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Improved survival of injured sciatic nerve Schwann cells in mice lacking the p75 receptor
Neuroscience Letters 272 (1999) 191±194 www.elsevier.com/locate/neulet
Improved survival of injured sciatic nerve Schwann cells in mice lacking the p75 receptor Catharine C. Ferri, Mark A. Bisby* Department of Physiology, Queen's University, Kingston, Ontario K7L 3N6, Canada Received 15 July 1999; received in revised form 19 July 1999; accepted 19 July 1999
Abstract Following nerve injury, there is a dramatic increase in the expression of the p75 neurotrophin receptor on Schwann cells (Heumann, R., Korsching, S., Bandtlow, C. and Thoenen, H., Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection. J. Cell Biol., 104 (1987) 1623-1631. Taniuchi, M., Clark, H.B., Schweitzer, J.B. and Johnson, E.M., Expression of nerve growth factor by Schwann cells of axotomized peripheral nerves: ultrastructural location, suppression by axonal contact, and binding properties. J. Neurosci., 8 (1988) 664681.), however the role of the p75 receptor following injury remains unclear. Previous studies have shown that the p75 receptor may play a role in the apoptosis of several cell types. To better understand the role of the p75 receptor in the events following nerve injury, we have compared apoptosis in injured sciatic nerves of adult mice lacking functional p75 receptors and Balb-C (wild-type) mice. Following sciatic nerve crush or resection injuries, we used a ¯uorescent FragEL DNA fragmentation method to examine the extent of cellular apoptosis in distal nerve segments 5 days, 21 days and 4 months later. Nerve injury induced large numbers of apoptotic nuclei in nerves of both strains, but in p75 knockout mice, the density of apoptotic cells was lower compared to Balb-C mice, 21 days following injury. The p75 receptor may promote apoptosis in Schwann cells when axons are regenerating into the denervated nerve stump. q 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Schwann cells; Apoptosis; p75 low-af®nity neurotrophin receptor; Sciatic nerve; Crush injury
Schwann cells play a critical role in peripheral nerve regeneration [2,6,10,13] and may be primary determinants of successful regeneration. Extensive Schwann cell mitosis occurs in the distal nerve stump following nerve injury but, in common with other proliferative tissues, actual cell numbers depend on a balance between proliferation and death. Schwann cell death may be in¯uenced by the p75 low-af®nity neurotrophin receptor, which becomes strongly upregulated in Schwann cells following nerve injury [11,16]. The p75 receptor has been implicated in promoting apoptosis in a number of cell types, including retinal neurons [9], oligodendrocytes [3], facial motoneurons [8], and Schwann cells [15]. To understand better the role of the p75 receptor on Schwann cells following nerve injury, sciatic nerve lesions were performed in mice lacking a functional p75 receptor (p75 knockout) and Balb-C (wild-type)
* Corresponding author. MRC of Canada, 1600 Scott Street, Postal Locator 3105A, Ottawa, Ontario, K1A 0W9, Canada. Tel.: 11-613-954-1959; fax: 11-613-952-2277. E-mail address: [email protected] (M.A. Bisby)
mice, and subsequent apoptotic cell densities were measured. Twenty-four adult male Balb-C mice (age 8±12 weeks) from Charles River, and 21 adult male p75 knockout (NGFRtm1Jae) mice (age 8±12 weeks), bred from p75 knockout mice obtained from Jackson Labs, were used. Mice were anaesthetized with metofane (methoxy¯urane) by nose cone and an incision was made on the left side at mid thigh level to expose the sciatic nerve via blunt dissection. In the short-term studies (5±21 days post-injury), crush injury was performed in 16 p75 knockout and 18 Balb-C mice by pulling the nerve against a blunt glass rod with a piece of thread for thirty seconds. Completeness of the lesion was assessed by inspection of the nerve. The crush site was then marked with forceps impregnated with charcoal. For mice examined 4 months following injury, the sciatic nerve was resected and the proximal end was de¯ected and sutured into muscle to prevent regeneration. In all mice following injury, muscle and connective tissue were realigned and the skin was closed with clips. Mice were killed at the following times after injury: 5
0304-3940/99/$ - see front matter q 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 61 8- 7
192
C.C. Ferri, M.A. Bisby / Neuroscience Letters 272 (1999) 191±194
C.C. Ferri, M.A. Bisby / Neuroscience Letters 272 (1999) 191±194
193
Table 1 Number of apoptotic cells/50 000 mm 2 in the sciatic nerve a
Balb-C p75 knockout
Uninjured sciatic nerve
Sciatic nerve 5 days after crush
Sciatic nerve 21 days after crush
Sciatic nerve 4 months after chronic denervation
0.08 ^ 0.08 (6) 0.08 ^ 0.08 (6)
6.66 ^ 2.0 (6) 7.96 ^ 1.2 (6)
63.7 ^ 5.4 (12) 31.9 ^ 6.1 (10)*
1.53 ^ 0.7 (6) 0.41 ^ 0.4 (5)
a Number of apoptotic cells/50 000 mm 2 in Balb-C and p75 knockout sciatic nerve, contralateral to injury, 5 days following crush, 21 days following crush, and 4 months following chronic denervation. Note that p75 knockout mice have fewer apoptotic cells, relative to Balb-C mice, 21 days following sciatic nerve crush injury. Means ^ SEM (n). *Signi®cant difference relative to Balb-C mice in the same experimental group (P , 0:05).
days (six p75 knockout and six Balb-C); 21 days (10 p75 knockout and 12 Balb-C); and 4 months (®ve p75 knockout and six Balb-C). Mice were deeply anaesthetized with sodium pentobarbitol, then perfused through the heart with phosphate-buffered saline (PBS), (pH 7.4). This was followed by perfusion with 4% paraformaldehyde in PBS. The right and left sciatic nerves were removed in all animals and placed in 30% sucrose in 4% paraformaldehyde overnight to cryoprotect them. Nerves were then frozen in isopentane cooled with dry ice. Ten-micrometer sections of the nerves were cut using a cryostat. A ¯uorescent FragEL DNA Fragmentation Detection Kit (Oncogene Research Products, QIA39) was used to examine the extent of cellular apoptosis in the distal sciatic nerve segment in uninjured, day 5, day 21 and 4 month post-injury sections. Intestine (Fig. 1I,J) and spleen from the same mice were also included in the analysis to serve as controls for detection of apoptosis. Sections were immersed in 4% formaldehyde in 0.1 M PBS for 15 min at room temperature. Sections were then washed with 0.1 M Tris buffered saline (TBS), followed by incubation with proteinase K in 10 mM Tris, (pH 8) (1:100) for 10 min at room temperature. Following a wash with 0.1 M TBS, sections were incubated with TdT (Terminal Deoxynucleotidyl Transferase) Equilibration buffer in dH2O (1:5) for 30 min. Sections were then incubated with TdT Enzyme in ¯uorescent-FragEL TdT Labeling Reaction Mix (1:19) for 1.5 h at 378C. Following washes with TBS, slides were coverslipped using ¯uorescent FragEL mounting media and edges were sealed with nailpolish. Fluorescent microscopy was used for examination. Slides were stored at 48C in the dark. An image (£312.5) of each section was obtained with a high-resolution CCD camera (WPI, Sarasota, FL) and was grabbed in computer memory. The number of intense red/orange ¯uorescent cells (Fig. 1) were counted manually in each section, and the area of the section was measured using Sigma Scan (SPSS, Chicago,
IL). The number of bright red ¯uorescent cells/50 000mm 2 was calculated and mean values were calculated for uninjured, day 5, day 21 and 4 month post-injury sciatic nerve in p75 knockout and Balb-C mice. Data were ®rst tested for normal distribution using Sigma Stat (Jandel Scienti®c Inc.), then a Student's t-test was used to determine signi®cant differences in the density of apoptotic cells between uninjured, day 5, day 21 and long-term p75 knockout and Balb-C mice. The number of apoptotic cells/50 000 mm 2 was examined in the uninjured sciatic nerve, 5 and 21 days following crush injury and 16 weeks following chronic denervation (Fig. 1; Table 1). Densities of apoptotic cells in both p75 knockout and Balb-C mice were increased in day 5 (Fig. 1C,D) and day 21 (Fig. 1E,F) sciatic nerve, compared with uninjured nerves, then decreased 16 weeks following chronic denervation (Fig. 1G,H). Density of apoptotic cells was signi®cantly lower in p75 knockout mice, than in Balb-C mice, 21 days following sciatic nerve crush (Fig. 1F). The density of apoptotic nuclei measured in the uninjured nerve segment of both p75 knockout and Balb-C mice was very low, indicating that little cell death occurs in the normal adult sciatic nerve of both p75 knockout and BalbC mice. The density of apoptotic cells in the distal nerve segment of both strains of mice 5 days following injury was higher than we expected, based on the results of Ekstrom [5]. This discrepancy may be due to increased sensitivity of the new ¯uorescent-FragEL DNA Fragmentation Detection technique used in the present studies, compared with the lesssensitive ISEL (in-situ end labeling) technique used by Ekstrom [5]. Control tissues revealed positive pro®les where apoptotic cells were expected, showing that this new technique is reliable. Our results show that the proliferation of Schwann cells in the distal nerve segment 5 day post-injury [4], is accompanied by signi®cant apoptosis [1]. Although ®broblasts and macrophages also proliferate
Fig. 1. Apoptotic cells in the sciatic nerve, longitudinal sections. (A,B) uninjured nerve of (A) Balb-C and (B) p75 knockout mice. (C,D) Five days following nerve crush in (C) Balb-C and (D) p75 knockout mice. (E,F) Twenty-one days following crush in (E) Balb-C and (F) p75 knockout mice. (G,H) Four months following chronic denervation in (G) Balb-C and (H) p75 knockout mice. (I,J) Apoptotic cells in intestinal villi (transverse section) of (I) Balb-C and (J) p75 knockout mice. In all sections, only intense red-orange ¯uorescent cells were considered apoptotic. Scale bar, 100 mm.
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C.C. Ferri, M.A. Bisby / Neuroscience Letters 272 (1999) 191±194
following nerve injury, Schwann cells remain the predominant cell type [14], hence the majority of apoptotic nuclei in the sciatic nerve probably represent Schwann cells. In both p75 knockout and Balb-C mice, an increased number of apoptotic cells are present in the sciatic nerve 21 days following injury, compared with any other time point. This may be related to the continuous increase in p75 expression, which in rat sciatic nerve reached a maximum one month after injury [18]. At 21 days following injury, p75 knockout mice nerves demonstrate signi®cantly less cell death, compared with Balb-C mice, indicating improved Schwann cell survival in the absence of the p75 receptor. The increased apoptosis observed at 21 days may be associated with the second phase of Schwann cell proliferation that occurs when regenerating axons grow into the denervated nerve stump [12], or with the pruning of excessive axon sprouts which occurs as regenerating axons successfully reinnervate their targets. In a study on human neuropathy tissue, it was noted that Schwann cell apoptosis appeared to be related to the pruning of regenerative sprouts [7]. In chronically denervated nerve, the density of apoptotic cells in the distal nerve segment had decreased, indicating that little cell death was occurring in p75 knockout and Balb-C mice. At 4 months following injury, Schwann cells may be replaced with connective tissue and ®broblasts [17], reducing the regenerative viability of the nerve. Although not statistically signi®cant, the number of apopototic nuclei was less in p75 knockout mice. S-100, a Schwann cell marker, was higher in p75 knockout than in Balb-C nerves, 4 months post-injury (unpublished results), suggesting that, consistent with a reduced rate of cell death, there may be more surviving Schwann cells in p75 knockout nerves. These results indicate that the p75 receptor is not critically involved in promoting apoptosis in Schwann cells during the initial wave of proliferation associated with Wallerian degeneration, but may play a role later on when axons are regenerating into the denervated nerve stump. Since Schwann cells are critical for peripheral nerve regeneration, improved Schwann cell survival may contribute to improved nerve regeneration in p75 knockout mice [8]. [1] Berciano, M.T., Calle, E., Fernandez, R. and Lafarga, M., Regulation of Schwann cell numbers in tellurium-induced neuropathy: apoptosis, supernumerary cells and internodal shortening. Acta. Neuropathol., 95 (1998) 269±279. [2] Bunge, R.P., Expanding roles for the Schwann cell:
[3]
[4]
[5] [6] [7] [8] [9] [10] [11]
[12] [13] [14]
[15]
[16]
[17] [18]
ensheathment, myelination, trophism and regeneration, Curr. Opin. Neurobiol., 3 (1993) 805±809. Casaccia-Bonne®l, P., Carter, B.D., Dobrowsky, R.T. and Chao, M.V., Death of Oligodendrocytes mediated by the interaction of nerve growth factor with its receptor p75. Nature, 383 (1996) 716±719. Clemence, A., Mirsky, R. and Jessen, K.R., Non-myelinforming Schwann cells proliferate rapidly during Wallerian degeneration in the rat sciatic nerve. J. Neurocytol., 18 (2) (1989) 185±192. Ekstrom, P.A.R., Neurones and glial cells of the mouse sciatic nerve undergo apoptosis after injury in vivo and in vitro. NeuroReport, 6 (1995) 1029±1032. Enver, M.K. and Hall, S.M., Are Schwann cells essential for axonal regeneration into muscle autografts? Neuropathol. Appl. Neurobiol., 20 (1994) 587±598. Erdem, S., Mendell, J.R. and Sahenk, Z., Fate of Schwann cells in CMT1A and HNPP: evidence for apoptosis. J. Neuropathol. Exp. Neurol., 57 (1998) 635±642. Ferri, C.C., Moore, F. and Bisby, M.A., Effects of facial nerve injury on mouse motoneurons lacking the p75 low-af®nity neurotrophin receptor. J. Neurobiol., 34 (1998) 1±9. Frade, J., Rodrigueztebar, A. and Barde, Y.A., Induction of cell death by endogenous nerve growth factor through its p75 receptor. Nature, 383 (1996) 166±168. Hall, S.M., Regeneration in cellular and acellular autografts in the peripheral nervous system. Neuropathol. Appl. Neurobiol., 12 (1986) 27±46. Heumann, R., Korsching, S., Bandtlow, C. and Thoenen, H., Changes of nerve growth factor synthesis in non-neuronal cells in response to sciatic nerve transection. J. Cell Biol., 104 (1987) 1623±1631. Pellegrino, R.G. and Spencer, P.S., Schwann cell mitosis in response to regenerating peripheral axons in vivo. Brain Res., 342 (1985) 16±25. Ramon y Cajal, S. and May, R.M., Degeneration and Regeneration in the Nervous System, Hafner, New York, 1928, p. 1. Salonen, V., Aho, H., Roytta, M. and Peltonen, J., Quanti®cation of Schwann cells and endoneurial ®broblast-like cells after experimental nerve trauma. Exp. Neurol., 149 (1988) 433±438. Soilu-Hanninen, M., Ekert, P., Bucci, T., Syroid, D., Bartlett, P.F. and Kilpatrick, T.J., Nerve growth factor signaling through p75 induces apoptosis in Schwann cells via a bcl2-independent pathway. J. Neurosci., 19 (1999) 4828±4838. Taniuchi, M., Clark, H.B., Schweitzer, J.B. and Johnson, E.M., Expression of nerve growth factor by Schwann cells of axotomized peripheral nerves: ultrastructural location, suppression by axonal contact, and binding properties. J. Neurosci., 8 (1988) 664±681. Weinberg, J.H. and Spencer, P.S., The fate of Schwann cells isolated from axonal contact. J. Neurocytol., 7 (1978) 555± 569. You, S., Petrov, T., Chung, P.H. and Gordon, T., The expression of the low af®nity nerve growth factor receptor in longterm denervated Schwann cells. Glia, 20 (1997) 87±100.
Improved survival of injured sciatic nerve Schwann cells in mice lacking the p75 receptor Catharine C. Ferri, Mark A. Bisby* Department of Physiology, Queen's University, Kingston, Ontario K7L 3N6, Canada Received 15 July 1999; received in revised form 19 July 1999; accepted 19 July 1999
Abstract Following nerve injury, there is a dramatic increase in the expression of the p75 neurotrophin receptor on Schwann cells (Heumann, R., Korsching, S., Bandtlow, C. and Thoenen, H., Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection. J. Cell Biol., 104 (1987) 1623-1631. Taniuchi, M., Clark, H.B., Schweitzer, J.B. and Johnson, E.M., Expression of nerve growth factor by Schwann cells of axotomized peripheral nerves: ultrastructural location, suppression by axonal contact, and binding properties. J. Neurosci., 8 (1988) 664681.), however the role of the p75 receptor following injury remains unclear. Previous studies have shown that the p75 receptor may play a role in the apoptosis of several cell types. To better understand the role of the p75 receptor in the events following nerve injury, we have compared apoptosis in injured sciatic nerves of adult mice lacking functional p75 receptors and Balb-C (wild-type) mice. Following sciatic nerve crush or resection injuries, we used a ¯uorescent FragEL DNA fragmentation method to examine the extent of cellular apoptosis in distal nerve segments 5 days, 21 days and 4 months later. Nerve injury induced large numbers of apoptotic nuclei in nerves of both strains, but in p75 knockout mice, the density of apoptotic cells was lower compared to Balb-C mice, 21 days following injury. The p75 receptor may promote apoptosis in Schwann cells when axons are regenerating into the denervated nerve stump. q 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Schwann cells; Apoptosis; p75 low-af®nity neurotrophin receptor; Sciatic nerve; Crush injury
Schwann cells play a critical role in peripheral nerve regeneration [2,6,10,13] and may be primary determinants of successful regeneration. Extensive Schwann cell mitosis occurs in the distal nerve stump following nerve injury but, in common with other proliferative tissues, actual cell numbers depend on a balance between proliferation and death. Schwann cell death may be in¯uenced by the p75 low-af®nity neurotrophin receptor, which becomes strongly upregulated in Schwann cells following nerve injury [11,16]. The p75 receptor has been implicated in promoting apoptosis in a number of cell types, including retinal neurons [9], oligodendrocytes [3], facial motoneurons [8], and Schwann cells [15]. To understand better the role of the p75 receptor on Schwann cells following nerve injury, sciatic nerve lesions were performed in mice lacking a functional p75 receptor (p75 knockout) and Balb-C (wild-type)
* Corresponding author. MRC of Canada, 1600 Scott Street, Postal Locator 3105A, Ottawa, Ontario, K1A 0W9, Canada. Tel.: 11-613-954-1959; fax: 11-613-952-2277. E-mail address: [email protected] (M.A. Bisby)
mice, and subsequent apoptotic cell densities were measured. Twenty-four adult male Balb-C mice (age 8±12 weeks) from Charles River, and 21 adult male p75 knockout (NGFRtm1Jae) mice (age 8±12 weeks), bred from p75 knockout mice obtained from Jackson Labs, were used. Mice were anaesthetized with metofane (methoxy¯urane) by nose cone and an incision was made on the left side at mid thigh level to expose the sciatic nerve via blunt dissection. In the short-term studies (5±21 days post-injury), crush injury was performed in 16 p75 knockout and 18 Balb-C mice by pulling the nerve against a blunt glass rod with a piece of thread for thirty seconds. Completeness of the lesion was assessed by inspection of the nerve. The crush site was then marked with forceps impregnated with charcoal. For mice examined 4 months following injury, the sciatic nerve was resected and the proximal end was de¯ected and sutured into muscle to prevent regeneration. In all mice following injury, muscle and connective tissue were realigned and the skin was closed with clips. Mice were killed at the following times after injury: 5
0304-3940/99/$ - see front matter q 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 61 8- 7
192
C.C. Ferri, M.A. Bisby / Neuroscience Letters 272 (1999) 191±194
C.C. Ferri, M.A. Bisby / Neuroscience Letters 272 (1999) 191±194
193
Table 1 Number of apoptotic cells/50 000 mm 2 in the sciatic nerve a
Balb-C p75 knockout
Uninjured sciatic nerve
Sciatic nerve 5 days after crush
Sciatic nerve 21 days after crush
Sciatic nerve 4 months after chronic denervation
0.08 ^ 0.08 (6) 0.08 ^ 0.08 (6)
6.66 ^ 2.0 (6) 7.96 ^ 1.2 (6)
63.7 ^ 5.4 (12) 31.9 ^ 6.1 (10)*
1.53 ^ 0.7 (6) 0.41 ^ 0.4 (5)
a Number of apoptotic cells/50 000 mm 2 in Balb-C and p75 knockout sciatic nerve, contralateral to injury, 5 days following crush, 21 days following crush, and 4 months following chronic denervation. Note that p75 knockout mice have fewer apoptotic cells, relative to Balb-C mice, 21 days following sciatic nerve crush injury. Means ^ SEM (n). *Signi®cant difference relative to Balb-C mice in the same experimental group (P , 0:05).
days (six p75 knockout and six Balb-C); 21 days (10 p75 knockout and 12 Balb-C); and 4 months (®ve p75 knockout and six Balb-C). Mice were deeply anaesthetized with sodium pentobarbitol, then perfused through the heart with phosphate-buffered saline (PBS), (pH 7.4). This was followed by perfusion with 4% paraformaldehyde in PBS. The right and left sciatic nerves were removed in all animals and placed in 30% sucrose in 4% paraformaldehyde overnight to cryoprotect them. Nerves were then frozen in isopentane cooled with dry ice. Ten-micrometer sections of the nerves were cut using a cryostat. A ¯uorescent FragEL DNA Fragmentation Detection Kit (Oncogene Research Products, QIA39) was used to examine the extent of cellular apoptosis in the distal sciatic nerve segment in uninjured, day 5, day 21 and 4 month post-injury sections. Intestine (Fig. 1I,J) and spleen from the same mice were also included in the analysis to serve as controls for detection of apoptosis. Sections were immersed in 4% formaldehyde in 0.1 M PBS for 15 min at room temperature. Sections were then washed with 0.1 M Tris buffered saline (TBS), followed by incubation with proteinase K in 10 mM Tris, (pH 8) (1:100) for 10 min at room temperature. Following a wash with 0.1 M TBS, sections were incubated with TdT (Terminal Deoxynucleotidyl Transferase) Equilibration buffer in dH2O (1:5) for 30 min. Sections were then incubated with TdT Enzyme in ¯uorescent-FragEL TdT Labeling Reaction Mix (1:19) for 1.5 h at 378C. Following washes with TBS, slides were coverslipped using ¯uorescent FragEL mounting media and edges were sealed with nailpolish. Fluorescent microscopy was used for examination. Slides were stored at 48C in the dark. An image (£312.5) of each section was obtained with a high-resolution CCD camera (WPI, Sarasota, FL) and was grabbed in computer memory. The number of intense red/orange ¯uorescent cells (Fig. 1) were counted manually in each section, and the area of the section was measured using Sigma Scan (SPSS, Chicago,
IL). The number of bright red ¯uorescent cells/50 000mm 2 was calculated and mean values were calculated for uninjured, day 5, day 21 and 4 month post-injury sciatic nerve in p75 knockout and Balb-C mice. Data were ®rst tested for normal distribution using Sigma Stat (Jandel Scienti®c Inc.), then a Student's t-test was used to determine signi®cant differences in the density of apoptotic cells between uninjured, day 5, day 21 and long-term p75 knockout and Balb-C mice. The number of apoptotic cells/50 000 mm 2 was examined in the uninjured sciatic nerve, 5 and 21 days following crush injury and 16 weeks following chronic denervation (Fig. 1; Table 1). Densities of apoptotic cells in both p75 knockout and Balb-C mice were increased in day 5 (Fig. 1C,D) and day 21 (Fig. 1E,F) sciatic nerve, compared with uninjured nerves, then decreased 16 weeks following chronic denervation (Fig. 1G,H). Density of apoptotic cells was signi®cantly lower in p75 knockout mice, than in Balb-C mice, 21 days following sciatic nerve crush (Fig. 1F). The density of apoptotic nuclei measured in the uninjured nerve segment of both p75 knockout and Balb-C mice was very low, indicating that little cell death occurs in the normal adult sciatic nerve of both p75 knockout and BalbC mice. The density of apoptotic cells in the distal nerve segment of both strains of mice 5 days following injury was higher than we expected, based on the results of Ekstrom [5]. This discrepancy may be due to increased sensitivity of the new ¯uorescent-FragEL DNA Fragmentation Detection technique used in the present studies, compared with the lesssensitive ISEL (in-situ end labeling) technique used by Ekstrom [5]. Control tissues revealed positive pro®les where apoptotic cells were expected, showing that this new technique is reliable. Our results show that the proliferation of Schwann cells in the distal nerve segment 5 day post-injury [4], is accompanied by signi®cant apoptosis [1]. Although ®broblasts and macrophages also proliferate
Fig. 1. Apoptotic cells in the sciatic nerve, longitudinal sections. (A,B) uninjured nerve of (A) Balb-C and (B) p75 knockout mice. (C,D) Five days following nerve crush in (C) Balb-C and (D) p75 knockout mice. (E,F) Twenty-one days following crush in (E) Balb-C and (F) p75 knockout mice. (G,H) Four months following chronic denervation in (G) Balb-C and (H) p75 knockout mice. (I,J) Apoptotic cells in intestinal villi (transverse section) of (I) Balb-C and (J) p75 knockout mice. In all sections, only intense red-orange ¯uorescent cells were considered apoptotic. Scale bar, 100 mm.
194
C.C. Ferri, M.A. Bisby / Neuroscience Letters 272 (1999) 191±194
following nerve injury, Schwann cells remain the predominant cell type [14], hence the majority of apoptotic nuclei in the sciatic nerve probably represent Schwann cells. In both p75 knockout and Balb-C mice, an increased number of apoptotic cells are present in the sciatic nerve 21 days following injury, compared with any other time point. This may be related to the continuous increase in p75 expression, which in rat sciatic nerve reached a maximum one month after injury [18]. At 21 days following injury, p75 knockout mice nerves demonstrate signi®cantly less cell death, compared with Balb-C mice, indicating improved Schwann cell survival in the absence of the p75 receptor. The increased apoptosis observed at 21 days may be associated with the second phase of Schwann cell proliferation that occurs when regenerating axons grow into the denervated nerve stump [12], or with the pruning of excessive axon sprouts which occurs as regenerating axons successfully reinnervate their targets. In a study on human neuropathy tissue, it was noted that Schwann cell apoptosis appeared to be related to the pruning of regenerative sprouts [7]. In chronically denervated nerve, the density of apoptotic cells in the distal nerve segment had decreased, indicating that little cell death was occurring in p75 knockout and Balb-C mice. At 4 months following injury, Schwann cells may be replaced with connective tissue and ®broblasts [17], reducing the regenerative viability of the nerve. Although not statistically signi®cant, the number of apopototic nuclei was less in p75 knockout mice. S-100, a Schwann cell marker, was higher in p75 knockout than in Balb-C nerves, 4 months post-injury (unpublished results), suggesting that, consistent with a reduced rate of cell death, there may be more surviving Schwann cells in p75 knockout nerves. These results indicate that the p75 receptor is not critically involved in promoting apoptosis in Schwann cells during the initial wave of proliferation associated with Wallerian degeneration, but may play a role later on when axons are regenerating into the denervated nerve stump. Since Schwann cells are critical for peripheral nerve regeneration, improved Schwann cell survival may contribute to improved nerve regeneration in p75 knockout mice [8]. [1] Berciano, M.T., Calle, E., Fernandez, R. and Lafarga, M., Regulation of Schwann cell numbers in tellurium-induced neuropathy: apoptosis, supernumerary cells and internodal shortening. Acta. Neuropathol., 95 (1998) 269±279. [2] Bunge, R.P., Expanding roles for the Schwann cell:
[3]
[4]
[5] [6] [7] [8] [9] [10] [11]
[12] [13] [14]
[15]
[16]
[17] [18]
ensheathment, myelination, trophism and regeneration, Curr. Opin. Neurobiol., 3 (1993) 805±809. Casaccia-Bonne®l, P., Carter, B.D., Dobrowsky, R.T. and Chao, M.V., Death of Oligodendrocytes mediated by the interaction of nerve growth factor with its receptor p75. Nature, 383 (1996) 716±719. Clemence, A., Mirsky, R. and Jessen, K.R., Non-myelinforming Schwann cells proliferate rapidly during Wallerian degeneration in the rat sciatic nerve. J. Neurocytol., 18 (2) (1989) 185±192. Ekstrom, P.A.R., Neurones and glial cells of the mouse sciatic nerve undergo apoptosis after injury in vivo and in vitro. NeuroReport, 6 (1995) 1029±1032. Enver, M.K. and Hall, S.M., Are Schwann cells essential for axonal regeneration into muscle autografts? Neuropathol. Appl. Neurobiol., 20 (1994) 587±598. Erdem, S., Mendell, J.R. and Sahenk, Z., Fate of Schwann cells in CMT1A and HNPP: evidence for apoptosis. J. Neuropathol. Exp. Neurol., 57 (1998) 635±642. Ferri, C.C., Moore, F. and Bisby, M.A., Effects of facial nerve injury on mouse motoneurons lacking the p75 low-af®nity neurotrophin receptor. J. Neurobiol., 34 (1998) 1±9. Frade, J., Rodrigueztebar, A. and Barde, Y.A., Induction of cell death by endogenous nerve growth factor through its p75 receptor. Nature, 383 (1996) 166±168. Hall, S.M., Regeneration in cellular and acellular autografts in the peripheral nervous system. Neuropathol. Appl. Neurobiol., 12 (1986) 27±46. Heumann, R., Korsching, S., Bandtlow, C. and Thoenen, H., Changes of nerve growth factor synthesis in non-neuronal cells in response to sciatic nerve transection. J. Cell Biol., 104 (1987) 1623±1631. Pellegrino, R.G. and Spencer, P.S., Schwann cell mitosis in response to regenerating peripheral axons in vivo. Brain Res., 342 (1985) 16±25. Ramon y Cajal, S. and May, R.M., Degeneration and Regeneration in the Nervous System, Hafner, New York, 1928, p. 1. Salonen, V., Aho, H., Roytta, M. and Peltonen, J., Quanti®cation of Schwann cells and endoneurial ®broblast-like cells after experimental nerve trauma. Exp. Neurol., 149 (1988) 433±438. Soilu-Hanninen, M., Ekert, P., Bucci, T., Syroid, D., Bartlett, P.F. and Kilpatrick, T.J., Nerve growth factor signaling through p75 induces apoptosis in Schwann cells via a bcl2-independent pathway. J. Neurosci., 19 (1999) 4828±4838. Taniuchi, M., Clark, H.B., Schweitzer, J.B. and Johnson, E.M., Expression of nerve growth factor by Schwann cells of axotomized peripheral nerves: ultrastructural location, suppression by axonal contact, and binding properties. J. Neurosci., 8 (1988) 664±681. Weinberg, J.H. and Spencer, P.S., The fate of Schwann cells isolated from axonal contact. J. Neurocytol., 7 (1978) 555± 569. You, S., Petrov, T., Chung, P.H. and Gordon, T., The expression of the low af®nity nerve growth factor receptor in longterm denervated Schwann cells. Glia, 20 (1997) 87±100.