Brain Research, 521 (1990) 343-346 Elsevier
343
BRES 24146
Differential induction of class I and II MHC antigen expression by degenerating myelinated and unmyelinated axons Ava M. Smetanka, Kathleen T. Yee and Raymond D. Lund Department of Neurobiology, Anatomy and Cell Science, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261 (U.S.A.)
(Accepted 13 March 1990)
Key words: Cerebellum; Microglia; Macrophage; Myelin; Degeneration The brain has long been considered an immunologically privileged site since immunogenetically mismatched grafts that are normally rejected peripherally, frequently survive for prolonged periods after transplantation to the central nervous system 4'5'9. One factor that may contribute to immune privilege in the brain is the absence under normal circumstances of cells expressing major histocompatability complex (MHC) antigens, which function to present foreign antigens to lymphocytes6'13A5-17. However, a variety of conditions have been identified in which cells of the brain are induced to express MHC antigens, and this can lead to rejection of neural grafts in the region of antigen-presenting cellss,n. A neural lesion is one of the stimuli that induces MHC antigen expression, not only around the site of the lesion itself2'3, but also along the course of the damaged fiber tracts and in terminal areas 12'18. Studies examining the induction of MHC antigen expression in the primary optic pathway following eye lesions have shown a differential distribution of MHC antigen expression. While class I MHC antigens were expressed on cells located both within the myelinated fiber region and in terminal distribution areas, class II MHC antigen expressing cells were mainly localized within the myelinated fiber region 12,18. This observation led to the hypothesis that class II MHC antigen expression may be associated with degenerating myelin. To test this hypothesis, we have examined the effects of small lesions of the cerebellar cortex. Superficial lesions were made to damage only the unmyelinated axons of the parallel fiber system. Deeper lesions were made to damage not only the superficial unmyelinated axons, but also the cerebellar white matter, permitting simultaneous examination of degenerating myelinated and unmyelinated pathways. If the hypothesis is correct, class II MHC antigens should not be induced on cells in the molecular layer by degenerating parallel fibers, but
should be induced in the white matter after deeper lesions. Adult Sprague-Dawley rats (4-8 months old, n = 50) received small lesions of the cerebellar cortex made in a sagittal plane with a 26 gauge needle. The rats were perfused with zinc-aldehyde fixative 1° after surviving for 1 to 9 days. The brains were cryoprotected in 30% sucrose in phosphate-buffered saline (PBS) and 40/~m-thick coronal sections were cut through the cerebellum. Selected sections were stained with Cresyl violet to localize the lesion site. Adjacent sections containing the lesion were then stained with the following antibodies: OX-6 (directed against class II MHC antigens), OX-18 (against class I MHC antigens), OX-42 (against microglia and monocytes) (Accurate Chemical and Scientific Corporation, Westbury, NY); ED-2 (against macrophages) (Bioproducts for Science, Inc., Indianapolis, IN); and GFAP (against fibriUar astrocytes) (Dako Corporation, Santa Barbara, CA). Antibody binding was demonstrated using a horseradish peroxidase-conjugated secondary antibody (American Qualex International, Inc., La Mirada, CA) and a nickel-intensified diaminobenzidine reaction 1. Equivalent sections of cerebellum from age-matched normal rats were stained with the same battery of antibodies to serve as controls. No MHC antigen expression was seen in the cerebellum of normal young adult rats. In all lesioned animals, class I MHC antigen expression was seen, both on cells around the lesion site and extending along the molecular layer up to a distance of 24 mm away from the lesion site (Fig. 1B). There were two principal types of cells expressing class I MHC antigens. One of these types morphologically resembled microglia (Fig. 2C), and was similar in appearance to those stained with OX-42 (Fig. 2D). The second type of cell was preferentially localized around the lesion site and resembled those cells stained
Correspondence: A.M. Smetanka, Department of Neurobiology, Anatomy and Cell Science, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261 (U.S.A.). 0006-8993/90/$03.50 (~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
344 with ED-2 (Fig. 2B). They were presumed to be macrophages. Class I MHC positive staining cells were seen within the white matter and in the deep cerebellar nuclei, after white matter lesions (Fig. 2F). After superficial lesions to the molecular layer, the staining of class lI MHC positive cells was very different from that seen with class I MHC antigen staining with respect to density, distribution and cell class. While large numbers of MHC class I staining cells were distributed for as much as 24 mm along the molecular layer (Fig. 1B), most MHC class II positive cells were localized close to the lesion site and were fewer in number (Fig. 1A). Most of the cells staining with MHC class II antibody were large elliptical cells with small processes arising from the cell body (Fig. 2A), and were thought to be macrophages because of their similar morphology to cells stained with ED-2 (Fig. 2B). These cells were localized mainly around the lesion site, but a few were seen in the molecular layer extending as far as 10 mm away from the lesion site, occasionally bordering the Purkinje cell layer. It was not clear whether cells along the Purkinje cell layer had migrated there, or were resident cells that had been induced to express MHC antigens because of damage to Purkinje cell axons. The distance at which stained cells could be seen from the lesion site was measured; OX-6 extent of staining (£ = 4.4 mm _+ 2.4), and OX-18 extent of staining (£ = 11.1 mm -+ 4.9). Using a paired t-test, a statistically significant difference was found between the extent of OX-6 and OX-18 staining (n = 50; P < 0.01). Cells in the molecular layer resembling microglia were not stained. Following a 5-day postlesion survival, a few cells with fine processes were seen; this type of cell was not present at any other survival time. These cells had processes that generally radiated away from the lesion site, rather than occupying the more vertical orientation typical of resident microglia, suggesting that they may be invading monocytes. These cells were generally not seen after day 6. Their disappearance on day 6 could be due to: (1) transformation to microglia and loss of specific antigenicity, (2) migration to the white matter, or (3) return to the bloodstream. When the lesion directly involved the white matter, class II MHC expressing cells were seen broadly dispersed in the underlying white matter, and in the deep cerebellar nuclei at all survival times (Fig. 2E). The results show that while degeneration of myelinated axons induces expression of class II MHC antigens, degeneration of unmyelinated axons does not. In contrast, class I MHC expression in the cerebellum, as in the primary optic pathway, occurs on cells associated with both myelinated and unmyelinated axonal degeneration. The differential expression of class II MHC antigens could be caused by a particular subclass of microglia, with
¢,,-
k,
1
Fig. 1. Adjacent sections of cerebellum stained with OX-6 for class II MHC antigen expression (A) and with OX-18 for class I MHC antigen expression (B) after a superficial lesion, indicated by the arrow. A difference is apparent in the extent of cell staining in the molecular layer. Bar signifies 500 urn.
the potential to express class II M H C antigens, or it may be due to a product of degenerating myelin. Oligodendrocytes are not involved since preliminary doublelabelling studies using an anti-transferrin receptor (against oligodendrocytes and endothelial cells) and OX-6 show that these two antibodies stain different cell types, and this is in accordance with previous studies v'14. The possibility of a unique subclass of microglial cells also seems unlikely, since cells of the molecular layer can express class II MHC antigens under a different set of circumstances. This has been shown in a recent study in which skin grafts induce both class I and class II MHC antigen expression by previously transplanted mouse glia which migrated to the molecular layer of the cerebellum (I.E Pollack and L.H.-C. Lee, unpublished observations). The cells in the molecular layer expressing MHC antigens have the typical morphology of resident microglia, showing that these cells are capable of expressing class II MHC antigens under the appropriate conditions. Therefore, it is unlikely that a unique subclass of microglia is causing the differential distribution of class lI
345
r~
. ~ 1 I1~
D t
\ o~
°.,,~
it Fig. 2. Cell types stained in the molecular layer ( A - D ) and in the white matter (E,F) of the cerebellum after superficial and deep lesi, A: OX-6 staining of small round cells in the molecular layer adjacent to the lesion site. B: cells in an adjacent section stained with ED-2 tor macrophages. C: OX-18 staining in the molecular layer distant from the lesion site showing microglia-like cells. D: an adjacent section stained with OX-42 for microglia. E: cells in the white matter stained with OX-6 after a deep lesion. F: an adjacent section to E stained with OX-18. Scale bar represents 30/~m in all photographs.
MHC antigen expression, based on the above observation. This finding lends support to the alternative hypothesis, namely, that products of degenerating myelin provide a specific stimulus for the induction of class II MHC antigen expression in a cell population exhibiting the morphological characteristics of microglia.
We wish to thank Dr. A. Like (University of Massachusetts, School of Pathology) for supplying crucial antibody-producing cell lines and Dr. Carl Lagenaur (University of Pittsburgh, School of Medicine) for maintaining these. We also wish to thank Robert Zatezalo and Robert Reinhart for their technical assistance and Frances Shagas for her photographic expertise. This research was supported by NIH Grants, EY05308 and HDO7343.
1 Adams, J.C., Heavy metal intensification of DAB based HRP reaction product, J. Histochem. Cytochem., 29 (1981) 775. 2 Akiyama, H., Itagaki, S. and McGeer, P.L., Major histocom-
patibility complex antigen expression on rat microglia following epidural kainic acid lesions, J. Neurosci. Res., 20 (1988) 147-157.
346 3 Akiyama, H. and McGeer, P.L., Microglial response to 6hydroxydopamine-induced substantia nigra lesions, Brain Research, 489 (1989) 247-253. 4 Barker, C.E and Billingham, R.E., Immunologically privileged sites, Adv. Immunol., 23 (1977) 1-54. 5 Head, J.R. and Billingham, R.E., Immunologically privileged sites in transplantation immunology and oncology, Perspect. Biol. Med., 29 (1985) 115-131. 6 Lampson, L.A. and Hickey, W.E, Monoclonal antibody analysis of MHC expression in human brain biopsies: tissues ranging from 'histologically normal' to that showing different levels of tumor involvement, J. Immunol., 136 (1986) 4054-4062. 7 Lee, S.C. and Raine, C.S., Multiple sclerosis: oligodendrocytes in active lesions do not express class II major histocompatibility complex molecules, J. Neuroimmunol., 25 (1989) 261-266. 8 Lund, R.D., Rao, K., Kunz, H.W. and Gill III, T.J., Instability of neural xenografts placed in neonatal rat brains, Transplantation, 46 (1988) 216-233. 9 Mason, D.W., Charlton, H.M., Jones, A.J., Lavy, C.B.D., Puklavec, M. and Simmonds, S.J., The fate of allogenic and xenogenic neuronal tissue transplanted into the third ventricle of rodents, Neuroscience, 19 (1986) 685-694. 10 Mugnaini, E. and Dahl, A.L., Zinc-aldehyde fixation for light microscopic immunocytochemistry of nervous tissues, J. Histochem. Cytochem., 31 (1983) 1435-1438. 11 Pollack, I.P., Lund, R.D. and Rao, K., MHC antigen expression in spontaneous and induced rejection of neural xenografts, Prog. Brain Research, 82, in press.
12 Rao, K. and Lund, R.D., Degeneration of optic axons induces the expression of major histocompatibility antigens, Brain Research, 488 (1989) 332-335. 13 Skoskiewicz, M.J., Colvin, R.C., Scheenberger, E.E. and Russell, ES., Widespread and selective induction of major histocompatibility complex-determined antigens in vivo by interferon, J. Exp. Med., 162 (1985) 1645-1664. 14 Suzumura, A., Silberberg, D.H. and Lisak, R.P., The expression of MHC antigens on oligodendrocytes: induction of polymorphic H-2 expression by lymphokines, J. Neuroimmunol., 11 (1986) 179-190. 15 Whelan, J.P., Eriksson, U. and Lampson, L.A., Expression of mouse fl:-microglobin in frozen and formaldehyde-fixed central nervous tissues: comparison of tissue behind the blood-brain barrier and tissue in a barrier-free region, J. lmmunol., 137 (1986) 2561-2566. 16 Williams, K.A., Hart, D.N.J., Fabre, J.W. and Morris, P.J., Distribution and quantitation of HLA-ABC and DR(Ia) antigens on human kidney and other tissues, Transplantation, 29 (1980) 274-279. 17 Wong, G.H.W., Bartlett, P.E, Clark-Lewis, I., McKimmBreschkim, J.L. and Schrader, J.W., Interferon induces the exwession of H-2 and Ia antigens on brain cells, J. Neuroimmunol., 7 (1985) 255-278. 18 Yee, K.T., Smetanka, A.M., Lund, R.D. and Rao, K., Development of major histocompatibility complex antigen expression in rat following eye removal at various postnatal ages, Soc. Neurosci. Abstr., 15 (1989) 689.
343
BRES 24146
Differential induction of class I and II MHC antigen expression by degenerating myelinated and unmyelinated axons Ava M. Smetanka, Kathleen T. Yee and Raymond D. Lund Department of Neurobiology, Anatomy and Cell Science, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261 (U.S.A.)
(Accepted 13 March 1990)
Key words: Cerebellum; Microglia; Macrophage; Myelin; Degeneration The brain has long been considered an immunologically privileged site since immunogenetically mismatched grafts that are normally rejected peripherally, frequently survive for prolonged periods after transplantation to the central nervous system 4'5'9. One factor that may contribute to immune privilege in the brain is the absence under normal circumstances of cells expressing major histocompatability complex (MHC) antigens, which function to present foreign antigens to lymphocytes6'13A5-17. However, a variety of conditions have been identified in which cells of the brain are induced to express MHC antigens, and this can lead to rejection of neural grafts in the region of antigen-presenting cellss,n. A neural lesion is one of the stimuli that induces MHC antigen expression, not only around the site of the lesion itself2'3, but also along the course of the damaged fiber tracts and in terminal areas 12'18. Studies examining the induction of MHC antigen expression in the primary optic pathway following eye lesions have shown a differential distribution of MHC antigen expression. While class I MHC antigens were expressed on cells located both within the myelinated fiber region and in terminal distribution areas, class II MHC antigen expressing cells were mainly localized within the myelinated fiber region 12,18. This observation led to the hypothesis that class II MHC antigen expression may be associated with degenerating myelin. To test this hypothesis, we have examined the effects of small lesions of the cerebellar cortex. Superficial lesions were made to damage only the unmyelinated axons of the parallel fiber system. Deeper lesions were made to damage not only the superficial unmyelinated axons, but also the cerebellar white matter, permitting simultaneous examination of degenerating myelinated and unmyelinated pathways. If the hypothesis is correct, class II MHC antigens should not be induced on cells in the molecular layer by degenerating parallel fibers, but
should be induced in the white matter after deeper lesions. Adult Sprague-Dawley rats (4-8 months old, n = 50) received small lesions of the cerebellar cortex made in a sagittal plane with a 26 gauge needle. The rats were perfused with zinc-aldehyde fixative 1° after surviving for 1 to 9 days. The brains were cryoprotected in 30% sucrose in phosphate-buffered saline (PBS) and 40/~m-thick coronal sections were cut through the cerebellum. Selected sections were stained with Cresyl violet to localize the lesion site. Adjacent sections containing the lesion were then stained with the following antibodies: OX-6 (directed against class II MHC antigens), OX-18 (against class I MHC antigens), OX-42 (against microglia and monocytes) (Accurate Chemical and Scientific Corporation, Westbury, NY); ED-2 (against macrophages) (Bioproducts for Science, Inc., Indianapolis, IN); and GFAP (against fibriUar astrocytes) (Dako Corporation, Santa Barbara, CA). Antibody binding was demonstrated using a horseradish peroxidase-conjugated secondary antibody (American Qualex International, Inc., La Mirada, CA) and a nickel-intensified diaminobenzidine reaction 1. Equivalent sections of cerebellum from age-matched normal rats were stained with the same battery of antibodies to serve as controls. No MHC antigen expression was seen in the cerebellum of normal young adult rats. In all lesioned animals, class I MHC antigen expression was seen, both on cells around the lesion site and extending along the molecular layer up to a distance of 24 mm away from the lesion site (Fig. 1B). There were two principal types of cells expressing class I MHC antigens. One of these types morphologically resembled microglia (Fig. 2C), and was similar in appearance to those stained with OX-42 (Fig. 2D). The second type of cell was preferentially localized around the lesion site and resembled those cells stained
Correspondence: A.M. Smetanka, Department of Neurobiology, Anatomy and Cell Science, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261 (U.S.A.). 0006-8993/90/$03.50 (~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
344 with ED-2 (Fig. 2B). They were presumed to be macrophages. Class I MHC positive staining cells were seen within the white matter and in the deep cerebellar nuclei, after white matter lesions (Fig. 2F). After superficial lesions to the molecular layer, the staining of class lI MHC positive cells was very different from that seen with class I MHC antigen staining with respect to density, distribution and cell class. While large numbers of MHC class I staining cells were distributed for as much as 24 mm along the molecular layer (Fig. 1B), most MHC class II positive cells were localized close to the lesion site and were fewer in number (Fig. 1A). Most of the cells staining with MHC class II antibody were large elliptical cells with small processes arising from the cell body (Fig. 2A), and were thought to be macrophages because of their similar morphology to cells stained with ED-2 (Fig. 2B). These cells were localized mainly around the lesion site, but a few were seen in the molecular layer extending as far as 10 mm away from the lesion site, occasionally bordering the Purkinje cell layer. It was not clear whether cells along the Purkinje cell layer had migrated there, or were resident cells that had been induced to express MHC antigens because of damage to Purkinje cell axons. The distance at which stained cells could be seen from the lesion site was measured; OX-6 extent of staining (£ = 4.4 mm _+ 2.4), and OX-18 extent of staining (£ = 11.1 mm -+ 4.9). Using a paired t-test, a statistically significant difference was found between the extent of OX-6 and OX-18 staining (n = 50; P < 0.01). Cells in the molecular layer resembling microglia were not stained. Following a 5-day postlesion survival, a few cells with fine processes were seen; this type of cell was not present at any other survival time. These cells had processes that generally radiated away from the lesion site, rather than occupying the more vertical orientation typical of resident microglia, suggesting that they may be invading monocytes. These cells were generally not seen after day 6. Their disappearance on day 6 could be due to: (1) transformation to microglia and loss of specific antigenicity, (2) migration to the white matter, or (3) return to the bloodstream. When the lesion directly involved the white matter, class II MHC expressing cells were seen broadly dispersed in the underlying white matter, and in the deep cerebellar nuclei at all survival times (Fig. 2E). The results show that while degeneration of myelinated axons induces expression of class II MHC antigens, degeneration of unmyelinated axons does not. In contrast, class I MHC expression in the cerebellum, as in the primary optic pathway, occurs on cells associated with both myelinated and unmyelinated axonal degeneration. The differential expression of class II MHC antigens could be caused by a particular subclass of microglia, with
¢,,-
k,
1
Fig. 1. Adjacent sections of cerebellum stained with OX-6 for class II MHC antigen expression (A) and with OX-18 for class I MHC antigen expression (B) after a superficial lesion, indicated by the arrow. A difference is apparent in the extent of cell staining in the molecular layer. Bar signifies 500 urn.
the potential to express class II M H C antigens, or it may be due to a product of degenerating myelin. Oligodendrocytes are not involved since preliminary doublelabelling studies using an anti-transferrin receptor (against oligodendrocytes and endothelial cells) and OX-6 show that these two antibodies stain different cell types, and this is in accordance with previous studies v'14. The possibility of a unique subclass of microglial cells also seems unlikely, since cells of the molecular layer can express class II MHC antigens under a different set of circumstances. This has been shown in a recent study in which skin grafts induce both class I and class II MHC antigen expression by previously transplanted mouse glia which migrated to the molecular layer of the cerebellum (I.E Pollack and L.H.-C. Lee, unpublished observations). The cells in the molecular layer expressing MHC antigens have the typical morphology of resident microglia, showing that these cells are capable of expressing class II MHC antigens under the appropriate conditions. Therefore, it is unlikely that a unique subclass of microglia is causing the differential distribution of class lI
345
r~
. ~ 1 I1~
D t
\ o~
°.,,~
it Fig. 2. Cell types stained in the molecular layer ( A - D ) and in the white matter (E,F) of the cerebellum after superficial and deep lesi, A: OX-6 staining of small round cells in the molecular layer adjacent to the lesion site. B: cells in an adjacent section stained with ED-2 tor macrophages. C: OX-18 staining in the molecular layer distant from the lesion site showing microglia-like cells. D: an adjacent section stained with OX-42 for microglia. E: cells in the white matter stained with OX-6 after a deep lesion. F: an adjacent section to E stained with OX-18. Scale bar represents 30/~m in all photographs.
MHC antigen expression, based on the above observation. This finding lends support to the alternative hypothesis, namely, that products of degenerating myelin provide a specific stimulus for the induction of class II MHC antigen expression in a cell population exhibiting the morphological characteristics of microglia.
We wish to thank Dr. A. Like (University of Massachusetts, School of Pathology) for supplying crucial antibody-producing cell lines and Dr. Carl Lagenaur (University of Pittsburgh, School of Medicine) for maintaining these. We also wish to thank Robert Zatezalo and Robert Reinhart for their technical assistance and Frances Shagas for her photographic expertise. This research was supported by NIH Grants, EY05308 and HDO7343.
1 Adams, J.C., Heavy metal intensification of DAB based HRP reaction product, J. Histochem. Cytochem., 29 (1981) 775. 2 Akiyama, H., Itagaki, S. and McGeer, P.L., Major histocom-
patibility complex antigen expression on rat microglia following epidural kainic acid lesions, J. Neurosci. Res., 20 (1988) 147-157.
346 3 Akiyama, H. and McGeer, P.L., Microglial response to 6hydroxydopamine-induced substantia nigra lesions, Brain Research, 489 (1989) 247-253. 4 Barker, C.E and Billingham, R.E., Immunologically privileged sites, Adv. Immunol., 23 (1977) 1-54. 5 Head, J.R. and Billingham, R.E., Immunologically privileged sites in transplantation immunology and oncology, Perspect. Biol. Med., 29 (1985) 115-131. 6 Lampson, L.A. and Hickey, W.E, Monoclonal antibody analysis of MHC expression in human brain biopsies: tissues ranging from 'histologically normal' to that showing different levels of tumor involvement, J. Immunol., 136 (1986) 4054-4062. 7 Lee, S.C. and Raine, C.S., Multiple sclerosis: oligodendrocytes in active lesions do not express class II major histocompatibility complex molecules, J. Neuroimmunol., 25 (1989) 261-266. 8 Lund, R.D., Rao, K., Kunz, H.W. and Gill III, T.J., Instability of neural xenografts placed in neonatal rat brains, Transplantation, 46 (1988) 216-233. 9 Mason, D.W., Charlton, H.M., Jones, A.J., Lavy, C.B.D., Puklavec, M. and Simmonds, S.J., The fate of allogenic and xenogenic neuronal tissue transplanted into the third ventricle of rodents, Neuroscience, 19 (1986) 685-694. 10 Mugnaini, E. and Dahl, A.L., Zinc-aldehyde fixation for light microscopic immunocytochemistry of nervous tissues, J. Histochem. Cytochem., 31 (1983) 1435-1438. 11 Pollack, I.P., Lund, R.D. and Rao, K., MHC antigen expression in spontaneous and induced rejection of neural xenografts, Prog. Brain Research, 82, in press.
12 Rao, K. and Lund, R.D., Degeneration of optic axons induces the expression of major histocompatibility antigens, Brain Research, 488 (1989) 332-335. 13 Skoskiewicz, M.J., Colvin, R.C., Scheenberger, E.E. and Russell, ES., Widespread and selective induction of major histocompatibility complex-determined antigens in vivo by interferon, J. Exp. Med., 162 (1985) 1645-1664. 14 Suzumura, A., Silberberg, D.H. and Lisak, R.P., The expression of MHC antigens on oligodendrocytes: induction of polymorphic H-2 expression by lymphokines, J. Neuroimmunol., 11 (1986) 179-190. 15 Whelan, J.P., Eriksson, U. and Lampson, L.A., Expression of mouse fl:-microglobin in frozen and formaldehyde-fixed central nervous tissues: comparison of tissue behind the blood-brain barrier and tissue in a barrier-free region, J. lmmunol., 137 (1986) 2561-2566. 16 Williams, K.A., Hart, D.N.J., Fabre, J.W. and Morris, P.J., Distribution and quantitation of HLA-ABC and DR(Ia) antigens on human kidney and other tissues, Transplantation, 29 (1980) 274-279. 17 Wong, G.H.W., Bartlett, P.E, Clark-Lewis, I., McKimmBreschkim, J.L. and Schrader, J.W., Interferon induces the exwession of H-2 and Ia antigens on brain cells, J. Neuroimmunol., 7 (1985) 255-278. 18 Yee, K.T., Smetanka, A.M., Lund, R.D. and Rao, K., Development of major histocompatibility complex antigen expression in rat following eye removal at various postnatal ages, Soc. Neurosci. Abstr., 15 (1989) 689.
