144
Brain Resear~h, ~ iAi ([gt;0) i44 l~l,~
["lsevic~ BRES 23982
A defic
y of axonal regeneration in C57BL/6J mice
Lu Xin 1, P.M. R i c h a r d s o n 1, E Gervais 2 and E. S k a m e n e 2 1Division of Neurosurgery and 2Centrefor the Study of Host Resistance, McGill University and Montreal General Hospital, Montreal (Canada)
(Accepted 14 November 1989) Key words." Peripheral nerve regeneration; Axonal regeneration; Spinal root; Mouse genetics; Macrophage
Axonal regeneration within peripheral nerves and dorsal spinal roots was investigated in inbred strains of mice with known differences in macrophage recruitment and inflammatory functions. During the second week after sciatic nerve crush, counts of regenerating newly myelinated fibres were significantly lower in C57BL/6J mice than in 4 other strains. After dorsal root crush with or without concomitant sciatic nerve transection to enhance regeneration, fibre counts in roots of C57BL/6J were one-fifth of those in A/J mice. Axonal regeneration is subnormal in C57BL/6J mice but this defect appears not to be linked to known deficiencies in macrophage function. The importance of macrophages during peripheral nerve regeneration is not fully defined. During Wallerian degeneration, macrophages are recruited into the endoneurium 5, scavenge axon and myelin debris 11, stimulate the proliferation of Schwann cells 1, and induce the synthesis of N G F (nerve growth factor) by Schwann cells 3. Also, the brisk proliferation of resident microglial cells in the anterior horn of the spinal cord after peripheral nerve injury 2'8'12 raises the possibility that this class of macrophages 6 might influence the responses of neurons to axotomy. Genetically controlled differences in macrophage functions have been observed among inbred strains of mice 4. For example, A/J and BALB/cJ mice show defective recruitment of macrophages to the peritoneum following inflammatory responses, C57BL/6J, DBA/1J, and B A L B / c J mice carry a mutant form of the Bcg (Ity, Lsh) gene that confers resistance to intracellular infection 9 and C 3 H / H e J mice are subnormally activated by lipopolysaccharide to release interleukin-11°. Of course, these strains of mice also show many other phenotypic differences unrelated to macrophages. To seek possible correlation between macrophage function and axonal regeneration, experiments were performed on female mice, 9 weeks old, of 5 different strains: C57BL/6J, A/J, DBA1/J, BALB/cJ or C3H/HeJ. U n d e r general anesthesia (ketamine 150 mg/kg, xylazine 10 mg/kg intraperitoneally), the right sciatic nerve was exposed at the level of the obturator tendon and crushed for 10 s with jeweller's forceps. Eleven days later, the mice were sacrificed and segments of the c o m m o n peroneal nerve 10 mm and 15 mm distal to the crush site were removed. Nerve specimens were fixed in mixed
aldehydes, post-fixed in osmium tetroxide, dehydrated, and embedded in plastic (Epon 812). Cross-sections, 1 p m thick, were stained with Toluidine blue and examined blindly under oil-immersion light microscopy to count the number of myelinated axons. In all cases, the myelin sheaths were thinner than normal with no suggestion of spared fibres. At both 10 and 15 m m from the crush site, fibre counts were lower for C57BL/6J mice than for the other 4 strains (Fig. 1). At 15 mm, counts were 1.9-2.9 times higher in DBA1/J, C3H/HeJ, A/J or B A L B / C J
E E
tt) tZ O O
LU n-
_m LI.
MOUSE
STRAIN
Fig. 1. Fibre counts in the common peroneal nerve. Thinly myeLinated fibres were counted in the common peroneal nerve 15 mm distal to the site of crushing 11 days earlier. Note that counts are significantlylower for C57BL/6J mice than for the other 4 strains (mean + S.E.M.; n 4--11; *P < 0.05, **P < 0.01 by Student's t-test). =
Correspondence." P.M. Richardson, Division of Neurosurgery, Montreal General Hospital, 1650 Cedar Avenue, Montreal, Canada H3G IA4.
0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
145 mice than in C57BL/6J mice. The differences between C57BL/6J mice and other strains were significant (P < 0.03 by the Student's t-test) in 7/8 comparisons: C57BL/
6J and DBA/1J mice did not differ significantly in counts at 10 mm from the crush site. For all comparisons among the strains of mice excluding C57BL/6J, the differences
I-Z :3 0 (3 LU rr
2 CRUSH
[]
A/J
[]
C57BL/6J
200-
El
U.
cUT
100-
nerve intact
nerve cut
Fig. 2. Regeneration in L 5 dorsal spinal roots. The L~ dorsal roots were crushed bilaterally and the right sciatic nerve was cut. After sacrifice, 14 days later, the progress of regeneration was assayed by fibre counts in the roots near their junction with the spinal cord. The histograms show a 5-fold difference between the two strains for regeneration with or without a conditioning lesion (mean _+ S.E.M.; n = 9-13; *P < 0,05, **P < 0.01 by Student's t-test). Photomicrographs show that thinly myelinated fibres are abundant in the root from an A/J mouse (right) but sparse for a C57BL/6J mouse (left). Note also that macrophages with a foamy cytoplasm are more frequent in the C57BL/6J spinal root (magnification x 960).
146 failed to reach statistical significance. By this measure of p e r i p h e r a l nerve regeneration, regrowth was slower for C57BL/6J mice than in 4 o t h e r strains. In a n o t h e r group of experiments, regeneration within crushed dorsal spinal roots was c o m p a r e d in C57BL/6J and A/J strains of mice (Fig. 2). This experimental p r e p a r a t i o n serves to assay the contribution of the nerve cell body to axonal regeneration because axonal regeneration in a dorsal root is accelerated by a 'conditioning' injury to the corresponding nerve 7. U n d e r general anesthesia and microsurgical conditions, both L 5 (fifth l u m b a r ) dorsal roots were exposed and crushed near their ganglia: the right sciatic nerve was transected at the hip. A f t e r sacrifice, 14 days later, each L 5 dorsal root was r e m o v e d and semi-thin cross-sections were p r e p a r e d from p l a s t i c - e m b e d d e d segments of the root near its junction with the spinal cord. A g a i n the n u m b e r of thinly m y e l i n a t e d axons was counted blindly. In all mice as previously described in rats 7, fibre counts were higher on the side (right) with sciatic nerve transection than on the side (left) without nerve injury. In comparison between strains of fibre counts on either the left or right side, A/J mice showed a p p r o x i m a t e l y 5-fold m o r e regenerating fibres than C57BL/6J mice on the same side ( P < 0.04 by S t u d e n t ' s t-test). The interstrain differences in dorsal root r e g e n e r a t i o n were even m o r e m a r k e d than those in p e r i p h e r a l nerve regeneration. C57BL/6J mice are here shown to be deficient in
regeneration of m y e l i n a t e d fibres in peripheral nerves and dorsal spinal roots. It remains to be proved that m o t o r neurons or unmyelinated fibres also regenerate less quickly in this mouse strain. R e t r o g r a d e signals to sensory neurons and neuronal responses are at least partially retained in C57BL/6J mice: perhaps the interactions between growing axons and their local environment are abnormal. W h e t h e r the primary defect involves neurons, Schwann cells or a n o t h e r cell type is unknown. The deficiency in axonal regeneration in C57BL/6J mice does not a p p e a r to be linked to any of the 3 specific abnormalities of m a c r o p h a g e function described earlier. B A L B / c J mice resemble C57BL/6J mice in carrying a defective allele of the Bcg (Ity, Lsh) gene but differ in regenerative behaviour. Mouse strains differing in their ability to recruit m a c r o p h a g e s to an inflammatory site or be activated by lipopolysaccharide show a similar rate of axonal regeneration. The C57BL/6J and A/J strains of mice were chosen in part because they are the progenitors of a large set of recombinant inbred mice. By analysis of these recombinant inbred strains, the genetic locus or loci regulating the p h e n o t y p i c differences in p e r i p h e r a l nerve regeneration can now be identified.
1 Beuche, W. and Friede, R.L., The role of non-resident cells in Wallerian degeneration, J. Neurocytol., 13 (1984) 767-796. 2 Blinzinger, K. and Kreutzberg, G., Displacement of synaptic terminals from regenerating motoneurons by microglial cells, Z. Zellforsch., 85 (1968) 145-157. 3 Heumann, R., Lindholm, D., Bandtlow, C., Meyer, M., Radeke, M.J., Misko, T.P., Shooter, E. and Thoenen, H., Differential regulation of mRNA encoding nerve growth factor and its receptor in rat sciatic nerve during development, degeneration, and regeneration: role of macrophages, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 8735-8739. 4 Nesbitt, M.N. and Skamene, E., Recombinant inbred mouse strains derived from A/J and C57BL/6J: A tool for the study of genetic mechanisms in host resistance to infection and malignancy, J. Leukocyte Biol., 36 (1984) 357-364. 5 Perry, V.H., Brown, M.C. and Gordon, S., The macrophage response to central and peripheral nerve injury, J. Exp. Med., 165 (1987) 1218-1223. 6 Perry, V.H., Hume, D.A. and Gordon, S., Immunohistochemical localization of macrophages and microglia in the adult and
developing mouse brain, Neuroscience, 15 (1985) 313-326. 7 Richardson, P.M. and Verge, V.M.K., Axonal regeneration in dorsal spinal roots is accelerated by peripheral axonal transection, Brain Research, 411 (1987) 406-408. 8 SjOstrand, J., Proliferative changes in glial cells during nerve regeneration, Z. Zellforsch., 68 (1965) 481-493. 9 Skamene, E., Gros, P.. Forget, A., Kongshavn, P.A.L., St. Charles, C. and Taylor, B.A., Genetic regulation of resistance to intracellular pathogens, Nature (Lond.), 297 (1982) 506-509. 10 Skamene, E. and Stevenson, M.M., Genetic control of macrophage response to infection. In R. Van Furth (Ed.), Mononuclear Phagocytes, Martinus Nijhoff, The Netherlands. 1985, pp. 647-653. 11 Thomas, P.K., Nerve injury. In R. Bellairs and E.G. Gray (Eds.), Essays on the Nervous System, Clarendon. Oxford. 1974, pp. 44-70. 12 Watson, W.E., An autoradiographic study of the incorporation of nucleic-acid precursors by neurones and glia during nerve regeneration, J. Physiol. (Lond.), 180 ~1965) 741-753.
This work was supported by the Rick Hansen Man in Motion Legacy Fund and the Canadian Paraplegic Association.
Brain Resear~h, ~ iAi ([gt;0) i44 l~l,~
["lsevic~ BRES 23982
A defic
y of axonal regeneration in C57BL/6J mice
Lu Xin 1, P.M. R i c h a r d s o n 1, E Gervais 2 and E. S k a m e n e 2 1Division of Neurosurgery and 2Centrefor the Study of Host Resistance, McGill University and Montreal General Hospital, Montreal (Canada)
(Accepted 14 November 1989) Key words." Peripheral nerve regeneration; Axonal regeneration; Spinal root; Mouse genetics; Macrophage
Axonal regeneration within peripheral nerves and dorsal spinal roots was investigated in inbred strains of mice with known differences in macrophage recruitment and inflammatory functions. During the second week after sciatic nerve crush, counts of regenerating newly myelinated fibres were significantly lower in C57BL/6J mice than in 4 other strains. After dorsal root crush with or without concomitant sciatic nerve transection to enhance regeneration, fibre counts in roots of C57BL/6J were one-fifth of those in A/J mice. Axonal regeneration is subnormal in C57BL/6J mice but this defect appears not to be linked to known deficiencies in macrophage function. The importance of macrophages during peripheral nerve regeneration is not fully defined. During Wallerian degeneration, macrophages are recruited into the endoneurium 5, scavenge axon and myelin debris 11, stimulate the proliferation of Schwann cells 1, and induce the synthesis of N G F (nerve growth factor) by Schwann cells 3. Also, the brisk proliferation of resident microglial cells in the anterior horn of the spinal cord after peripheral nerve injury 2'8'12 raises the possibility that this class of macrophages 6 might influence the responses of neurons to axotomy. Genetically controlled differences in macrophage functions have been observed among inbred strains of mice 4. For example, A/J and BALB/cJ mice show defective recruitment of macrophages to the peritoneum following inflammatory responses, C57BL/6J, DBA/1J, and B A L B / c J mice carry a mutant form of the Bcg (Ity, Lsh) gene that confers resistance to intracellular infection 9 and C 3 H / H e J mice are subnormally activated by lipopolysaccharide to release interleukin-11°. Of course, these strains of mice also show many other phenotypic differences unrelated to macrophages. To seek possible correlation between macrophage function and axonal regeneration, experiments were performed on female mice, 9 weeks old, of 5 different strains: C57BL/6J, A/J, DBA1/J, BALB/cJ or C3H/HeJ. U n d e r general anesthesia (ketamine 150 mg/kg, xylazine 10 mg/kg intraperitoneally), the right sciatic nerve was exposed at the level of the obturator tendon and crushed for 10 s with jeweller's forceps. Eleven days later, the mice were sacrificed and segments of the c o m m o n peroneal nerve 10 mm and 15 mm distal to the crush site were removed. Nerve specimens were fixed in mixed
aldehydes, post-fixed in osmium tetroxide, dehydrated, and embedded in plastic (Epon 812). Cross-sections, 1 p m thick, were stained with Toluidine blue and examined blindly under oil-immersion light microscopy to count the number of myelinated axons. In all cases, the myelin sheaths were thinner than normal with no suggestion of spared fibres. At both 10 and 15 m m from the crush site, fibre counts were lower for C57BL/6J mice than for the other 4 strains (Fig. 1). At 15 mm, counts were 1.9-2.9 times higher in DBA1/J, C3H/HeJ, A/J or B A L B / C J
E E
tt) tZ O O
LU n-
_m LI.
MOUSE
STRAIN
Fig. 1. Fibre counts in the common peroneal nerve. Thinly myeLinated fibres were counted in the common peroneal nerve 15 mm distal to the site of crushing 11 days earlier. Note that counts are significantlylower for C57BL/6J mice than for the other 4 strains (mean + S.E.M.; n 4--11; *P < 0.05, **P < 0.01 by Student's t-test). =
Correspondence." P.M. Richardson, Division of Neurosurgery, Montreal General Hospital, 1650 Cedar Avenue, Montreal, Canada H3G IA4.
0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
145 mice than in C57BL/6J mice. The differences between C57BL/6J mice and other strains were significant (P < 0.03 by the Student's t-test) in 7/8 comparisons: C57BL/
6J and DBA/1J mice did not differ significantly in counts at 10 mm from the crush site. For all comparisons among the strains of mice excluding C57BL/6J, the differences
I-Z :3 0 (3 LU rr
2 CRUSH
[]
A/J
[]
C57BL/6J
200-
El
U.
cUT
100-
nerve intact
nerve cut
Fig. 2. Regeneration in L 5 dorsal spinal roots. The L~ dorsal roots were crushed bilaterally and the right sciatic nerve was cut. After sacrifice, 14 days later, the progress of regeneration was assayed by fibre counts in the roots near their junction with the spinal cord. The histograms show a 5-fold difference between the two strains for regeneration with or without a conditioning lesion (mean _+ S.E.M.; n = 9-13; *P < 0,05, **P < 0.01 by Student's t-test). Photomicrographs show that thinly myelinated fibres are abundant in the root from an A/J mouse (right) but sparse for a C57BL/6J mouse (left). Note also that macrophages with a foamy cytoplasm are more frequent in the C57BL/6J spinal root (magnification x 960).
146 failed to reach statistical significance. By this measure of p e r i p h e r a l nerve regeneration, regrowth was slower for C57BL/6J mice than in 4 o t h e r strains. In a n o t h e r group of experiments, regeneration within crushed dorsal spinal roots was c o m p a r e d in C57BL/6J and A/J strains of mice (Fig. 2). This experimental p r e p a r a t i o n serves to assay the contribution of the nerve cell body to axonal regeneration because axonal regeneration in a dorsal root is accelerated by a 'conditioning' injury to the corresponding nerve 7. U n d e r general anesthesia and microsurgical conditions, both L 5 (fifth l u m b a r ) dorsal roots were exposed and crushed near their ganglia: the right sciatic nerve was transected at the hip. A f t e r sacrifice, 14 days later, each L 5 dorsal root was r e m o v e d and semi-thin cross-sections were p r e p a r e d from p l a s t i c - e m b e d d e d segments of the root near its junction with the spinal cord. A g a i n the n u m b e r of thinly m y e l i n a t e d axons was counted blindly. In all mice as previously described in rats 7, fibre counts were higher on the side (right) with sciatic nerve transection than on the side (left) without nerve injury. In comparison between strains of fibre counts on either the left or right side, A/J mice showed a p p r o x i m a t e l y 5-fold m o r e regenerating fibres than C57BL/6J mice on the same side ( P < 0.04 by S t u d e n t ' s t-test). The interstrain differences in dorsal root r e g e n e r a t i o n were even m o r e m a r k e d than those in p e r i p h e r a l nerve regeneration. C57BL/6J mice are here shown to be deficient in
regeneration of m y e l i n a t e d fibres in peripheral nerves and dorsal spinal roots. It remains to be proved that m o t o r neurons or unmyelinated fibres also regenerate less quickly in this mouse strain. R e t r o g r a d e signals to sensory neurons and neuronal responses are at least partially retained in C57BL/6J mice: perhaps the interactions between growing axons and their local environment are abnormal. W h e t h e r the primary defect involves neurons, Schwann cells or a n o t h e r cell type is unknown. The deficiency in axonal regeneration in C57BL/6J mice does not a p p e a r to be linked to any of the 3 specific abnormalities of m a c r o p h a g e function described earlier. B A L B / c J mice resemble C57BL/6J mice in carrying a defective allele of the Bcg (Ity, Lsh) gene but differ in regenerative behaviour. Mouse strains differing in their ability to recruit m a c r o p h a g e s to an inflammatory site or be activated by lipopolysaccharide show a similar rate of axonal regeneration. The C57BL/6J and A/J strains of mice were chosen in part because they are the progenitors of a large set of recombinant inbred mice. By analysis of these recombinant inbred strains, the genetic locus or loci regulating the p h e n o t y p i c differences in p e r i p h e r a l nerve regeneration can now be identified.
1 Beuche, W. and Friede, R.L., The role of non-resident cells in Wallerian degeneration, J. Neurocytol., 13 (1984) 767-796. 2 Blinzinger, K. and Kreutzberg, G., Displacement of synaptic terminals from regenerating motoneurons by microglial cells, Z. Zellforsch., 85 (1968) 145-157. 3 Heumann, R., Lindholm, D., Bandtlow, C., Meyer, M., Radeke, M.J., Misko, T.P., Shooter, E. and Thoenen, H., Differential regulation of mRNA encoding nerve growth factor and its receptor in rat sciatic nerve during development, degeneration, and regeneration: role of macrophages, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 8735-8739. 4 Nesbitt, M.N. and Skamene, E., Recombinant inbred mouse strains derived from A/J and C57BL/6J: A tool for the study of genetic mechanisms in host resistance to infection and malignancy, J. Leukocyte Biol., 36 (1984) 357-364. 5 Perry, V.H., Brown, M.C. and Gordon, S., The macrophage response to central and peripheral nerve injury, J. Exp. Med., 165 (1987) 1218-1223. 6 Perry, V.H., Hume, D.A. and Gordon, S., Immunohistochemical localization of macrophages and microglia in the adult and
developing mouse brain, Neuroscience, 15 (1985) 313-326. 7 Richardson, P.M. and Verge, V.M.K., Axonal regeneration in dorsal spinal roots is accelerated by peripheral axonal transection, Brain Research, 411 (1987) 406-408. 8 SjOstrand, J., Proliferative changes in glial cells during nerve regeneration, Z. Zellforsch., 68 (1965) 481-493. 9 Skamene, E., Gros, P.. Forget, A., Kongshavn, P.A.L., St. Charles, C. and Taylor, B.A., Genetic regulation of resistance to intracellular pathogens, Nature (Lond.), 297 (1982) 506-509. 10 Skamene, E. and Stevenson, M.M., Genetic control of macrophage response to infection. In R. Van Furth (Ed.), Mononuclear Phagocytes, Martinus Nijhoff, The Netherlands. 1985, pp. 647-653. 11 Thomas, P.K., Nerve injury. In R. Bellairs and E.G. Gray (Eds.), Essays on the Nervous System, Clarendon. Oxford. 1974, pp. 44-70. 12 Watson, W.E., An autoradiographic study of the incorporation of nucleic-acid precursors by neurones and glia during nerve regeneration, J. Physiol. (Lond.), 180 ~1965) 741-753.
This work was supported by the Rick Hansen Man in Motion Legacy Fund and the Canadian Paraplegic Association.
