J. Comp. Path. 1991Vol. 104
Carbonic Anhydrase II Expression in Fibrous Astrocytes of the Sheep M. Jeffrey, G. A. H. Wells* and A. W. Bridges* Lasswade Veterinary Laboratory, Bush Estate, Penicuik, Midlothian EH26 OSA, and *Pathology Department, Central Veterinary Laboratory, New Haw, Weybridge, Surrey KT15 3NB, U.K.
Summary Immunohistochemical expression of carbonic anhydrase isoenzyme II (CAII) was demonstrated in a population of fibrous astrocytes in a young lamb and an adult sheep. Such cells were identified by co-expression of CAII and glial fibrillary acidic protein, nuclear morphology and their contribution of glial fibrillary acidic protein reactive processes to the glial limitans. Similar cells were not identified in neonatal lambs. As in man and mouse, CAII was also expressed in ehoroid plexus epithelium occurring in neonate, young and adult sheep brain. In contrast, however, to man and mouse, CAII was not expressed in sheep oligodendrocytes.
Introduction Carbonic anhydrase is a widespread catalyst of reversible hydration of carbon dioxide and is thought to be involved in the control of the ion, fluid and acidbase balance in various organs. In the CNS, the carbonic anhydrase isoenzyme II (CAII) is generally regarded as a glial specific enzyme and is considered to be a useful marker for oligodendroglia (Ghandour, Langley, Vincendon, Gombos, Fillippi, Limozin, Dalmassa and Laurent, 1980; Cammer and Tansey, 1988a). As a preliminary investigation to the study ofgliogenesis in Border disease, a dysmyelinating disease of sheep (Barlow and Patterson, 1982), the cellular localization of glial fibrillary acidic protein (GFAP), CAII and myelin basic protein (MBP) was investigated in the normal sheep (Jeffrey, Wells and Bridges, 1990). The distribution of GFAP and MBP was found to be similar to that reported for some other mammals but the distribution and cellular localization of CAII differed fi'om that of previous accounts. We report here the localization of CAII in fibrous astrocytes of a 29-day-old lamb and an adult sheep, but absence of such expression in the neonate lamb.
Materials and Methods Brains and spinal cords were removed from six sheep which showed no clinical or pathological evidence of central nervous system abnormality, and were immediately placed in fixative. Details of the breeds, ages and fixatives used are shown in Table 1. 0021-9975/91/040337+ 07 $03.00/0
338
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et al.
Table 1
Ages and breeds o f s h e e p , t i s s u e f i x a t i v e s a n d l o c a l i z a t i o n o f C A I I Age
Breed
Fixative
1 day I clay
Suffolk C h e v i o t cross
1 day* 1 day 29 d a y s adult
Dorset Border Dorset Dorset
i 0 per c e n t neutral formalin 4 per cent p a r a f o r m a l d e h y d e / 0'1 p e r cent g l u t a r a l d e h y d e forrnol s u b l i m a t e C a r n o y ' s fluid Carnoy's fluid Carnoy's fluid
horn Leicester horn horn
Localization of CAII Chornid plexus Glia +
-
+ + +
+ +
* D i e d at b i r t h following dystokia.
All the sheep, but for one lamb, which died at birth following dystokia, were killed by intravenous injection of pentobarbitone sodium. Tissues from three animals were placed in Carnoy's fluid and, after 1 h, the cerebellum was removed and cut parasagittally; the remaining brain and spinal cord segments C3, T~ and Ls_~ were cut coronally at 0"5 cm intervals and all tissues were returned to fixative. After a further 2 h fixation, tissues were trimmed and placed in fresh fixative for an additional 2 h before routine processing and embedding in paraffin wax. Tissues from three other animals were placed in phosphate-buffered neutral 10 per cent formalin, formol sublimate or 4- per cent paraformaldehyde with 0" 1 per cent glutaraldehyde, respectively, retained whole and fixed for 48 h to 3 weeks prior to processing and paraffin wax embedding. Commercially available monoclonal GFAP antiserum (ICN Ltd) was used for double immunostaining. CAII antiserum was a gift from P. V. Steart. Its production and testing have been published previously (Weller, Steart and Moore, 1986). Sections 5 g m thick were cut and stained for the presence of" CAII by the peroxidase-antiperoxidase technique (PAP). Brain fixed in Carnoy's fluid from 4-week-old mice was used as positive control for CAII immunostaining. Double staining for GFAP and CAII was performed on selected sections of Carnoy's fixed tissues. With this technique, staining for CAII was by the PAP method and staining for GFAP was by an alkaline phosphatase-anti-atkaline phosphatase (APAAP) method. Endogenous phosphatase activity was inhibited by levamisole and APAAP antibody complex was visualized by fast blue. Sections were mounted in glycergel (Dako U K Ltd.).
Results C A I I expression could be identified in tissues fixed either in C a r n o y ' s fluid o r in 4 p e r cent p a r a f o r m a l d e h y d e a n d 0" 1 per cent g l u t a r a l d e h y d e b u t staining was of g r e a t e r intensity in tissues fixed in the former. C A I I expression was n o t s e e n in b u f f e r e d formalin or formol sublimate fixed tissues. C A I I was p r e s e n t in t h e c y t o p l a s m of choroid plexus epithelium in all ages o f sheep e x a m i n e d (Fig. 1). I n the n e o n a t a l tissues fixed in C a r n o y ' s fluid, C A I I was n o t found in the g r e y m a t t e r n e u r o p i l or white m a t t e r . I n the 29-day-old l a m b , weak C A I I e x p r e s sion was p r e s e n t in glial cell processes a n d neuropil of the v e n t r a l h o r n g r e y m a t t e r a n d in processes and perinuclear cytoplasm o f some glial cells in v e n t r a l a n d lateral funiculi o f the spinal cord. I n white m a t t e r , the processes o f s t a i n e d
CAII i n S h e e p
339
Fig. 1. 29-day-old sheep. CAII expression in choroid plexus epithelium. PAP CAII stain x 1280. Fig. 2.
Adult sheep spinal cord, CAII expression in a white matter glial cell. PAP CAII stain x 1680,
340
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CAII in Sheep
341
cells were single, or numbered up to four and were sharply delineated, u n b r a n c h e d and tapered from the cell body. Nuclei remained unstained. In the adult sheep, strong CAII expression was present in glial cells of white matter throughout the brain and spinal cord (Fig. 2). The CAII-expressing processes of these cells were contiguous with CAII-positive pericapillary glial limitans. As in the 29-day-old, lamb CAII expression was present in glial cell processes and neuropil of the ventral horns. Mouse brain CAII expression was similar to that previously described ( G h a n d o u r et al., 1980). In double-stained preparations of mouse brain, glial cells expressed either GFAP or CAII (Fig. 3) but in similar preparations of the 29-day-old and adult sheep brains, co-expression of CAII and GFAP was evident in some white matter glial cells (Figs 4a and b). In grey matter, glia that were G F A P expressing contained no demonstrable CAII. Co-expression of C A I I and GFAP was a feature of some glial processes in spinal cord ventral grey m a t t e r while some other similar glial processes expressed only CAII. Discussion
These results demonstrate that the CNS enzyme CAII is present in sheep and that its cellular localization differs from that previously described for man and rodents. T h e preservation of CAII expression according to the fixatives employed was similar to that previously reported (Ghandour el aL, 1980; K u m p u l a i n e n and Korhonen, 1982). The occurrence of CAII in choroid plexus epithelium of the sheep concurs with observations in h u m a n and mouse brain (Kumpulainen and Korhonen, 1982; K u m p u l a i n e n and Nystrom, 1981; Ghandour et al., 1980). However, in man and the mouse, CAII is also demonstrated in outer lamellae &myelin and in cells with light microscopical, ultrastructural and immunocytochemical characteristics of oligodendroglia (Langley, Ghandour, Vincendon and Gombos, 1980; G h a n d o u r el al., 1980). There is some controversy as to whether CAII occurs in astrocytes in the rodent brain. G h a n d o u r et al. (1980), by a combined peroxidase and immunofluorescence technique, showed discrete, non-overlapping populations of GFAP- or CAII-expressing glia in adult mouse brain, but C a m m e r and Tansey (1988a) have demonstrated co-expression of CAII and GFAP in glia of adult rat cerebral cortex. In 3- to 4-week-old mice, simultaneous expression of GFAP and C A I I has been demonstrated in glia of grey matter (presumed protoplas-
Fig. 3.
Mouse cerebrum. Double immunohistochemical stain showing peroxidase-labelled GAII-expressing cells (brown) and alkaline phosphatase-labelled GFAP-expressing cells (blue), PAP and APAAP. CAII and GFAP stain x 600.
Fig. 4a.
Adult sheep cerebellum. Double immunohistochemical stain showing GFAP-expressing ceils (blue) of the granule cell layer and cells of white matter showing co-localization of reaction products (blue-black or black). PAP and APAAP. CAII and GFAP stain x 500.
Fig. 4b.
Adult sheep spinal cord. Detail of CAII- and GFAP-expressing cell. x 1050.
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M. Jeffrey et al.
mic astrocytes) of the cerebral cortex but not in fibrous astrocytes of white matter (Cammer and Tansey, 1988b,c). CAII has also been demonstrated in embryonic mouse brain in cells thought to be derived from radial glia (Benjelloun, Delaunoy, Gomes, De-Vitry, Langui and Dupouey, 1986) but the glial cell lineage for which these cells were destined was not determined. Radial glial cells potentially give rise to both oligodendrocytes and astrocytes (Choi, 1988; Hirano and Goldman, 1988). Reactive astrocytes (Cammer and Tansey, 1988c) and other modified astrocytes such as Mfiller cells of the retina (Kumpulainen and Korhonen, 1982) may also express CAII. I n the present study the immunoperoxidase method of double labelling applied to mduse brain showed GFAP expressing cells which were morphologically similar to astrocytes and distinct from CAII expressing cells which resembled oligodendrocytes, confirming the previous results of Ghandour et al. (1980). In sheep brain, CAII and GFAP were simultaneously expressed by a proportion of white matter glia which were morphologically similar to astroeytes and contributed processes to the glial limitans and were thus interpreted as fibrous astrocytes. Reactive astroglia were not observed in this material. That CAII expression was present in glia of the 29-day-old lamb and adult sheep but absent from glia of newborn lambs is surprising. Previous studies of rodents brains have shown that galactocerebroside, myelin basic protein and proteolipid protein are sequentially expressed by myelinating oligodendroglia during development (Bologa-Sandru, Siegrist, Z'Graggen, Hoffmann, Wiesmann, Dahl and Herschkowitz, 1981; Hartman, Agrawal, Agrawal and Kalmbach, 1982), with CAII expression occurring only in mature oligodendroglia (Ghandour et al., 1980; Kumpulainen and Korhonen, 1982). The apparent absence of expression of CAII in glia of newborn sheep may also be interpreted to suggest that some maturation of fibrous astrocytes may be required before CAII is fnlly expressed in developing white matter of the sheep. Additional studies of sheep should be undertaken to confirm the observation that ovine fibrous astrocytes express CAII. Nevertheless, the observations that ovine oligodendroglia do not express CAII and the apparent expression of CAII in ovine fibrous astrocytes, taken in conjunction with observations of expression of CAII in glial progenitor cells (Benjelloun et al., 1986), protoplasmic astrocytes (Cammer and Tansey 1988c) and modified astrocytes (Kumpulainen and Korhonen, 1982) as well as in oligodendrocytes of rodents and man, suggest that expression of this glial marker enzyme is controlled by epigenetic factors which determine the secondary local environmental conditions in which glial cells develop, The cellular origins of CAII expression in ventral horn grey matter was not determined. Some processes which expressed both GFAP and CAII were probably those of protoplasmic astrocytes. Astroeytes of dorsal horn grey matter invariably expressed only GFAP.
Acknowledgment We are grateful to P. V. Steart, Southampton General Hospital, for the kind donation of CAII antiserum.
CAII in Sheep
343
References Barlow, R. M. and Patterson, D. S. P. (1982). Border disease of sheep: a virus-induced teratogenic disorder. Advances in Veterinary Medicine, 36, 1-80. Benjelloun, S., Delaunoy, J. P., Gomes, D., De-Vitry, F., Langui, D. and Dupouey, P. (1986). Early expression of Carbonic Anhydrase II (CAll) in transitory glial cells of the developing murine nervous system. DevelopmentalNeuroscience, 8, 150-159. Bologa-Sandru, L., Siegrist, H. P., Z'Graggen, A., Hofmann, K., Wiesmann, U., Dahl, D. and Herschkowitz, N. (1981). Expression of antigenic markers during the development of oligodendrocytes in mouse brain cultures. Brain Research, 210, 217-229. Cammer, W. and Tansey, F. A. (1988a). Carbonic anhydrase immunostaining in astrocytes in the rat cerebral cortex. Journal of Neurochemistry, 50, 319-322. Cammer, W. and Tansey, F. A. (1988b). The astrocyte as a locus of carbonic anhydrase in the brains of normal and dysmyelinating mutant mice. Journal of Comparative Neurology, 275, 65-75. Cammer, W. and Tansey, F. A. (1988c). Localization ofglial cell antigens in the brains of young, normal mice and the dysmyelinating mutant mice, jimpy and shiverer. Journal of Neuroscience Research, 20, 23-31. Choi, B. H. (1988). Prenatal gliogenesis in the developing cerebrum of the mouse. Glia, 1, 308-316. Ghandour, M. S., Langley, O. K., Vincendon, G., Gombos, G., Fillippi, D., Limozin, N., Dalmassa, D. and Laurent, G. (1980). Immunochemical and immunohistochemical study of carbonic anhydrase II in adult rat cerebellum. A marker for oligodendrocytes. Neuroscience, 5, 559-571. Hartman, B. K., Agrawal, H. C., Agrawal, D. and Kalrnbach, S. (1982). Development and maturation of central nervous system myelin: comparison of immunohistochemical localisation of proteolipid protein and basic protein in myelin and oligodendrocytes. Proceedingsof the National Academy of Science USA, 79, 4217--4220. Hirano, M. and Goldman, J. E. (1988). Gliogenesis in rat spinal cord: Evidence for origin of astrocytes and oligodendrocytes from radial precursors. Journal of
Neuroscience Research, 21, 155- 157. Jeffrey, M., Wells, G. A. FI. and Bridges, A. W. (1990). An immunohistochemical study of the topography and cellular localization of three nervous system proteins in ovine central and peripheral nervous systems. Journal of Comparative Pathology, 1037 23-35. Kumpulainen, T. and Nystrom, S. H. M. (1981). Immunohistochemical localisation of carbonic anhydrase isoenzyme C in human brain. Brain Research, 220, 220-225. Kumpulainen, T. and Korhonen, L. K. (1982). Immunochemical locaIisation of carbonic anhydrase isoenzyme within the central and peripheral nervous system of the mouse. Journal of Histochemistry and Cytochemistry, 30, 283-292. Langley, O. K., Ghandour, M. S., Vincendon, G. and Gombos, G. (1980). Carbonic anhydrase: an uhrastructural study in rat cerebellum. Journal ofHistochemistry, 12~ 473-483. Weller, R. O., Steart, P. V. and Moore, I. E. (1986). Carbonic anhydrase C as a marker antigen in the diagnosis of choroid plexus papillomas: an immunoperoxidase study. In: Biology of Brain Tumours. M. D. Walker and D. G. T. Thomas, Eds. Martinus Nijhoff, Boston, pp. 115-120.
February 12th, 1990"] Accepted, May 19th, 1990 J EReceived,
Carbonic Anhydrase II Expression in Fibrous Astrocytes of the Sheep M. Jeffrey, G. A. H. Wells* and A. W. Bridges* Lasswade Veterinary Laboratory, Bush Estate, Penicuik, Midlothian EH26 OSA, and *Pathology Department, Central Veterinary Laboratory, New Haw, Weybridge, Surrey KT15 3NB, U.K.
Summary Immunohistochemical expression of carbonic anhydrase isoenzyme II (CAII) was demonstrated in a population of fibrous astrocytes in a young lamb and an adult sheep. Such cells were identified by co-expression of CAII and glial fibrillary acidic protein, nuclear morphology and their contribution of glial fibrillary acidic protein reactive processes to the glial limitans. Similar cells were not identified in neonatal lambs. As in man and mouse, CAII was also expressed in ehoroid plexus epithelium occurring in neonate, young and adult sheep brain. In contrast, however, to man and mouse, CAII was not expressed in sheep oligodendrocytes.
Introduction Carbonic anhydrase is a widespread catalyst of reversible hydration of carbon dioxide and is thought to be involved in the control of the ion, fluid and acidbase balance in various organs. In the CNS, the carbonic anhydrase isoenzyme II (CAII) is generally regarded as a glial specific enzyme and is considered to be a useful marker for oligodendroglia (Ghandour, Langley, Vincendon, Gombos, Fillippi, Limozin, Dalmassa and Laurent, 1980; Cammer and Tansey, 1988a). As a preliminary investigation to the study ofgliogenesis in Border disease, a dysmyelinating disease of sheep (Barlow and Patterson, 1982), the cellular localization of glial fibrillary acidic protein (GFAP), CAII and myelin basic protein (MBP) was investigated in the normal sheep (Jeffrey, Wells and Bridges, 1990). The distribution of GFAP and MBP was found to be similar to that reported for some other mammals but the distribution and cellular localization of CAII differed fi'om that of previous accounts. We report here the localization of CAII in fibrous astrocytes of a 29-day-old lamb and an adult sheep, but absence of such expression in the neonate lamb.
Materials and Methods Brains and spinal cords were removed from six sheep which showed no clinical or pathological evidence of central nervous system abnormality, and were immediately placed in fixative. Details of the breeds, ages and fixatives used are shown in Table 1. 0021-9975/91/040337+ 07 $03.00/0
338
M. Jeffrey
et al.
Table 1
Ages and breeds o f s h e e p , t i s s u e f i x a t i v e s a n d l o c a l i z a t i o n o f C A I I Age
Breed
Fixative
1 day I clay
Suffolk C h e v i o t cross
1 day* 1 day 29 d a y s adult
Dorset Border Dorset Dorset
i 0 per c e n t neutral formalin 4 per cent p a r a f o r m a l d e h y d e / 0'1 p e r cent g l u t a r a l d e h y d e forrnol s u b l i m a t e C a r n o y ' s fluid Carnoy's fluid Carnoy's fluid
horn Leicester horn horn
Localization of CAII Chornid plexus Glia +
-
+ + +
+ +
* D i e d at b i r t h following dystokia.
All the sheep, but for one lamb, which died at birth following dystokia, were killed by intravenous injection of pentobarbitone sodium. Tissues from three animals were placed in Carnoy's fluid and, after 1 h, the cerebellum was removed and cut parasagittally; the remaining brain and spinal cord segments C3, T~ and Ls_~ were cut coronally at 0"5 cm intervals and all tissues were returned to fixative. After a further 2 h fixation, tissues were trimmed and placed in fresh fixative for an additional 2 h before routine processing and embedding in paraffin wax. Tissues from three other animals were placed in phosphate-buffered neutral 10 per cent formalin, formol sublimate or 4- per cent paraformaldehyde with 0" 1 per cent glutaraldehyde, respectively, retained whole and fixed for 48 h to 3 weeks prior to processing and paraffin wax embedding. Commercially available monoclonal GFAP antiserum (ICN Ltd) was used for double immunostaining. CAII antiserum was a gift from P. V. Steart. Its production and testing have been published previously (Weller, Steart and Moore, 1986). Sections 5 g m thick were cut and stained for the presence of" CAII by the peroxidase-antiperoxidase technique (PAP). Brain fixed in Carnoy's fluid from 4-week-old mice was used as positive control for CAII immunostaining. Double staining for GFAP and CAII was performed on selected sections of Carnoy's fixed tissues. With this technique, staining for CAII was by the PAP method and staining for GFAP was by an alkaline phosphatase-anti-atkaline phosphatase (APAAP) method. Endogenous phosphatase activity was inhibited by levamisole and APAAP antibody complex was visualized by fast blue. Sections were mounted in glycergel (Dako U K Ltd.).
Results C A I I expression could be identified in tissues fixed either in C a r n o y ' s fluid o r in 4 p e r cent p a r a f o r m a l d e h y d e a n d 0" 1 per cent g l u t a r a l d e h y d e b u t staining was of g r e a t e r intensity in tissues fixed in the former. C A I I expression was n o t s e e n in b u f f e r e d formalin or formol sublimate fixed tissues. C A I I was p r e s e n t in t h e c y t o p l a s m of choroid plexus epithelium in all ages o f sheep e x a m i n e d (Fig. 1). I n the n e o n a t a l tissues fixed in C a r n o y ' s fluid, C A I I was n o t found in the g r e y m a t t e r n e u r o p i l or white m a t t e r . I n the 29-day-old l a m b , weak C A I I e x p r e s sion was p r e s e n t in glial cell processes a n d neuropil of the v e n t r a l h o r n g r e y m a t t e r a n d in processes and perinuclear cytoplasm o f some glial cells in v e n t r a l a n d lateral funiculi o f the spinal cord. I n white m a t t e r , the processes o f s t a i n e d
CAII i n S h e e p
339
Fig. 1. 29-day-old sheep. CAII expression in choroid plexus epithelium. PAP CAII stain x 1280. Fig. 2.
Adult sheep spinal cord, CAII expression in a white matter glial cell. PAP CAII stain x 1680,
340
M. Jeffrey e t al.
CAII in Sheep
341
cells were single, or numbered up to four and were sharply delineated, u n b r a n c h e d and tapered from the cell body. Nuclei remained unstained. In the adult sheep, strong CAII expression was present in glial cells of white matter throughout the brain and spinal cord (Fig. 2). The CAII-expressing processes of these cells were contiguous with CAII-positive pericapillary glial limitans. As in the 29-day-old, lamb CAII expression was present in glial cell processes and neuropil of the ventral horns. Mouse brain CAII expression was similar to that previously described ( G h a n d o u r et al., 1980). In double-stained preparations of mouse brain, glial cells expressed either GFAP or CAII (Fig. 3) but in similar preparations of the 29-day-old and adult sheep brains, co-expression of CAII and GFAP was evident in some white matter glial cells (Figs 4a and b). In grey matter, glia that were G F A P expressing contained no demonstrable CAII. Co-expression of C A I I and GFAP was a feature of some glial processes in spinal cord ventral grey m a t t e r while some other similar glial processes expressed only CAII. Discussion
These results demonstrate that the CNS enzyme CAII is present in sheep and that its cellular localization differs from that previously described for man and rodents. T h e preservation of CAII expression according to the fixatives employed was similar to that previously reported (Ghandour el aL, 1980; K u m p u l a i n e n and Korhonen, 1982). The occurrence of CAII in choroid plexus epithelium of the sheep concurs with observations in h u m a n and mouse brain (Kumpulainen and Korhonen, 1982; K u m p u l a i n e n and Nystrom, 1981; Ghandour et al., 1980). However, in man and the mouse, CAII is also demonstrated in outer lamellae &myelin and in cells with light microscopical, ultrastructural and immunocytochemical characteristics of oligodendroglia (Langley, Ghandour, Vincendon and Gombos, 1980; G h a n d o u r el al., 1980). There is some controversy as to whether CAII occurs in astrocytes in the rodent brain. G h a n d o u r et al. (1980), by a combined peroxidase and immunofluorescence technique, showed discrete, non-overlapping populations of GFAP- or CAII-expressing glia in adult mouse brain, but C a m m e r and Tansey (1988a) have demonstrated co-expression of CAII and GFAP in glia of adult rat cerebral cortex. In 3- to 4-week-old mice, simultaneous expression of GFAP and C A I I has been demonstrated in glia of grey matter (presumed protoplas-
Fig. 3.
Mouse cerebrum. Double immunohistochemical stain showing peroxidase-labelled GAII-expressing cells (brown) and alkaline phosphatase-labelled GFAP-expressing cells (blue), PAP and APAAP. CAII and GFAP stain x 600.
Fig. 4a.
Adult sheep cerebellum. Double immunohistochemical stain showing GFAP-expressing ceils (blue) of the granule cell layer and cells of white matter showing co-localization of reaction products (blue-black or black). PAP and APAAP. CAII and GFAP stain x 500.
Fig. 4b.
Adult sheep spinal cord. Detail of CAII- and GFAP-expressing cell. x 1050.
342
M. Jeffrey et al.
mic astrocytes) of the cerebral cortex but not in fibrous astrocytes of white matter (Cammer and Tansey, 1988b,c). CAII has also been demonstrated in embryonic mouse brain in cells thought to be derived from radial glia (Benjelloun, Delaunoy, Gomes, De-Vitry, Langui and Dupouey, 1986) but the glial cell lineage for which these cells were destined was not determined. Radial glial cells potentially give rise to both oligodendrocytes and astrocytes (Choi, 1988; Hirano and Goldman, 1988). Reactive astrocytes (Cammer and Tansey, 1988c) and other modified astrocytes such as Mfiller cells of the retina (Kumpulainen and Korhonen, 1982) may also express CAII. I n the present study the immunoperoxidase method of double labelling applied to mduse brain showed GFAP expressing cells which were morphologically similar to astrocytes and distinct from CAII expressing cells which resembled oligodendrocytes, confirming the previous results of Ghandour et al. (1980). In sheep brain, CAII and GFAP were simultaneously expressed by a proportion of white matter glia which were morphologically similar to astroeytes and contributed processes to the glial limitans and were thus interpreted as fibrous astrocytes. Reactive astroglia were not observed in this material. That CAII expression was present in glia of the 29-day-old lamb and adult sheep but absent from glia of newborn lambs is surprising. Previous studies of rodents brains have shown that galactocerebroside, myelin basic protein and proteolipid protein are sequentially expressed by myelinating oligodendroglia during development (Bologa-Sandru, Siegrist, Z'Graggen, Hoffmann, Wiesmann, Dahl and Herschkowitz, 1981; Hartman, Agrawal, Agrawal and Kalmbach, 1982), with CAII expression occurring only in mature oligodendroglia (Ghandour et al., 1980; Kumpulainen and Korhonen, 1982). The apparent absence of expression of CAII in glia of newborn sheep may also be interpreted to suggest that some maturation of fibrous astrocytes may be required before CAII is fnlly expressed in developing white matter of the sheep. Additional studies of sheep should be undertaken to confirm the observation that ovine fibrous astrocytes express CAII. Nevertheless, the observations that ovine oligodendroglia do not express CAII and the apparent expression of CAII in ovine fibrous astrocytes, taken in conjunction with observations of expression of CAII in glial progenitor cells (Benjelloun et al., 1986), protoplasmic astrocytes (Cammer and Tansey 1988c) and modified astrocytes (Kumpulainen and Korhonen, 1982) as well as in oligodendrocytes of rodents and man, suggest that expression of this glial marker enzyme is controlled by epigenetic factors which determine the secondary local environmental conditions in which glial cells develop, The cellular origins of CAII expression in ventral horn grey matter was not determined. Some processes which expressed both GFAP and CAII were probably those of protoplasmic astrocytes. Astroeytes of dorsal horn grey matter invariably expressed only GFAP.
Acknowledgment We are grateful to P. V. Steart, Southampton General Hospital, for the kind donation of CAII antiserum.
CAII in Sheep
343
References Barlow, R. M. and Patterson, D. S. P. (1982). Border disease of sheep: a virus-induced teratogenic disorder. Advances in Veterinary Medicine, 36, 1-80. Benjelloun, S., Delaunoy, J. P., Gomes, D., De-Vitry, F., Langui, D. and Dupouey, P. (1986). Early expression of Carbonic Anhydrase II (CAll) in transitory glial cells of the developing murine nervous system. DevelopmentalNeuroscience, 8, 150-159. Bologa-Sandru, L., Siegrist, H. P., Z'Graggen, A., Hofmann, K., Wiesmann, U., Dahl, D. and Herschkowitz, N. (1981). Expression of antigenic markers during the development of oligodendrocytes in mouse brain cultures. Brain Research, 210, 217-229. Cammer, W. and Tansey, F. A. (1988a). Carbonic anhydrase immunostaining in astrocytes in the rat cerebral cortex. Journal of Neurochemistry, 50, 319-322. Cammer, W. and Tansey, F. A. (1988b). The astrocyte as a locus of carbonic anhydrase in the brains of normal and dysmyelinating mutant mice. Journal of Comparative Neurology, 275, 65-75. Cammer, W. and Tansey, F. A. (1988c). Localization ofglial cell antigens in the brains of young, normal mice and the dysmyelinating mutant mice, jimpy and shiverer. Journal of Neuroscience Research, 20, 23-31. Choi, B. H. (1988). Prenatal gliogenesis in the developing cerebrum of the mouse. Glia, 1, 308-316. Ghandour, M. S., Langley, O. K., Vincendon, G., Gombos, G., Fillippi, D., Limozin, N., Dalmassa, D. and Laurent, G. (1980). Immunochemical and immunohistochemical study of carbonic anhydrase II in adult rat cerebellum. A marker for oligodendrocytes. Neuroscience, 5, 559-571. Hartman, B. K., Agrawal, H. C., Agrawal, D. and Kalrnbach, S. (1982). Development and maturation of central nervous system myelin: comparison of immunohistochemical localisation of proteolipid protein and basic protein in myelin and oligodendrocytes. Proceedingsof the National Academy of Science USA, 79, 4217--4220. Hirano, M. and Goldman, J. E. (1988). Gliogenesis in rat spinal cord: Evidence for origin of astrocytes and oligodendrocytes from radial precursors. Journal of
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February 12th, 1990"] Accepted, May 19th, 1990 J EReceived,
