PI PE SULF CB-OH CB CH PH GL
6.2 18.2 10.3 15.3 5.1 15.5 10.1 19.2 49.9 30.8
• • • • • _ • • • •
c (3)
T (4) 0.5 1.5 1.1 1.0 0.1 1.3 0.9 3.7 2.4 2.1
4.2 16.3 7.9 12.5 4.2 19.6 11.6 23.2 40.9 35.4
• 0.6* -+ 2.7 -- 1.4'* • 2.4** _ 1.1 • 0.9* • 2.6 • 5.9 +_ 3.4*** _+ 4.0
3.6 18.8 8.4 12.5 4.2 18.9 8.4 25.3 43.4 31.4
-+ • -+ • -+ • +_ -+ •
1.0 5.8 1.8 1.8 0.7 7.6 3.0 4.3 7.5 10.1
T (4) 5.5 20.9 7.9 11.2 4.2 19.4 7.3 23.4 45.5 31.0
• 1.3 -. 2.4 +- 1.4 -+ 3.6 +_ 1.4 - 3.4 - 2.5 - 4.7 • 6.9 -+ 5.0
C, Control; T, EAE Values (means -+ SD) are expressed as percentage of total lipids. Numbers in parentheses represent number of cases * p < 0.05; ** p < 0.5; *** p < 0.1; (Student's t test).
REFERENCES 1. Gozzo, S., OIiverio, A., Salvati, S., Serlupi Crescenzi, G., Tagliamonte, B. and Tomassi, G. 1978. Nutritional studies of the lipid fraction of n-alkane grown yeast. IV Effects on behavioral development. Nutr. Rep. Intern. 17:357-366. 2. Gozzo, S., Oliverio, A., Salvati, S., Serlupi Crescenzi, G., Tagliamonte, B. and Tomassi, G. 1982. Effects of dietary phospholipids and odd-chain fatty acids on the behavioral maturation of mice. Food Chem. Toxicol. 20:151-157. 3. Salvati, S., Conti DeVergiliis, L., Di Felice, M., de Gier, J., DemeI, R. A., Serlupi Crescenzi, G., Tomassi, G. and Tagliamonte, B. 1984. Morphological and biochemical changes in myelin subfractions of developing rats fed microbial lipids. J. Neurochem. 42:634-643. 4. Agrawal, H. C., Trotter, L L., Burton, R. M. and Mitchell, R. F. 1974. Metabolic studies on myelin. Evidence of a precursor role of a myelin subfraction. Biochem. J. 140:99-109. 5. Salvati, S., D'Urso, D., Conti DeVergiliis, L., Serlupi Crescenzi, G. 1986. Biochemical changes in central nervous system membranes il~ experimental allergic encephalomyelitis, J. Neurochem. 47:239-24,~. 6. Ganser, A. L., .,nd Kirschner, D. A. 1980. Myelin structure in the absence of basic protein in the shiverer mouse: Pages 171176, in Neurological mutations affecting myelination (N. Baumann ed.) Elsevier-NortbHolland Biomedical Press. 7. Levine, S., and Wenk, E. J. 1965. A hyperacute form of allergic encephalomyelitis. Am. J. Pathol. 47:61-88.
8. Norton, W. T. and Poduslo, S. E. 1973. Myelination in rat brain: method of myelin isolation. J. Neurochem. 21:749-757. 9. Folch, J., Lees, M., and Sloane-Stanley, G. H. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. 10. Di Blase, A., Salvati, S., and Serlupi Crescenzi, G. 1989. Analysis of brain and myelin lipids by high-performance thin layer chromatography and densitometry. Neurochem. Res. 14:153-156. 11. Salvati, S., Confaloni, A., Di Blase, A., Attorri, L., and Serlupi Crescenzi, G. 1990. Biochemical changes in central nervous system membranes in chronic-relapsing experimental allergic encephalomyelitis. Mol. Chem. Neuropathol. in press. 12. Ludwin, S. K. 1988. Remyelination in the central nervous system and the peripheral nervous system. Adv. Neurol. 47:215-254. 13. London, Y., and Wenk, E. J. (1973) Specific interaction of central nervous system myelin basic protein with lipids. Biochim. Biophys. Acta 307:478-482. 14. Tennekoon, G., Zavenba, M., and Walinsky, J. 1983. Topography of cerebroside sulfatransferase in Golgi-enriched vesicles from rat brain. 97:1107-1112. 15. Omlin, F. X., Webster, H. deF., Polkovits, C. G. and Cohen, S. R. 1982. ImmunocytochemicaI localization of basic protein in major dense line regions of central and peripheral myelin. J. Cell Biol. 95:242-247. 16. Rumsby, M. G. 1987. Structural organization and stability of central nervous system myelin. Pages 111-132, in A multidisciplinary approach to myelin diseases (Serlupi-Crescenzi, G. ed.), Vol. 142, Nato ASI series plenum press N.Y.
Lipid Changes in Central Nervous System Membranes in Experimental Allergic Encephalomyelitis (EAE) Serafina Salvati 1,2, Lucilla Attorri 1, Annamaria Confaloni 1, and Antonella Di Biase I (AcceptedAugust 1, 1990)
Lipid composition of myelin fractions isolated from Lewis rats during the early stage of the development of experimental allergic encephalomyelitis (EAE) were determined by high-performance thin layer chromatography (HPTLC). When comparing the myelin fractions of EAE-affected animals with those of controls, the main differences were observed in the light fraction, where a decrease in the percentage of phospholipids (PH) relative to the total lipids was observed. These findings give further support that the light myelin fraction being the most sensitive at the onset of clinical symptoms must play a key role in demyelinating process. KEY WORDS: Myelin; EAE; CNS; rat.
also affect the insertion of basic proteins in the membrane and consequently its structure. In Lewis rats, with experimental allergic encephalomyelitis (EAE), an autoimmune demyelinating disease, we found in the light fraction, isolated at the onset of clinical symptoms, a morphological aspect similar to that observed in the nutritional experiments described above. In opposition to those experiments, the amount of BP in test animals was lower compared to controls (5). These observations strengthen the opinion that BP does not have a structural role (6). In this paper we analyze the lipid composition of myelin fractions isolated from EAE-affected rats in order to study their role in the morphological and biochemical changes observed.
INTRODUCTION Our previous studies (1,2) have shown that manipulation of dietary lipids induces an accelerated behavioral development. The alterations in functional patterns were related to biochemical and morphological changes in the light myelin fraction (3) isolated by density gradient centrifugation according to Agrawal (4). Electron microscopy observations showed that in test animals this fraction is composed of small vesicles, whereas in the controls the light fraction is composed mainly of large multilayered membranes. Biochemical data showed that the amount of basic protein relative to total proteins increases in test animals. Monolayer experiments (3) have suggested that dietary lipids by influencing the chemical composition of myelin lipids can t Istituto Superioredi Sanita, Roma, Italy. To whom to address reprint requests. NeurochemistrySection, Dept. Metabolismand PathoIogicalBiochemistry,IstitutoSuperioredi Sanitfi, Viale Regina Elena, 299,00161, Roma, Italy. Abbreviations used: EAE, experimentalallergicencephalomyelitis; PH, phospholipids;CH, cholesterol;GL, galactolipid; PE, phosphatidylethanolamine; SPM, sphingomyelin;PC, phosphatidylcholine;PS, phosphatidylserine; PI, phosphatidylinositol;CB, cerebroside;CB-OH, hydroxy-cerebroside;SULF, sulfatides; BP, basic proteins
E X P E R I M E N T A L PROCEDURE Animals. Ten adultmale Lewisrats (CharlesRiver)weighing200250 g at the beginningof the experimentwere used. To induct EAE, 5 rats were inoculatedwith injectionsof a) 0.2 ml of emulsionof Sprague-Dawleyrat spinal cord in completeFreund's adjuvant into the footpadsof the hindlimbs, and b) 0.1 ml of Bordetella
1051 0364-3190/90/1100-1051506.00/0 9 1990 Plenum Publishing Corporation
1052 pertussis culture (20• 10 cells/ml; Institut Merieux, Lyon France), also into the hindlimbs. Five control rats concurrently received footpad injections of complete Freund's adjuvant alone and Bordetella pertussis
(7). All rats were killed by decapitation I0 days postinoculation. The brains were quickly removed and weighed. Four independent fractions experiments were conducted. Isolation of Myelin Fractions. Myelin preparations were isolated from fresh tissue essentially by the procedure of Norton and Poduslo (8). Fractions of the whole myelin was done according to Agrawal (4). The myelin pellet was dispersed in 0.32 M sucrose and distributed equally into freshly made discontinuous gradients, consisting of 0.55 M sucrose layered over 0.75 M, 0.85 M and 0.88 M sucrose solution. The myelin was then centrifuged at 75,000g per 60 min, using a SW 27.2 rotor in a Beckman L8-70 ultracentrifuge. Three bands and a pellet were obtained: a light fraction floating on 0.55 M sucrose, a heavy fraction on 0.75 M sucrose, a membrane fraction on 0.85 M sucrose and a pellet. The corresponding fractions from each of the mixture were combined, dispersed in distilled water, and centrifuged at 12,000 g during 20 rain, to remove the sucrose. This last step was repeated three times. Lyophilized subfractions were stored at 20~ Lipid analysis. Lipids were extracted from myelin fractions with 20 vol of chloroform-methanol (2:1 v/v) according to Folch et al. (9). Lipids were separated in classes by one-dimensional HPTLC and quantified by in situ laser densitometry (md.XL-LKB-Sweden) after staining with specific reagents or phospholipids (PH), cholesterol (CH), and galactolipid (GL) as previously described (10). Peak areas were obtained by an internal digital integrator.
RESULTS AND DISCUSSION The majority of the EAE-affected rats (80%) develop clinical signs at 10 days p.i. The neurological signs involve prevalently a distal tail weakness and a mild paraparesis of hindlimbs. There are no body-weight differences between the test animals and controls. Thus, the biochemical changes observed are solely due to EAE process and other side-effects such as starvation are to be excluded. The lipid composition of myelin fractions is shown in Table I. The major changes are present in the light fraction of EAE-affected rats. In this myelin fraction the phospholipids percentage is reduced because of the significant decrease of SPM, PS + PI, PE. These results confirm our previous biochemical study on CR-EAE affected rats, where the PH changes were mainly located in the light fraction of myelin isolated at various time points. But on the contrary, our previous experiments, the PH changes were due only to the decrease of PE. In this present study, we observe that the lower PH is in relation with the decrease of all PH classes. In EAE the alteration in PH composition is probably so marked because of the acute monophasic nature of the disease. In the CR-EAE experimental model, characterized by recovery and relapse periods, the changes are
Salvati, Attorri, Confaloni, and Di Biase less pronounced, since the remyelinating process that occurs during the course of the disease (12) can balance the demyetinating process. The light fraction shows differences also in galactolipids content. In the test group the percentage is higher because of the significant increase of CB-OH. The decrease of sulfatides percentage is not significant. Although monolayer experiments showed high specific interaction between sulfatides and BP (13), our experimental data make it difficult to correlate the low amount of BP with the slight decrease of sulfatides. These data support the paper of Tennekoon et al. (14) in demonstrating that the sulfatides are probably not the acidic lipids with which BP interacts in vivo, since they have different topological distribution in the membrane. Sulfatides are located at the external surface, while the BP is at the cytoplasmatic side (15). Immunochemical studies have provided evidence that also PE, PS, and PI are located at the cytoplasmatic side (16). Since the lipid changes in EAE-affected rats are so marked in PH percentage, we therefore propose that in vivo their charged polar groups may be important for the interaction with BP. Modification of some of this myelin constituents, probably perturbing intermolecular reactions and ionic permeability, may affect the structure and the function of membranes. These findings support the hypothesis that the morphological aspect is not dependent on the single myelin constituents, but rather on an alteration of lipid/protein or lipid/lipid ratio. No differences are present in cholesterol percentage in both fractions. The percentage of PH and GL in EAE-affected rats does not show significant differences in the heavy fraction when compared to controls. In this study it is difficult to suggest if the observed differences in EAE may be the first consequence of specific disruptive responses of cellular or humoral immunological agents originated in response to encephalitogenic mixture. We can only strengthen the opinion that the light fraction being the most sensitive at the early stages of the disease, must play a key role in triggering demyelinating disease.
ACKNOWLEDGMENTS We gratefully acknowledge the excellent technical assistance of L. Malvezzi Campeggi. This work was supported in part by CNR through "Progetto Finalizzato Invecchiamento".
Changes in Light Myelin Fraction in EAE
1053
Table I. Lipid Composition of Myelin Fractions
Light
Heavy
c (3) SPM PC PS + PI PE SULF CB-OH CB CH PH GL
6.2 18.2 10.3 15.3 5.1 15.5 10.1 19.2 49.9 30.8
• • • • • _ • • • •
c (3)
T (4) 0.5 1.5 1.1 1.0 0.1 1.3 0.9 3.7 2.4 2.1
4.2 16.3 7.9 12.5 4.2 19.6 11.6 23.2 40.9 35.4
• 0.6* -+ 2.7 -- 1.4'* • 2.4** _ 1.1 • 0.9* • 2.6 • 5.9 +_ 3.4*** _+ 4.0
3.6 18.8 8.4 12.5 4.2 18.9 8.4 25.3 43.4 31.4
-+ • -+ • -+ • +_ -+ •
1.0 5.8 1.8 1.8 0.7 7.6 3.0 4.3 7.5 10.1
T (4) 5.5 20.9 7.9 11.2 4.2 19.4 7.3 23.4 45.5 31.0
• 1.3 -. 2.4 +- 1.4 -+ 3.6 +_ 1.4 - 3.4 - 2.5 - 4.7 • 6.9 -+ 5.0
C, Control; T, EAE Values (means -+ SD) are expressed as percentage of total lipids. Numbers in parentheses represent number of cases * p < 0.05; ** p < 0.5; *** p < 0.1; (Student's t test).
REFERENCES 1. Gozzo, S., OIiverio, A., Salvati, S., Serlupi Crescenzi, G., Tagliamonte, B. and Tomassi, G. 1978. Nutritional studies of the lipid fraction of n-alkane grown yeast. IV Effects on behavioral development. Nutr. Rep. Intern. 17:357-366. 2. Gozzo, S., Oliverio, A., Salvati, S., Serlupi Crescenzi, G., Tagliamonte, B. and Tomassi, G. 1982. Effects of dietary phospholipids and odd-chain fatty acids on the behavioral maturation of mice. Food Chem. Toxicol. 20:151-157. 3. Salvati, S., Conti DeVergiliis, L., Di Felice, M., de Gier, J., DemeI, R. A., Serlupi Crescenzi, G., Tomassi, G. and Tagliamonte, B. 1984. Morphological and biochemical changes in myelin subfractions of developing rats fed microbial lipids. J. Neurochem. 42:634-643. 4. Agrawal, H. C., Trotter, L L., Burton, R. M. and Mitchell, R. F. 1974. Metabolic studies on myelin. Evidence of a precursor role of a myelin subfraction. Biochem. J. 140:99-109. 5. Salvati, S., D'Urso, D., Conti DeVergiliis, L., Serlupi Crescenzi, G. 1986. Biochemical changes in central nervous system membranes il~ experimental allergic encephalomyelitis, J. Neurochem. 47:239-24,~. 6. Ganser, A. L., .,nd Kirschner, D. A. 1980. Myelin structure in the absence of basic protein in the shiverer mouse: Pages 171176, in Neurological mutations affecting myelination (N. Baumann ed.) Elsevier-NortbHolland Biomedical Press. 7. Levine, S., and Wenk, E. J. 1965. A hyperacute form of allergic encephalomyelitis. Am. J. Pathol. 47:61-88.
8. Norton, W. T. and Poduslo, S. E. 1973. Myelination in rat brain: method of myelin isolation. J. Neurochem. 21:749-757. 9. Folch, J., Lees, M., and Sloane-Stanley, G. H. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. 10. Di Blase, A., Salvati, S., and Serlupi Crescenzi, G. 1989. Analysis of brain and myelin lipids by high-performance thin layer chromatography and densitometry. Neurochem. Res. 14:153-156. 11. Salvati, S., Confaloni, A., Di Blase, A., Attorri, L., and Serlupi Crescenzi, G. 1990. Biochemical changes in central nervous system membranes in chronic-relapsing experimental allergic encephalomyelitis. Mol. Chem. Neuropathol. in press. 12. Ludwin, S. K. 1988. Remyelination in the central nervous system and the peripheral nervous system. Adv. Neurol. 47:215-254. 13. London, Y., and Wenk, E. J. (1973) Specific interaction of central nervous system myelin basic protein with lipids. Biochim. Biophys. Acta 307:478-482. 14. Tennekoon, G., Zavenba, M., and Walinsky, J. 1983. Topography of cerebroside sulfatransferase in Golgi-enriched vesicles from rat brain. 97:1107-1112. 15. Omlin, F. X., Webster, H. deF., Polkovits, C. G. and Cohen, S. R. 1982. ImmunocytochemicaI localization of basic protein in major dense line regions of central and peripheral myelin. J. Cell Biol. 95:242-247. 16. Rumsby, M. G. 1987. Structural organization and stability of central nervous system myelin. Pages 111-132, in A multidisciplinary approach to myelin diseases (Serlupi-Crescenzi, G. ed.), Vol. 142, Nato ASI series plenum press N.Y.
