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Astrocytes in the aged rat spinal cord fail to increase GFAP mRNA following sciatic nerve axotomy
Brain Research 759 Ž1997. 163–165
Short communication
Astrocytes in the aged rat spinal cord fail to increase GFAP mRNA following sciatic nerve axotomy Cynthia J.M. Kane ) , Terry J. Sims, Shirley A. Gilmore Department of Anatomy, UniÕersity of Arkansas for Medical Sciences, 4301 W. Markham, Slot 510, Little Rock, AR 72205, USA Accepted 11 March 1997
Abstract Aging in the brain is associated with specific changes in the astrocyte population. The present study establishes that similar changes occur in the aging spinal cord. The levels of glial fibrillary acidic protein ŽGFAP. mRNA were significantly increased 0.4-fold in aged 8to 17-month-old rats compared to young 2-month-old rats. The ability of astrocytes in the aging spinal cord to respond to a non-invasive CNS injury was compared to young rats 4 days following sciatic nerve axotomy. The level of GFAP mRNA was significantly increased 0.5-fold in the young rats in response to axotomy. In contrast, the level of GFAP mRNA in aged rats did not increase following injury above that present in non-axotomized rats of the same age. Keywords: Neuroglia; Astrocyte; Aging; Glial fibrillary acidic protein; Gene expression; Sciatic axotomy
The astrocyte population of the brain undergoes marked changes with age across species which include increases in the intermediate filament glial fibrillary acidic protein ŽGFAP.. Several-fold increases in GFAP mRNA and protein have been detected in the brain of rodents w6,9,11– 13,15x in association with aging. Analysis of human hippocampus and cerebral cortex revealed a 2- to 4-fold increase in GFAP mRNA independent of degenerative pathology in individuals over 60 years old w11x compared with younger individuals. The astrocyte response is of central importance following CNS injury and can either promote or deter recovery of neural function w1–3,10x. It is not clear if or how aging affects the ability of astrocytes to respond to CNS injury. Scheff et al. w14x suggest a less robust astrocytic response in older animals. In contrast, Goss and Morgan w7x suggest the astrocytic response is greater, but delayed, in older animals. The present study examines whether astrocytes in the aging, but otherwise normal, spinal cord of the rat undergo changes similar to those reported in the aging brain. The levels of GFAP mRNA were compared quantitatively in the lumbar spinal cord of young Ž2 months. and old Ž8, 11
)
Corresponding author. Fax: q1 Ž501. 686-6382; e-mail: [email protected]
or 17 months. Charles River CD rats. Fresh lumbar spinal cord centered on segments L4-L6 was dissected from anesthetized animals. RNA was isolated and GFAP mRNA was quantified by Northern hybridization with 32 P-labeled cDNA ŽFig. 1.. The level of GFAP mRNA was increased 0.36 " 0.02-fold in rats aged 8, 11, or 17 months compared to rats 2 months of age Ž P - 0.01.. There was no difference in the amount of GFAP mRNA in the lumbar spinal cord of the three age groups Ž8, 11 or 17 months. designated as ‘‘old’’ animals; therefore, animals of these ages were grouped together for further analysis as old animals G 8 months of age. The ability of astrocytes to respond to non-invasive injury with increased GFAP mRNA expression was compared in young and old rats following sciatic nerve axotomy. Previous work from this laboratory has revealed that axotomy of the sciatic nerve induces increased GFAP immunoreactivity in the spinal cord in young rats w4x, but the astrocytic response in old rats was unexplored. The right sciatic nerve was transected near the interface between the gluteal region and the thigh under anesthesia. The distal stump of the nerve was positioned so that an obvious gap was present between the cut ends. On the fourth post-operative day, or at an equivalent age in nonaxotomized controls, the L4-L6 region of spinal cord giving rise to the sciatic nerve was removed. As shown in Fig. 1, a significant 0.47 " 0.03-fold induction of GFAP mRNA was detected in the spinal cord of 2-month-old rats
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 3 5 9 - 4
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C.J.M. Kane et al.r Brain Research 759 (1997) 163–165
following sciatic axotomy compared to non-injured controls of the same age Ž P - 0.001.. In contrast, the level of GFAP mRNA in the spinal cord of animals 8, 11 or 17 months of age did not increase, but remained stable. The approximately 0.5-fold increased level of GFAP mRNA induced by injury in the spinal cord of young animals was similar to the approximately 0.4-fold constitutively increased level of GFAP mRNA in non-injured spinal cord of older animals. This study demonstrates that the level of GFAP mRNA is increased significantly in the lumbar spinal cord of aged rats compared to young rats. This is consistent with observations in the brain of humans and rodents w6,9,11–13,15x in which GFAP mRNA and protein increase with age.
Interestingly, the rat spinal cord developed stable, age-related changes in the astrocyte population at a less advanced age than reported for the brain. The present observations allow extension of the hypothesis proposed in the brain to the spinal cord, such that normal aging involves alterations in astrocytes similar to those associated with their activation following injury. Increased levels of GFAP expression by astrocytes appear to represent a generalized CNS phenomenon associated with aging. The absence of astrocytic response in the spinal cord of older animals following injury was striking. Several mechanisms may underlie this observation. First, the constitutively elevated expression of GFAP in the spinal cord of older animals, as demonstrated by mRNA levels in the
Fig. 1. GFAP mRNA levels in the lumbar spinal cord of young and old rats. A: RNA was isolated from the lumbar spinal cord of rats 2, 8, 11, or 17 months of age Ž‘‘y’’ non-axotomized control. and from ‘‘q’’ axotomized littermates of the same age 4 days after sciatic nerve transection. The 2.3 kb GFAP mRNA was detected by Northern blot hybridization and autoradiography of all samples in parallel. Each lane represents RNA from a single animal. B: the relative levels of GFAP mRNA were quantified by densitometry. The mean and standard error are indicated. ) P - 0.001; ) ) P - 0.01.
C.J.M. Kane et al.r Brain Research 759 (1997) 163–165
present study, may represent the maximum capacity of the astrocyte to synthesize GFAP. Achievement of maximum expression may preclude further enhancement in GFAP expression following injury. Second, the ability of astrocytes to respond to injury may be impaired or suppressed in older animals as suggested in the brain by Scheff et al. w14x. Third, the temporal course of the astrocytic response may be slower or delayed in older animals as suggested in the brain by Goss and Morgan w7x. It is noteworthy that the astrocytic response to sciatic axotomy analyzed in the present study did not involve a proliferation of astrocytes w5,8x. If proliferation occurred, enhanced expression of GFAP could result from an increase in the number of astrocytes within the tissue. Thus, the absence of enhanced GFAP mRNA in astrocytes in the aged spinal cord following sciatic nerve axotomy was independent of potential age-associated changes in astrocyte proliferation. Transection of the sciatic nerve in the present study elicited an astrocytic response in the spinal cord without invasion of the parenchyma of the CNS. This avoided potential complications in interpretation associated with an invasive injury. This is important because it allowed analysis of the astrocytic response to neuronal damage in the absence of direct trauma to CNS glia. Further, the lesion did not compromise the pial or glial barrier and did not disrupt the cerebrospinal fluid or the CNS microvasculature. Thus, the impaired response of astrocytes in the spinal cord of aged rats to injury did not involve potential age-associated changes in these aspects of CNS homeostasis. Since the sciatic axotomy lesion used in this investigation does not invade the parenchyma of the CNS and does not involve astrocyte proliferation, it will be a useful model to elucidate the mechanisms which underlie the differential astrocytic responses observed in young and aged animals.
Acknowledgements This work was funded by NIH ŽNS04761 and AG12411..
165
References w1x A. Bignami, D. Dahl, Gliosis, in: H. Kettenmann, B.R. Ransom ŽEds.., Neuroglia, Oxford University Press, New York, 1995, pp. 843–858. w2x M. Eddleston, L. Mucke, Molecular profile of reactive astrocytes – implications for their role in neurologic disease, Neuroscience 54 Ž1993. 15–36. w3x L.F. Eng, A.C.H. Yu, Y.L. Lee, Astrocytic response to injury, Prog. Brain Res. 94 Ž1992. 353–365. w4x S.A. Gilmore, T.J. Sims, J.E. Leiting, Astrocytic reactions in spinal gray matter following sciatic axotomy, Glia 3 Ž1990. 342–349. w5x S.A. Gilmore, R.C. Walls, Patterns of labeling of intraspinal reactive cells in rats injected with w 3 Hxthymidine prior to or following sciatic axotomy, Brain Res. 218 Ž1981. 1–13. w6x J.R. Goss, C.E. Finch, D.G. Morgan, Age-related changes in glial fibrillary acidic protein mRNA in the mouse brain, Neurobiol. Aging 12 Ž1991. 165–170. w7x J.R. Goss, D.G. Morgan, Enhanced glial fibrillary acidic protein RNA response to fornix transection in aged mice, J. Neurochem. 64 Ž1995. 1351–1360. w8x M.B. Graeber, W. Tetzlaff, W.J. Streit, G.W. Kreutzberg, Microglial cells but not astrocytes undergo mitosis following rat facial nerve axotomy, Neurosci. Lett. 85 Ž1988. 317–321. w9x D. Linnemann, T. Skarsfelt, Regional changes in expression of NCAM, GFAP, and S100 in aging rat brain, Neurobiol. Aging 15 Ž1994. 651–655. w10x R.J. McKeon, J. Silver, Functional significance of glial-derived matrix during development and regeneration, in: H. Kettenman, B.R. Ransom ŽEds.., Neuroglia, Oxford University Press, New York, 1995, pp. 398–410. w11x N.R. Nichols, J.R. Day, N.J. Laping, S.A. Johnson, C.E. Finch, GFAP mRNA increases with age in rat and human brain, Neurobiol. Aging 14 Ž1993. 421–429. w12x J.P. O’Callaghan, D.B. Miller, The concentration of glial fibrillary acidic protein increases with age in the mouse and rat brain, Neurobiol. Aging 12 Ž1991. 171–174. w13x H. Riol, C. Fages, M. Tardy, Transcriptional regulation of glial fibrillary acidic protein ŽGFAP.-mRNA expression during postnatal development of mouse brain, J. Neurosci. Res. 32 Ž1992. 79–85. w14x S.W. Scheff, L.S. Bernardo, C.W. Cotman, Decline in reactive fiber growth in the dentate gyrus of aged rats compared to young adult rats following entorhinal cortex removal, Brain Res. 199 Ž1980. 21–38. w15x A.P. Wagner, G. Reck, D. Platt, Evidence that Vq fibronectin, GFAP and S100b mRNAs are increased in the hippocampus of aged rats, Exp. Gerontol. 28 Ž1993. 135–143.
Short communication
Astrocytes in the aged rat spinal cord fail to increase GFAP mRNA following sciatic nerve axotomy Cynthia J.M. Kane ) , Terry J. Sims, Shirley A. Gilmore Department of Anatomy, UniÕersity of Arkansas for Medical Sciences, 4301 W. Markham, Slot 510, Little Rock, AR 72205, USA Accepted 11 March 1997
Abstract Aging in the brain is associated with specific changes in the astrocyte population. The present study establishes that similar changes occur in the aging spinal cord. The levels of glial fibrillary acidic protein ŽGFAP. mRNA were significantly increased 0.4-fold in aged 8to 17-month-old rats compared to young 2-month-old rats. The ability of astrocytes in the aging spinal cord to respond to a non-invasive CNS injury was compared to young rats 4 days following sciatic nerve axotomy. The level of GFAP mRNA was significantly increased 0.5-fold in the young rats in response to axotomy. In contrast, the level of GFAP mRNA in aged rats did not increase following injury above that present in non-axotomized rats of the same age. Keywords: Neuroglia; Astrocyte; Aging; Glial fibrillary acidic protein; Gene expression; Sciatic axotomy
The astrocyte population of the brain undergoes marked changes with age across species which include increases in the intermediate filament glial fibrillary acidic protein ŽGFAP.. Several-fold increases in GFAP mRNA and protein have been detected in the brain of rodents w6,9,11– 13,15x in association with aging. Analysis of human hippocampus and cerebral cortex revealed a 2- to 4-fold increase in GFAP mRNA independent of degenerative pathology in individuals over 60 years old w11x compared with younger individuals. The astrocyte response is of central importance following CNS injury and can either promote or deter recovery of neural function w1–3,10x. It is not clear if or how aging affects the ability of astrocytes to respond to CNS injury. Scheff et al. w14x suggest a less robust astrocytic response in older animals. In contrast, Goss and Morgan w7x suggest the astrocytic response is greater, but delayed, in older animals. The present study examines whether astrocytes in the aging, but otherwise normal, spinal cord of the rat undergo changes similar to those reported in the aging brain. The levels of GFAP mRNA were compared quantitatively in the lumbar spinal cord of young Ž2 months. and old Ž8, 11
)
Corresponding author. Fax: q1 Ž501. 686-6382; e-mail: [email protected]
or 17 months. Charles River CD rats. Fresh lumbar spinal cord centered on segments L4-L6 was dissected from anesthetized animals. RNA was isolated and GFAP mRNA was quantified by Northern hybridization with 32 P-labeled cDNA ŽFig. 1.. The level of GFAP mRNA was increased 0.36 " 0.02-fold in rats aged 8, 11, or 17 months compared to rats 2 months of age Ž P - 0.01.. There was no difference in the amount of GFAP mRNA in the lumbar spinal cord of the three age groups Ž8, 11 or 17 months. designated as ‘‘old’’ animals; therefore, animals of these ages were grouped together for further analysis as old animals G 8 months of age. The ability of astrocytes to respond to non-invasive injury with increased GFAP mRNA expression was compared in young and old rats following sciatic nerve axotomy. Previous work from this laboratory has revealed that axotomy of the sciatic nerve induces increased GFAP immunoreactivity in the spinal cord in young rats w4x, but the astrocytic response in old rats was unexplored. The right sciatic nerve was transected near the interface between the gluteal region and the thigh under anesthesia. The distal stump of the nerve was positioned so that an obvious gap was present between the cut ends. On the fourth post-operative day, or at an equivalent age in nonaxotomized controls, the L4-L6 region of spinal cord giving rise to the sciatic nerve was removed. As shown in Fig. 1, a significant 0.47 " 0.03-fold induction of GFAP mRNA was detected in the spinal cord of 2-month-old rats
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 3 5 9 - 4
164
C.J.M. Kane et al.r Brain Research 759 (1997) 163–165
following sciatic axotomy compared to non-injured controls of the same age Ž P - 0.001.. In contrast, the level of GFAP mRNA in the spinal cord of animals 8, 11 or 17 months of age did not increase, but remained stable. The approximately 0.5-fold increased level of GFAP mRNA induced by injury in the spinal cord of young animals was similar to the approximately 0.4-fold constitutively increased level of GFAP mRNA in non-injured spinal cord of older animals. This study demonstrates that the level of GFAP mRNA is increased significantly in the lumbar spinal cord of aged rats compared to young rats. This is consistent with observations in the brain of humans and rodents w6,9,11–13,15x in which GFAP mRNA and protein increase with age.
Interestingly, the rat spinal cord developed stable, age-related changes in the astrocyte population at a less advanced age than reported for the brain. The present observations allow extension of the hypothesis proposed in the brain to the spinal cord, such that normal aging involves alterations in astrocytes similar to those associated with their activation following injury. Increased levels of GFAP expression by astrocytes appear to represent a generalized CNS phenomenon associated with aging. The absence of astrocytic response in the spinal cord of older animals following injury was striking. Several mechanisms may underlie this observation. First, the constitutively elevated expression of GFAP in the spinal cord of older animals, as demonstrated by mRNA levels in the
Fig. 1. GFAP mRNA levels in the lumbar spinal cord of young and old rats. A: RNA was isolated from the lumbar spinal cord of rats 2, 8, 11, or 17 months of age Ž‘‘y’’ non-axotomized control. and from ‘‘q’’ axotomized littermates of the same age 4 days after sciatic nerve transection. The 2.3 kb GFAP mRNA was detected by Northern blot hybridization and autoradiography of all samples in parallel. Each lane represents RNA from a single animal. B: the relative levels of GFAP mRNA were quantified by densitometry. The mean and standard error are indicated. ) P - 0.001; ) ) P - 0.01.
C.J.M. Kane et al.r Brain Research 759 (1997) 163–165
present study, may represent the maximum capacity of the astrocyte to synthesize GFAP. Achievement of maximum expression may preclude further enhancement in GFAP expression following injury. Second, the ability of astrocytes to respond to injury may be impaired or suppressed in older animals as suggested in the brain by Scheff et al. w14x. Third, the temporal course of the astrocytic response may be slower or delayed in older animals as suggested in the brain by Goss and Morgan w7x. It is noteworthy that the astrocytic response to sciatic axotomy analyzed in the present study did not involve a proliferation of astrocytes w5,8x. If proliferation occurred, enhanced expression of GFAP could result from an increase in the number of astrocytes within the tissue. Thus, the absence of enhanced GFAP mRNA in astrocytes in the aged spinal cord following sciatic nerve axotomy was independent of potential age-associated changes in astrocyte proliferation. Transection of the sciatic nerve in the present study elicited an astrocytic response in the spinal cord without invasion of the parenchyma of the CNS. This avoided potential complications in interpretation associated with an invasive injury. This is important because it allowed analysis of the astrocytic response to neuronal damage in the absence of direct trauma to CNS glia. Further, the lesion did not compromise the pial or glial barrier and did not disrupt the cerebrospinal fluid or the CNS microvasculature. Thus, the impaired response of astrocytes in the spinal cord of aged rats to injury did not involve potential age-associated changes in these aspects of CNS homeostasis. Since the sciatic axotomy lesion used in this investigation does not invade the parenchyma of the CNS and does not involve astrocyte proliferation, it will be a useful model to elucidate the mechanisms which underlie the differential astrocytic responses observed in young and aged animals.
Acknowledgements This work was funded by NIH ŽNS04761 and AG12411..
165
References w1x A. Bignami, D. Dahl, Gliosis, in: H. Kettenmann, B.R. Ransom ŽEds.., Neuroglia, Oxford University Press, New York, 1995, pp. 843–858. w2x M. Eddleston, L. Mucke, Molecular profile of reactive astrocytes – implications for their role in neurologic disease, Neuroscience 54 Ž1993. 15–36. w3x L.F. Eng, A.C.H. Yu, Y.L. Lee, Astrocytic response to injury, Prog. Brain Res. 94 Ž1992. 353–365. w4x S.A. Gilmore, T.J. Sims, J.E. Leiting, Astrocytic reactions in spinal gray matter following sciatic axotomy, Glia 3 Ž1990. 342–349. w5x S.A. Gilmore, R.C. Walls, Patterns of labeling of intraspinal reactive cells in rats injected with w 3 Hxthymidine prior to or following sciatic axotomy, Brain Res. 218 Ž1981. 1–13. w6x J.R. Goss, C.E. Finch, D.G. Morgan, Age-related changes in glial fibrillary acidic protein mRNA in the mouse brain, Neurobiol. Aging 12 Ž1991. 165–170. w7x J.R. Goss, D.G. Morgan, Enhanced glial fibrillary acidic protein RNA response to fornix transection in aged mice, J. Neurochem. 64 Ž1995. 1351–1360. w8x M.B. Graeber, W. Tetzlaff, W.J. Streit, G.W. Kreutzberg, Microglial cells but not astrocytes undergo mitosis following rat facial nerve axotomy, Neurosci. Lett. 85 Ž1988. 317–321. w9x D. Linnemann, T. Skarsfelt, Regional changes in expression of NCAM, GFAP, and S100 in aging rat brain, Neurobiol. Aging 15 Ž1994. 651–655. w10x R.J. McKeon, J. Silver, Functional significance of glial-derived matrix during development and regeneration, in: H. Kettenman, B.R. Ransom ŽEds.., Neuroglia, Oxford University Press, New York, 1995, pp. 398–410. w11x N.R. Nichols, J.R. Day, N.J. Laping, S.A. Johnson, C.E. Finch, GFAP mRNA increases with age in rat and human brain, Neurobiol. Aging 14 Ž1993. 421–429. w12x J.P. O’Callaghan, D.B. Miller, The concentration of glial fibrillary acidic protein increases with age in the mouse and rat brain, Neurobiol. Aging 12 Ž1991. 171–174. w13x H. Riol, C. Fages, M. Tardy, Transcriptional regulation of glial fibrillary acidic protein ŽGFAP.-mRNA expression during postnatal development of mouse brain, J. Neurosci. Res. 32 Ž1992. 79–85. w14x S.W. Scheff, L.S. Bernardo, C.W. Cotman, Decline in reactive fiber growth in the dentate gyrus of aged rats compared to young adult rats following entorhinal cortex removal, Brain Res. 199 Ž1980. 21–38. w15x A.P. Wagner, G. Reck, D. Platt, Evidence that Vq fibronectin, GFAP and S100b mRNAs are increased in the hippocampus of aged rats, Exp. Gerontol. 28 Ž1993. 135–143.