- Email: [email protected]
Reactive astrocytes in lesioned rat spinal cord: Effect of neural transplants
Bruin Research Bulletin, Vol. 22, pp. 81-87. 0 Pergamon Press plc, 1989. Printed in the U.S.A.
$3.00 + .OO
0361-9230/89
Reactive Astrocytes in Lesioned Rat Spinal Cord: Effect of Neural Transplants R. PREDY* AND S. K. MALHOTRAt’
~e~~rt~e~ts of ~at~o~o~* and Zoology, t U~jversity of Alberta, Edmonton, Alberia, Canada
PREDY, R. AND S. K. MALHOTRA. Reactive mtrocytes in lesioned rat spinalcord: Effect of neural tr~nspi~nts. BRAIN RES BULL 22(f) 81-87, 1989.-The astroglial reaction following a laceration-type surgical lesion of rat spinal cord is recognized by hypertrophy of astrocytes. This phenomenon can be readily demonstrated by enhanced immunoreactivity for glial fibril-
lary acidic protein (GFAP) and a recently discovered 30 kD protein (51-31 antigen). The results reported in this article lead to the conclusion that the astroglial reaction is not influenced significantly by the transplantation of embryonic neocortical tissue to the cavitv of a laceration-tvne . . lesion. These observations could be relevant to the assessment of strategies for treatment of spinal cord-injury. Reactive gliosis
30 kD protein
Monoclonal antibody
cytes, retinal Muller cells, tanycytes and ciliated ependymal cells (7, 10, 12, 13). In an early study, intense immuno~uorescence staining owing to MAb 51-31 was detected in cryostat sections of MS tissue (cervical spinal cord). This suggested the possibility that reactive astrocytes, which are known to be associated with MS plaques (9), might show enhanced expression of Jl31 antigen. However, the MS tissue then available was poorly preserved and therefore, not suitable for histological study. Consequently, we undertook to investigate the expression of Jl31 antigen by reactive astrocytes using a model system for gliosis, namely laceration-type surgical lesion of the rat spinal cord. Using this experimental system, we were able to show that reactive astrocytes exhibit enhanced immunofluorescence staining for Jl-31 antigen (11). When MS tissue suitable for histological study subsequently became available, it was shown that MS plaques exhibit enhanced staining for 51-31 antigen, as compared to adjacent “apparently normal” white matter (8). Thus as the expression of J l-3 1 antigen appears to increase during transformation to the reactive state, MAb 51-31 can be employed to probe the appearance of reactive astrocytes. This article reports on a pilot study undertaken to assess the effect of embryonic neocortical transplants on the astroglial response following a laceration-tie surgical lesion of rat spinal cord. This study is based on immunofluorescence microscopy using MAb 51-31, and antiserum to glial fibrillary acidic protein [GFAP, a recognized marker protein for astrocytes; (2)].
of cells or tissue into the adult central nervous system (CNS) is being investigated as a potential treatment strategy for alleviation of neurotransmitter deficits, such as that responsible for Parkinson’s disease, as well as for optimization of functional recovery following traumatic injury to spinal cord (1). So far, this strategy has shown only limited success in the treatment of Parkinson’s disease (14). However, neural transplantation has shown promise as a treatment for spinal cord injury. Grafting of embryonic brain tissue into the cavity of a laceration-type surgical lesion of rat spinal cord appears to prevent the protracted degeneration of those nerve fiber tracts spared direct injury (3,4). As CNS neurons exhibit limited capacity for axonal regeneration, functional recovery is restricted to axonal tracts left intact following injury. Prevention of degenerative changes affecting these spared axons is essential for the optimi~tion of functionai recovery foliowing injury. We are involved in studies on astrocytes and their response to CNS injury. This project evolved out of research aimed at detecting unique or characteristic antigens relevant to multiple sclerosis (MS), which is a demyelinating disease of the CNS. For this purpose, mouse monoclonal antibodies (MAbs) were raised against autopsy sampIes of CNS tissue from MS patients. This led to the isolation of a hybridoma clone secreting MAb J l-3 1 [isotype IgG 2b; (7)] which recognizes an intracellular protein [Jl-31 antigen, 30 kD; (13)] expressed by astroTRANSPLANTATION
‘Requests for reprints should be addressed to Dr. S. K. Malhotra, Department of Zoology, University of Alberta, Edmonton, 2E9, Canada.
81
Alberta T6G
82
PREDY
We report that the appearance of reactive astrocytes (which exhibit enhanced staining for 51-31 antigen) is similar in the presence and absence of embryonic transplants. This result may be of significance in assessing the effectiveness of neural transplantation as a treatment strategy for spinal cord injury, especially in light of work implicating reactive astrocytes in the inhibition of axonal regeneration observed in the CNS (6). METHOD
Laceration-type surgical lesions were made in the spinal cords (lumbar level L3-L4) of laboratory bred Long-Evans hooded rats (3-6 months old). This procedure involved cutting of the spinal cord as well as removal of the neural tissue from the site of the lesion, resulting in surgical cavities in the rostro-caudal axis. The lesions extended for one segment in the longitudinal plane, and 50% in the transverse plane. These shallow lesions did not induce any paraplegic syndrome, although they did result in some transient autonomic dysfunctions. Neocortical tissue, from 14.5-day-old rat embryos, was grafted into the cavity of the lesion. At the end of the surgery, the incision was sutured and the animals were placed in their cages. The surgical details and animal care adapted are as previously published (3). Specimens of lesioned spinal cords, bearing transplants, have been collected at intervals of 6 days, 15 days, 6 weeks, and 3 months following surgery. These survival periods were chosen to assess the time course of the astrocytic response following surgical trauma. At each of these intervals, one specimen was examined (N=l). Rats were etherized and perfused with 4% paraformaldehyde (0.2 M phosphate buffer; pH 7.2). The operated spinal cord segments (L3-L4) were removed and further fixed in paraformaldehyde for 48-72 hr during transit from Purdue University (Indiana) to the University of Alberta, Alberta. The tissue was washed with phosphate buffered saline (PBS) and cryoprotected in 30% sucrose (12-18 hr, 4°C) prior to freezing at -20°C. The tissue was embedded in Tissue Tek II O.C.T. compound and sagittal sections were cut on a cryostat at 8-12 pm thickness, mounted on rubber cement-coated coverglasses, and dried at 4°C overnight. Indirect immunofluorescence staining was carried out using MAb Jl-31 culture supernatant; supernatant from NS-1 myeloma cells served as control. Sections were double-labeled with rabbit antiserum to cow GFAP (Dakopatts, Dimension Labs Inc., Mississauga, Ontario; 1:lOOO dilution); normal rabbit serum (NRS; Sigma, St. Louis, MO) served as control. Antibodies and normal sera were used in dilutions made-up in PBS. The protocol for the immunostaining was as follows: The sections were first washed with PBS, incubated in 30% goat serum (Sigma) for 30 min at 20°C (room temperature), and then incubated with the primary antibody for 12-18 hr at 4°C. Then the sections were washed with PBS and incubated with the appropriate fluorochrome conjugated secondary antibody for 1 hr at 20°C. Goat antimouse IgG conjugated to fluorescein isothiocyanate (FITC; Sigma) and goat antirabbit IgG conjugated to tetramethylrhodamine isothiocyanate (TRITC; Sigma) were used at dilutions of 1:lOO and 1:SOO respectively. Double-labeled sections were prepared following a repetition of the procedure using the second set of primary and secondary antibodies. Following washes with PBS and distilled water, sections were mounted in buffered glycerol containing p-phenylenediamine (5) and viewed by epifluorescence microscopy. Sections were also examined after routine staining with hematoxylin and eosin.
AND MALHOTRA
KESlJLTS
Immunofluorescence
Preparations
In transplant specimens collected 3 months (Fig. 1A and B), 6 weeks (Fig. 2A and B) and 15 days following surgery, astrocytes adjacent to the lesion show intense immunofluorescence staining for 51-31 antigen (Fig. 1A and Fig. 2A) and GFAP (Fig. 1B and Fig. 2B). In adjacent apparently uninjured tissue (nonlesion region), astrocytes show reduced staining for 51-31 antigen (Fig. 1C) and GFAP (Fig. ID). The situation in transplant specimens is comparable to that observed in lesioned specimens in the absence of neural transplants, where staining for 51-31 antigen and GFAP is intense adjacent to the lesion, and much reduced in the nonlesion region (11). In the transplant specimen collected 6 days following surgery, staining for 51-31 antigen adjacent to the lesion (Fig. 2C) was not appreciably greater than that observed in the nonlesion region. Staining for GFAP was intense in the region adjacent to the lesion (Fig. 2D) and served as a guide to detect weak staining for 51-31 antigen (Fig. 2C). In control sections incubated with culture supernatant from NS-1 myeloma cells and NRS, there was no staining of reactive astrocytes comparable to that observed in sections incubated with antibodies.
Hematoxylin
and Eosin Stained Sections
In cryostat sections stained with hematoxylin and eosin (H and E), we detected astroglial reaction (reactive gliosis), leading to formation of a glial scar, in the specimens collected 15 days (Fig. 3C and D), 6 weeks (Fig. 4A and B) and 3 months (Fig. 4C and D) following surgery. It is remarked that quality of histological preservation in our frozen (cryostat) sections is poor, and that the fragile transplant tissue (embryonic) did not withstand the rigors of the H and E staining procedure. Nevertheless, there is clear evidence of glial scar formation in the host tissue adjacent to the lesion site (most evident at high magnification: Figs. 3D, 4B and 4D). These glial scars exhibit hypercellularity and reactive gliosis. Numerous darkly staining nuclei, predominantly of astrocytes, are evident within the glial scars (Figs. 3D, 4B and 4D). Six day transplant. In this specimen, the host tissue adjacent to the cavity of the lesion appears to be infiltrated by blood cells, erythrocytes and leucocytes (Fig. 3A and B). This specimen does not show clear evidence of glial scar formation, as compared to the specimens collected at longer intervals following surgery. Fifteen day transplant. In this specimen, fibrous gliosis is most pronounced in the extreme dorsal white matter (Fig. 3C) as is clearly evident at higher magnification (Fig. 3D). Ventral to this region, white matter in close apposition to the transplant exhibits minimal gliosis (Fig. 3C). Six week transplant. In this specimen, there is a space or cavity (top of Fig. 4A) which is devoid of transplant tissue. The host tissue adjacent to this cavity exhibits a pronounced glial reaction as is evident at higher magnification (Fig. 4B). Three month transplant. There is a pronounced glial reaction evident in this specimen even though the host tissue is in close apposition to the transplant (Fig. 4C and D). DISCUSSION
Enhanced staining for J l-3 1 antigen has been observed in reactive astrocytes which arise following surgical injury to rat spinal cord (11) and cerebral cortex (unpublished), as well as in association with multiple sclerosis plaques (8). Therefore, en-
ASTROCYTES
FOLLOWING
83
TRANSPLANTATION
FIG. 1. Cryostat section of lacerated rat spinal cord bearing embryonic neocortical transplant, 3 months following surgery. In the dorsal white matter immediately adjacent to the lesion, astrocytes exhibit intense immunofluorescence staining for 51-31 antigen (A, FITC) and GFAP (B, TRITC). In the dorsal white matter distant from the lesion, staining for 51-31 antigen (C) and GFAP (D) is much reduced (x304; 1.5 cm=50 pm).
hance astrog emplc study
,d Sl:aining for Jl-31 antigen reveals a new aspect of the ;lial response to injury. Consequently, MAb 51-31 can be ‘Yed to probe the appearance of reactive astrocytes. In this ii: i:s reported that the astroglial response following sur-
gical lesion of rat spinal cord (laceration-type) does n ot appear to be affected by neural transplantation to the lesion Si ite. The astroglial response in the absence of such transplants h as been described previously (11). The assessment of this re:SF jonse is
PREDY AND MALHOTRA
FIG. 2. A and B. Cryostat section of lacerated rat spinal cord bearing embryonic neocortical transplant, 6 weeks following surgery. Large reactive astrocytes, ventral to the cavity (see Fig. 4A), exhibit intense immunofluorescence staining for both 51-31 antigen (A, FITC) and GFAP (B, TRITC). C and D. Cryostat section of lacerated rat spinal cord bearing embryonic neocortical transplant, 6 days following surgery. Astrocytes ventral to the lesion (see Fig. 3A), exhibit weak staining for 31-31 antigen (C, FITC). Staining for GFAP is intense in this region (D, TRITC) and serves as a guide to detect staining for 51-31 antigen (x312; 1.6 cm=50 pm).
ASTROCYTES
FOLLOWING
TRANSPLANTATION
FIG. 3. A and B. Transplant specimen, 6 days following surgery. The grey matter (evident along the left hand border of A) is poorly preserved in this cryostat section. At high magnification, the host tissue adjacent to the lesion site appears to be infiltrated by blood cells, erythrocytes and leucocytes. This specimen does not show clear evidence of glial scar formation, as compared to the specimens collected at longer intervals following surgery. C and D. Transplant specimen, 15 days following surgery. A pronounced glial reaction is evident in the extreme dorsal white matter adjacent to the lesion (C and D). Ventral to this region, white matter in close apposition to the transplant exhibits minima1 gliosis (C). All hematoxylin and eosin stained preparations were photographed through a green filter. (A and C: x51; 1.0 cm=200 pm)/(B and D: x 128; 1.3 cm= 100 pm).
85
PREDY AND MALHOTRA
FIG. 4. A and B. Transplant specimen, 6 weeks following surgery. In this specimen, there is a cavity which is devoid of transplant tissue (top of A). The host tissue adjacent to this cavity exhibits a pronounced glial reaction, as is clearly evident at higher magnification (B). The narrow space in the grey matter (left hand side of A) is a histological artifact. C and D. Transplant specimen, 3 months following surgery. In this specimen, the host tissue is in close apposition to the transplant. The host tissue exhibits a pronounced gliai reaction which is clearly evident at higher magnification (D). (A and C: x51; 1.0 cm=200 pm)/(B and D: x128; 1.3 cm=100 rm).
ASTROCYTES
FOLLOWING
87
TRANSPLANTATION
based on immunofluorescence staining for 51-31 antigen and GFAP, as well as hematoxylin and eosin stained sections (cryostat). It has been suggested that reactive astrocytes are responsible for the inhibition of axonal regeneration observed in the CNS (6). In light of results presented in this paper, grafts of embryonic neocortical tissue do not inhibit the formation of reactive astrocytes following a spinal cord injury. However, such transplants do appear to prevent the protracted degeneration of those fiber tracts spared direct injury, possibly by preventing the infiltration of loose connective tissue and other foreign materials into the lesion site (3,4). In summary, the astroglial reaction following injury presum-
ably results from trauma to the spinal cord. This effect of trauma does not appear to be inhibited by the transplantation of embryonic neocortical tissue to the lesion site. These observations may be relevant to the assessment of strategies for treatment of spinal cord injury. ACKNOWLEDGEMENTS
Dr. G. D. Das (Purdue University, West Lafayette, Indiana) kindly provided spinal cord specimens bearing transplants. R. P. is registered as a graduate student in the Department of Pathology. The research was supported by grants awarded by NSERC of Canada, and gifts from Dr. K. B. Newbound and Dr. N. R. Thomas.
REFERENCES 1. Azmitia, E. C.; Bjorklund, A. Cell and tissue transplantation into the adult brain. Ann. NY Acad. Sci. 495:810; 1987. 2. Bienami. A.: Dahl. D. Soecificitv of the glial fibrillary acidic prote& for astroglia. J. Histochem: Cytochim. 25:466-469; 1977. 3. Das, G. D. Neural transplantation in spinal cord under different conditions of lesions and their functional significance. In: Das, 0. D.; Wallace, R. B., eds. Neural transplantation and regeneration. Proc. Life Sci. New York: Springer-Verlag; 1986:1-61. 4. Das, G. D. Neural transplantation in normal and traumatized spinal cord. Ann. NY Acad. Sci. 495:53-70; 1987. 5. Johnson, G. D.; de C. Nogueria Araujo, G. M. A simple method of reducing the fading of immunofluorescence during microscopy. J. Immunol. Methods 43:349-350; 1981. 6. Liuzzi, F. J.; Lasek, R. J.; Astrocytes block axonal regeneration in mammals by activating the physiological stop pathway. Science 237~642-645; 1987. 7. Malhotra, S. K.; Wong, F.; Cumming, P.; Ross, S. D.; Shnitka, T. K.; Manickavel, V.; Warren, K. G.; Jeffrey, V. A monoclonal antibody for cytoskeletal antigenic determinant(s) distinguishable from glial fibrillary acidic protein in astrocytes. Microbios Lett. 26: 151-157; 1984.
8. Malhotra, S. K.; Predy, R.; Johnson, E. S.; Singh, R.; Leeuw, K. 9. 10.
11.
12.
13. 14.
A novel astrocytic protein in multiple sclerosis plaques. J. Neurosci. Res. 22:36-49; 1989. Perier, 0.; Gregoire, A. Electron microscopic features of multiple sclerosis lesions. Brain 88:937-952; 1965. Predy, R.; Singh, D.; Bhatnagar, R.; Singh, R.; Malhotra, S. K. A new protein (51-31 antigen, 30 kD) is expressed by astrocytes, Muller glia and ependyma. Biosci. Rep. 7:491-502; 1987. Predy, R.; Malhotra, S. K.; Das, G. D. Enhanced expression of a protein antigen (J l-3 1 antigen, 30 Kilodaltions) by reactive astrocytes in lacerated spinal cord. J. Neurosci. Res. 19:397-404; 1988. Schroder, H.; Malhotra, S. K. Characterization of rodent pineal astrocytes by immunofluorescence microscopy using a monoclonal antibody (51-31). Cell Tissue Res. 248:607-610; 1987. Singh, R.; Singh, B.; Malhotra, S. K. A new “marker” protein for astrocytes. Biosci. Rep. 6:73-79; 1986. Sladek, J. R.; Shoulson, I. Neural transplantation: A call for patience rather than patients. Science 240:1386-1388; 1988.
$3.00 + .OO
0361-9230/89
Reactive Astrocytes in Lesioned Rat Spinal Cord: Effect of Neural Transplants R. PREDY* AND S. K. MALHOTRAt’
~e~~rt~e~ts of ~at~o~o~* and Zoology, t U~jversity of Alberta, Edmonton, Alberia, Canada
PREDY, R. AND S. K. MALHOTRA. Reactive mtrocytes in lesioned rat spinalcord: Effect of neural tr~nspi~nts. BRAIN RES BULL 22(f) 81-87, 1989.-The astroglial reaction following a laceration-type surgical lesion of rat spinal cord is recognized by hypertrophy of astrocytes. This phenomenon can be readily demonstrated by enhanced immunoreactivity for glial fibril-
lary acidic protein (GFAP) and a recently discovered 30 kD protein (51-31 antigen). The results reported in this article lead to the conclusion that the astroglial reaction is not influenced significantly by the transplantation of embryonic neocortical tissue to the cavitv of a laceration-tvne . . lesion. These observations could be relevant to the assessment of strategies for treatment of spinal cord-injury. Reactive gliosis
30 kD protein
Monoclonal antibody
cytes, retinal Muller cells, tanycytes and ciliated ependymal cells (7, 10, 12, 13). In an early study, intense immuno~uorescence staining owing to MAb 51-31 was detected in cryostat sections of MS tissue (cervical spinal cord). This suggested the possibility that reactive astrocytes, which are known to be associated with MS plaques (9), might show enhanced expression of Jl31 antigen. However, the MS tissue then available was poorly preserved and therefore, not suitable for histological study. Consequently, we undertook to investigate the expression of Jl31 antigen by reactive astrocytes using a model system for gliosis, namely laceration-type surgical lesion of the rat spinal cord. Using this experimental system, we were able to show that reactive astrocytes exhibit enhanced immunofluorescence staining for Jl-31 antigen (11). When MS tissue suitable for histological study subsequently became available, it was shown that MS plaques exhibit enhanced staining for 51-31 antigen, as compared to adjacent “apparently normal” white matter (8). Thus as the expression of J l-3 1 antigen appears to increase during transformation to the reactive state, MAb 51-31 can be employed to probe the appearance of reactive astrocytes. This article reports on a pilot study undertaken to assess the effect of embryonic neocortical transplants on the astroglial response following a laceration-tie surgical lesion of rat spinal cord. This study is based on immunofluorescence microscopy using MAb 51-31, and antiserum to glial fibrillary acidic protein [GFAP, a recognized marker protein for astrocytes; (2)].
of cells or tissue into the adult central nervous system (CNS) is being investigated as a potential treatment strategy for alleviation of neurotransmitter deficits, such as that responsible for Parkinson’s disease, as well as for optimization of functional recovery following traumatic injury to spinal cord (1). So far, this strategy has shown only limited success in the treatment of Parkinson’s disease (14). However, neural transplantation has shown promise as a treatment for spinal cord injury. Grafting of embryonic brain tissue into the cavity of a laceration-type surgical lesion of rat spinal cord appears to prevent the protracted degeneration of those nerve fiber tracts spared direct injury (3,4). As CNS neurons exhibit limited capacity for axonal regeneration, functional recovery is restricted to axonal tracts left intact following injury. Prevention of degenerative changes affecting these spared axons is essential for the optimi~tion of functionai recovery foliowing injury. We are involved in studies on astrocytes and their response to CNS injury. This project evolved out of research aimed at detecting unique or characteristic antigens relevant to multiple sclerosis (MS), which is a demyelinating disease of the CNS. For this purpose, mouse monoclonal antibodies (MAbs) were raised against autopsy sampIes of CNS tissue from MS patients. This led to the isolation of a hybridoma clone secreting MAb J l-3 1 [isotype IgG 2b; (7)] which recognizes an intracellular protein [Jl-31 antigen, 30 kD; (13)] expressed by astroTRANSPLANTATION
‘Requests for reprints should be addressed to Dr. S. K. Malhotra, Department of Zoology, University of Alberta, Edmonton, 2E9, Canada.
81
Alberta T6G
82
PREDY
We report that the appearance of reactive astrocytes (which exhibit enhanced staining for 51-31 antigen) is similar in the presence and absence of embryonic transplants. This result may be of significance in assessing the effectiveness of neural transplantation as a treatment strategy for spinal cord injury, especially in light of work implicating reactive astrocytes in the inhibition of axonal regeneration observed in the CNS (6). METHOD
Laceration-type surgical lesions were made in the spinal cords (lumbar level L3-L4) of laboratory bred Long-Evans hooded rats (3-6 months old). This procedure involved cutting of the spinal cord as well as removal of the neural tissue from the site of the lesion, resulting in surgical cavities in the rostro-caudal axis. The lesions extended for one segment in the longitudinal plane, and 50% in the transverse plane. These shallow lesions did not induce any paraplegic syndrome, although they did result in some transient autonomic dysfunctions. Neocortical tissue, from 14.5-day-old rat embryos, was grafted into the cavity of the lesion. At the end of the surgery, the incision was sutured and the animals were placed in their cages. The surgical details and animal care adapted are as previously published (3). Specimens of lesioned spinal cords, bearing transplants, have been collected at intervals of 6 days, 15 days, 6 weeks, and 3 months following surgery. These survival periods were chosen to assess the time course of the astrocytic response following surgical trauma. At each of these intervals, one specimen was examined (N=l). Rats were etherized and perfused with 4% paraformaldehyde (0.2 M phosphate buffer; pH 7.2). The operated spinal cord segments (L3-L4) were removed and further fixed in paraformaldehyde for 48-72 hr during transit from Purdue University (Indiana) to the University of Alberta, Alberta. The tissue was washed with phosphate buffered saline (PBS) and cryoprotected in 30% sucrose (12-18 hr, 4°C) prior to freezing at -20°C. The tissue was embedded in Tissue Tek II O.C.T. compound and sagittal sections were cut on a cryostat at 8-12 pm thickness, mounted on rubber cement-coated coverglasses, and dried at 4°C overnight. Indirect immunofluorescence staining was carried out using MAb Jl-31 culture supernatant; supernatant from NS-1 myeloma cells served as control. Sections were double-labeled with rabbit antiserum to cow GFAP (Dakopatts, Dimension Labs Inc., Mississauga, Ontario; 1:lOOO dilution); normal rabbit serum (NRS; Sigma, St. Louis, MO) served as control. Antibodies and normal sera were used in dilutions made-up in PBS. The protocol for the immunostaining was as follows: The sections were first washed with PBS, incubated in 30% goat serum (Sigma) for 30 min at 20°C (room temperature), and then incubated with the primary antibody for 12-18 hr at 4°C. Then the sections were washed with PBS and incubated with the appropriate fluorochrome conjugated secondary antibody for 1 hr at 20°C. Goat antimouse IgG conjugated to fluorescein isothiocyanate (FITC; Sigma) and goat antirabbit IgG conjugated to tetramethylrhodamine isothiocyanate (TRITC; Sigma) were used at dilutions of 1:lOO and 1:SOO respectively. Double-labeled sections were prepared following a repetition of the procedure using the second set of primary and secondary antibodies. Following washes with PBS and distilled water, sections were mounted in buffered glycerol containing p-phenylenediamine (5) and viewed by epifluorescence microscopy. Sections were also examined after routine staining with hematoxylin and eosin.
AND MALHOTRA
KESlJLTS
Immunofluorescence
Preparations
In transplant specimens collected 3 months (Fig. 1A and B), 6 weeks (Fig. 2A and B) and 15 days following surgery, astrocytes adjacent to the lesion show intense immunofluorescence staining for 51-31 antigen (Fig. 1A and Fig. 2A) and GFAP (Fig. 1B and Fig. 2B). In adjacent apparently uninjured tissue (nonlesion region), astrocytes show reduced staining for 51-31 antigen (Fig. 1C) and GFAP (Fig. ID). The situation in transplant specimens is comparable to that observed in lesioned specimens in the absence of neural transplants, where staining for 51-31 antigen and GFAP is intense adjacent to the lesion, and much reduced in the nonlesion region (11). In the transplant specimen collected 6 days following surgery, staining for 51-31 antigen adjacent to the lesion (Fig. 2C) was not appreciably greater than that observed in the nonlesion region. Staining for GFAP was intense in the region adjacent to the lesion (Fig. 2D) and served as a guide to detect weak staining for 51-31 antigen (Fig. 2C). In control sections incubated with culture supernatant from NS-1 myeloma cells and NRS, there was no staining of reactive astrocytes comparable to that observed in sections incubated with antibodies.
Hematoxylin
and Eosin Stained Sections
In cryostat sections stained with hematoxylin and eosin (H and E), we detected astroglial reaction (reactive gliosis), leading to formation of a glial scar, in the specimens collected 15 days (Fig. 3C and D), 6 weeks (Fig. 4A and B) and 3 months (Fig. 4C and D) following surgery. It is remarked that quality of histological preservation in our frozen (cryostat) sections is poor, and that the fragile transplant tissue (embryonic) did not withstand the rigors of the H and E staining procedure. Nevertheless, there is clear evidence of glial scar formation in the host tissue adjacent to the lesion site (most evident at high magnification: Figs. 3D, 4B and 4D). These glial scars exhibit hypercellularity and reactive gliosis. Numerous darkly staining nuclei, predominantly of astrocytes, are evident within the glial scars (Figs. 3D, 4B and 4D). Six day transplant. In this specimen, the host tissue adjacent to the cavity of the lesion appears to be infiltrated by blood cells, erythrocytes and leucocytes (Fig. 3A and B). This specimen does not show clear evidence of glial scar formation, as compared to the specimens collected at longer intervals following surgery. Fifteen day transplant. In this specimen, fibrous gliosis is most pronounced in the extreme dorsal white matter (Fig. 3C) as is clearly evident at higher magnification (Fig. 3D). Ventral to this region, white matter in close apposition to the transplant exhibits minimal gliosis (Fig. 3C). Six week transplant. In this specimen, there is a space or cavity (top of Fig. 4A) which is devoid of transplant tissue. The host tissue adjacent to this cavity exhibits a pronounced glial reaction as is evident at higher magnification (Fig. 4B). Three month transplant. There is a pronounced glial reaction evident in this specimen even though the host tissue is in close apposition to the transplant (Fig. 4C and D). DISCUSSION
Enhanced staining for J l-3 1 antigen has been observed in reactive astrocytes which arise following surgical injury to rat spinal cord (11) and cerebral cortex (unpublished), as well as in association with multiple sclerosis plaques (8). Therefore, en-
ASTROCYTES
FOLLOWING
83
TRANSPLANTATION
FIG. 1. Cryostat section of lacerated rat spinal cord bearing embryonic neocortical transplant, 3 months following surgery. In the dorsal white matter immediately adjacent to the lesion, astrocytes exhibit intense immunofluorescence staining for 51-31 antigen (A, FITC) and GFAP (B, TRITC). In the dorsal white matter distant from the lesion, staining for 51-31 antigen (C) and GFAP (D) is much reduced (x304; 1.5 cm=50 pm).
hance astrog emplc study
,d Sl:aining for Jl-31 antigen reveals a new aspect of the ;lial response to injury. Consequently, MAb 51-31 can be ‘Yed to probe the appearance of reactive astrocytes. In this ii: i:s reported that the astroglial response following sur-
gical lesion of rat spinal cord (laceration-type) does n ot appear to be affected by neural transplantation to the lesion Si ite. The astroglial response in the absence of such transplants h as been described previously (11). The assessment of this re:SF jonse is
PREDY AND MALHOTRA
FIG. 2. A and B. Cryostat section of lacerated rat spinal cord bearing embryonic neocortical transplant, 6 weeks following surgery. Large reactive astrocytes, ventral to the cavity (see Fig. 4A), exhibit intense immunofluorescence staining for both 51-31 antigen (A, FITC) and GFAP (B, TRITC). C and D. Cryostat section of lacerated rat spinal cord bearing embryonic neocortical transplant, 6 days following surgery. Astrocytes ventral to the lesion (see Fig. 3A), exhibit weak staining for 31-31 antigen (C, FITC). Staining for GFAP is intense in this region (D, TRITC) and serves as a guide to detect staining for 51-31 antigen (x312; 1.6 cm=50 pm).
ASTROCYTES
FOLLOWING
TRANSPLANTATION
FIG. 3. A and B. Transplant specimen, 6 days following surgery. The grey matter (evident along the left hand border of A) is poorly preserved in this cryostat section. At high magnification, the host tissue adjacent to the lesion site appears to be infiltrated by blood cells, erythrocytes and leucocytes. This specimen does not show clear evidence of glial scar formation, as compared to the specimens collected at longer intervals following surgery. C and D. Transplant specimen, 15 days following surgery. A pronounced glial reaction is evident in the extreme dorsal white matter adjacent to the lesion (C and D). Ventral to this region, white matter in close apposition to the transplant exhibits minima1 gliosis (C). All hematoxylin and eosin stained preparations were photographed through a green filter. (A and C: x51; 1.0 cm=200 pm)/(B and D: x 128; 1.3 cm= 100 pm).
85
PREDY AND MALHOTRA
FIG. 4. A and B. Transplant specimen, 6 weeks following surgery. In this specimen, there is a cavity which is devoid of transplant tissue (top of A). The host tissue adjacent to this cavity exhibits a pronounced glial reaction, as is clearly evident at higher magnification (B). The narrow space in the grey matter (left hand side of A) is a histological artifact. C and D. Transplant specimen, 3 months following surgery. In this specimen, the host tissue is in close apposition to the transplant. The host tissue exhibits a pronounced gliai reaction which is clearly evident at higher magnification (D). (A and C: x51; 1.0 cm=200 pm)/(B and D: x128; 1.3 cm=100 rm).
ASTROCYTES
FOLLOWING
87
TRANSPLANTATION
based on immunofluorescence staining for 51-31 antigen and GFAP, as well as hematoxylin and eosin stained sections (cryostat). It has been suggested that reactive astrocytes are responsible for the inhibition of axonal regeneration observed in the CNS (6). In light of results presented in this paper, grafts of embryonic neocortical tissue do not inhibit the formation of reactive astrocytes following a spinal cord injury. However, such transplants do appear to prevent the protracted degeneration of those fiber tracts spared direct injury, possibly by preventing the infiltration of loose connective tissue and other foreign materials into the lesion site (3,4). In summary, the astroglial reaction following injury presum-
ably results from trauma to the spinal cord. This effect of trauma does not appear to be inhibited by the transplantation of embryonic neocortical tissue to the lesion site. These observations may be relevant to the assessment of strategies for treatment of spinal cord injury. ACKNOWLEDGEMENTS
Dr. G. D. Das (Purdue University, West Lafayette, Indiana) kindly provided spinal cord specimens bearing transplants. R. P. is registered as a graduate student in the Department of Pathology. The research was supported by grants awarded by NSERC of Canada, and gifts from Dr. K. B. Newbound and Dr. N. R. Thomas.
REFERENCES 1. Azmitia, E. C.; Bjorklund, A. Cell and tissue transplantation into the adult brain. Ann. NY Acad. Sci. 495:810; 1987. 2. Bienami. A.: Dahl. D. Soecificitv of the glial fibrillary acidic prote& for astroglia. J. Histochem: Cytochim. 25:466-469; 1977. 3. Das, G. D. Neural transplantation in spinal cord under different conditions of lesions and their functional significance. In: Das, 0. D.; Wallace, R. B., eds. Neural transplantation and regeneration. Proc. Life Sci. New York: Springer-Verlag; 1986:1-61. 4. Das, G. D. Neural transplantation in normal and traumatized spinal cord. Ann. NY Acad. Sci. 495:53-70; 1987. 5. Johnson, G. D.; de C. Nogueria Araujo, G. M. A simple method of reducing the fading of immunofluorescence during microscopy. J. Immunol. Methods 43:349-350; 1981. 6. Liuzzi, F. J.; Lasek, R. J.; Astrocytes block axonal regeneration in mammals by activating the physiological stop pathway. Science 237~642-645; 1987. 7. Malhotra, S. K.; Wong, F.; Cumming, P.; Ross, S. D.; Shnitka, T. K.; Manickavel, V.; Warren, K. G.; Jeffrey, V. A monoclonal antibody for cytoskeletal antigenic determinant(s) distinguishable from glial fibrillary acidic protein in astrocytes. Microbios Lett. 26: 151-157; 1984.
8. Malhotra, S. K.; Predy, R.; Johnson, E. S.; Singh, R.; Leeuw, K. 9. 10.
11.
12.
13. 14.
A novel astrocytic protein in multiple sclerosis plaques. J. Neurosci. Res. 22:36-49; 1989. Perier, 0.; Gregoire, A. Electron microscopic features of multiple sclerosis lesions. Brain 88:937-952; 1965. Predy, R.; Singh, D.; Bhatnagar, R.; Singh, R.; Malhotra, S. K. A new protein (51-31 antigen, 30 kD) is expressed by astrocytes, Muller glia and ependyma. Biosci. Rep. 7:491-502; 1987. Predy, R.; Malhotra, S. K.; Das, G. D. Enhanced expression of a protein antigen (J l-3 1 antigen, 30 Kilodaltions) by reactive astrocytes in lacerated spinal cord. J. Neurosci. Res. 19:397-404; 1988. Schroder, H.; Malhotra, S. K. Characterization of rodent pineal astrocytes by immunofluorescence microscopy using a monoclonal antibody (51-31). Cell Tissue Res. 248:607-610; 1987. Singh, R.; Singh, B.; Malhotra, S. K. A new “marker” protein for astrocytes. Biosci. Rep. 6:73-79; 1986. Sladek, J. R.; Shoulson, I. Neural transplantation: A call for patience rather than patients. Science 240:1386-1388; 1988.