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Insulin-like growth factor I (IGF-I) stimulates regeneration of the rat sciatic nerve
Brain Research, 486 (1989) 396-398
396
Elsevier BRE 23492
Insulin-like growth factor I (IGF-I) stimulates regeneration of the rat sciatic nerve Martin Kanje 1, Anna Skottner 2, Jacob Sj6berg and G6ran Lundborg 3 1Department of Zoophysiology, University of Lund, Lurid (Sweden), 2KabiVitrum AB, Stockholm (Sweden) and 3Department of Handsurgery, Malm6 Allmiinna Sjukhus, MalmO (Sweden) (Accepted 24 January 1989)
Key words: Rat; Somatomedin; Growth factor; Insulin-like growth factor I; Nerve regeneration; Sciatic nerve; Nerve growth factor; Local administration
The effect of insulin-like growth factor I (IGF-I) was tested on regeneration of the rat sciatic nerve after a crush lesion. IGF-I was administered via miniosmotic pumps to the dorsal root ganglia or locally around the crush lesion. Regeneration of sensory fibers was measured after 3 or 4 days superfusion by pinching. IGF-I stimulated regeneration in both administration paradigms. Regeneration was inhibited if the nerve was perfused with specific antibodies to native IGF-I. The results suggest that endogenous extracellular IGF-I plays an important role during regeneration of peripheral nerve fibers.
R e c e n t studies suggest that insulin-like growth factors ( I G F - I and I G F - I I ) possess neurotrophic activities 2-5'1°'11. In response to I G F - I I cultured n e u r o b l a s t o m a cells extend neurites and exhibit characteristics of the differentiated neuron 1°'11 and I G F - I has been shown to p r o m o t e the survival of cultured CNS neurons 3. Hansson et al. 4 r e p o r t e d that I G F - I accumulated in the rat sciatic nerve after transection and suggested that I G F - I could be involved in peripheral nerve regeneration. We have d e v e l o p e d a m e t h o d which allows local superfusion of the regenerating rat sciatic nerve with drugs or growth factors for several days 6. This m e t h o d was used to test the effects of I G F - I and antibodies to 1GF-I on regeneration of the rat sciatic nerve after a crush lesion. H e r e we r e p o r t that I G F - I superfusion stimulates nerve regeneration and that endogenous extracellular I G F - I appears to be involved in regeneration of sensory nerve fibers. S p r a g u e - D a w ley ( A l a b , Sweden) rats weighing approximately 200 g were used throughout the experiments. Rats were anaesthetized with an intraperitoneal injection of 0.3 ml of a mixture of p e n t o b a r b i t a l (60 mg/ml), diazepam (5 mg/ml) and 0.9% NaC1 in 1:2:1 volume
proportions. The sciatic nerve was e x p o s e d along the thigh and the nerve was transected at the level of the knee. The nerve was then crushed a p p r o x i m a t e l y 10 mm proximal to the transection with specially designed pliers. The site of the lesion was labelled by attaching a 9:0 epineural suture. The crush lesion and the distal segment of the proximal stump were then enclosed in a t h r e e - p o r t silicone tube c h a m b e r (STC) as previously described 6. T h e STC was filled with Ringer solution with or without growth factors and/or antibodies. The STC was attached via a catheter to a miniosmotic p u m p , filled with Ringer solution with or without growth factors or antibodies. The p u m p was i m p l a n t e d subcutaneously on the a b d o m e n of the rat. The wounds were then closed by sutures. R e g e n e r a t i o n was evaluated 3 o r 4 days later. The animals were reanesthetized and consecutive segments (0.5 m m apart) of the sciatic nerve were pinched, starting from the distal end. W h e n the regenerated sensory axons were crushed they elicited a reflex response noted as a contraction of muscles on the back of the rat. The distance b e t w e e n the site of the first reflex response and that of the original crush was m e a s u r e d with calipers under a
Correspondence: M. Kanje, Department of Zoophysiology, University of Lund, Helgonav/igen 3 B, S-223 62 Lund, Sweden. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
397 TABLE I
Effects of lGF-I on regeneration of the rat sciatic nerve Concentration as m e a s u r e d in the miniosmotic p u m p . Values are m e a n + S.E.M. with the n u m b e r given in parentheses. R e g e n e r a t i o n was m e a s u r e d 3 days after crushing the sciatic nerve.
Treatment
Concentration (l~g/ml)
Rdgeneration distance (mm )
Control IGF-I
100
4.7 + 0.5 (6) 7.0 + 0.1" (3)
* Student's t-test, P < 0.05.
dissection microscope and regarded as the regeneration distance. Regeneration distances obtained with this method are similar to those obtained when regeneration is estimated by other methods including axonal transport of radiolabelled proteins or staining for neurofilaments in the regenerating fibers 12. In some experiments both sciatic nerves were crushed and IGF-I was administered, unilaterally, to the dorsal root ganglia via osmotic pumps. Recombinant IGF-I produced in yeast was used. The batches were 2104 and 2111 with specific activities of 14,900 U/mg and 13,600 U/mg, respectively, according to radio receptor assay. Antibodies to IGF-I were raised in rabbits. Antibody K624 was made against human serum derived IGF-I and kindly provided by Johnny Brandt. Antibody 863/5 was raised against recombinant IGF-I and kindly provided by Dr. P. Gluckman, Auckland, New Zealand. Antibodies to insulin was obtained from Milab, Maim6, Sweden, Nerve growth factor (2.5 S) was purchased from Sigma. Superfusion of the nerve with IGF-I at a pump concentration of 100/tg/ml for 3 days increased the regeneration distance by 49% as compared to nerve perfused with Ringer solution alone (Table I). Superfusion with lower concentrations did not affect the regeneration distance (Table III). A stimulatory effect of IGF-I was also observed when the dorsal root ganglia were perfused (Table II). Superfusion of the STC with NGF (2.5 S) for 4 days had no effect (not shown). To test the specificity of the IGF-I response and to study if endogenous IGF-I was important for regeneration the STC surrounded nerves were perfused with antibodies to IGF-I (Table III). Superfusion with either of two different
antisera K624 and 863/5 significantly inhibited regeneration during a 3-day superfusion period. This inhibition was neutralized by addition of IGF-I at 5 or 25 /zg/ml to the miniosmotic pump. At these concentrations IGF-I alone does not stimulate regeneration. In contrast, superfusion with non-immune serum diluted 1/25 or antibodies to insulin failed to affect the regeneration distances. The present results showed that local administration of IGF-I to the regenerating rat sciatic nerve stimulated regeneration. Superfusion with specific antibodies to IGF-I inhibited regeneration, suggesting that extracellular IGF-I plays a role during regeneration of peripheral sensory fibers. We assumed that the antibodies acted by reducing the level of extracellular IGF-I since they, due to their size, are unlikely to gain access to the intracellular compartments. A role for IGF-I during peripheral nerve regeneration is also supported by immunohistochemical data which shows that IGF-I accumulate in Schwann cells in the regenerating rat sciatic nerve 4. Our observation 7 that human growth hormone which partly regulates the synthesis of IGF-I, stimulated regeneration of the rat sciatic nerve, when given systemically, lends further support to this idea. The IGF-I response appeared to be specific since neither NGF nor antibodies to insulin affected regeneration. We do not know if IGF-I acts via its own receptors, or via cross-reaction with other receptors. However, we have found that the sciatic
T A B L E II
Effects of ganglionic application of lGF-I Values are m e a n S.D. R e g e n e r a t i o n was m e a s u r e d 4 days after crushing of the sciatic nerve.
Rat I Rat 2 Rat 3
IGF-I contralateral IGF-I contralateral IGF-I contralateral
Student's t test, P < 0.05.
Regeneration (mm)
Ratio IGF-1/ contralateral
8.9 7.6 8.6 7.8 9.8 7.9 Mean
1.17 1.10 1.24 1.17 + 0.07
398 TABLE III Effects of antibodies to 1GF-I on regeneration
Values are mean + S.D. with the number given in parentheses. Dunnett's test was used for test of significance. The antibodies were diluted to give the same binding capacity. Treatment
Dilution
Regeneration distance (ram) a 3 Days
4 Days
Preimmune serum Preimmune serum + IGF-I Preimmune serum + IGF-I
1:25 1:25 + 5/~g/ml 1:25 + 25/~g/ml
3.8 + 0.4 (6) 4.0 + 0.2 (4) -
8.0_+ 0.6 (5) 7.0+1.6 (6) 8.2_+0.3 (3)
Antibody K624 Antibody K624 + IGF-I Antibody 863/5 Anti-insulin
1:25 1:25 + 5/~g/ml 1:1000 1:25
2.3 + 1.0" (5) 1.8 + 0.6* (4) -
4.8 _+2.2* (4) 7.9 _+0.4 (3) 2.1 (2) 8.3 (2)
P < 0.05.
nerve possesses receptors for I G F - I as measured by specific binding of 125I-IGF-I (unpublished results). The present result showed that I G F - I acted at the site of the crush lesion but also at the level of the dorsal root ganglia. It is possible that locally synthesized IGF-I, analogous to N G F 1'8'9'13, is picked up by the regenerating fibers, retrogradely transported t o t h e cell body where it exerts its action. The finding that I G F - I is axonally transported 5 lends some support to this idea. It is therefore tempting to
1 Abrahamson, I.K., Bridges, D. and Rush, R.A., Transport of endogenous nerve growth factor in the proximal stump of sectioned nerves, J. Neurocytol., 16 (1987) 417-422. 2 Burgess, S.K., Jacobs, S., Cuatrecasas, P. and Sahyoun, N., Characterization of a neuronal subtype of insulin-like growth factor I receptor, J. Biol. Chem., 262 (1987) 1618-1622. 3 Fellows, R., AI-Hadar, A. and Kadle, R., IGF-I supports survival and differentiation of fetal rat brain neurons in serum-free hormone free defined medium, Soc. Neurosci. Abstr., 13 (1987) 1615. 4 Hansson, H.-A., Dahlin, L.B., Danielsen, N., Fryklund, L., Nachemson, A.K., Polleryd, P., Rozell, B., Skottner, A., Stemme, S. and Lundborg, G., Evidence indicating trophic importance of IGF-I in regenerating peripheral nerve, Acta Physiol. Scand., 126 (1986) 609-614. 5 Hansson, H.-A., Rozell, B. and Skottner, A., Rapid axoplasmic transport of insulin-like growth factor, Cell Tissue Res., 247 (1987) 241-247. 6 Kanje, M., Lundborg, G. and Edstr6m, A., A new method for studies of the effects of locally applied drugs on peripheral nerve regeneration in vivo, Brain Research, 439 (1988) 116-121. 7 Kanje, M., Skottner, A. and Lundborg, G., Effects of
suggest that locally produced and secreted I G F - I could be taken up by the growth cones and reach the cell body by retrograde axonal transport where it could trigger or increase regenerative changes. The present study was supported by grants from Swedish Natural Research Council and K a b i V i t r u m AB. Thanks are due to Marie A d l e r Maihofer, Eva L6fberg and Vibeke Arrhenius-Nyberg for expert technical assistance.
8 9
10
11
12 13
growth hormone treatment on the regeneration of rat sciatic nerve, Brain Research, 475 (1988) 254-258. Levi-Montalcini, R., The nerve growth factor 35 years later, Science, 237 (1987) 1154-1162. Paravicini, U., St6ckel, K. and Thoenen, H., Biological importance of retrograde axonal transport of nerve growth factor in adrenergic neurons, Brain Research, 84 (1975) 279-291. Recio-Pinto, E., Lang, EE and Ishii, D.N., Insulin and insulin-like growth factor II permit nerve growth factor binding and the neurite formation response in cultured human neuroblastoma cells, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 2562-2566. Recio-Pinto, E. and Ishii, D.N., Effects of insulin, insulinlike growth factor II and nerve growth factor on neurite outgrowth in cultured human neuroblastoma cells, Brain Research, 302 (1984) 323-334. Sj6berg, J., Kanje, M. and Edstr6m, E., Influence of non-neuronalcells on regeneration of the rat sciatic nerve, Brain Reseach, 453 (1988) 221-226. St6ckel, K., Paravicini, U. and Thoenen, H., Specificity of the retrograde axonal transport of nerve growth factor, Brain Research, 76 (1974) 413-421.
396
Elsevier BRE 23492
Insulin-like growth factor I (IGF-I) stimulates regeneration of the rat sciatic nerve Martin Kanje 1, Anna Skottner 2, Jacob Sj6berg and G6ran Lundborg 3 1Department of Zoophysiology, University of Lund, Lurid (Sweden), 2KabiVitrum AB, Stockholm (Sweden) and 3Department of Handsurgery, Malm6 Allmiinna Sjukhus, MalmO (Sweden) (Accepted 24 January 1989)
Key words: Rat; Somatomedin; Growth factor; Insulin-like growth factor I; Nerve regeneration; Sciatic nerve; Nerve growth factor; Local administration
The effect of insulin-like growth factor I (IGF-I) was tested on regeneration of the rat sciatic nerve after a crush lesion. IGF-I was administered via miniosmotic pumps to the dorsal root ganglia or locally around the crush lesion. Regeneration of sensory fibers was measured after 3 or 4 days superfusion by pinching. IGF-I stimulated regeneration in both administration paradigms. Regeneration was inhibited if the nerve was perfused with specific antibodies to native IGF-I. The results suggest that endogenous extracellular IGF-I plays an important role during regeneration of peripheral nerve fibers.
R e c e n t studies suggest that insulin-like growth factors ( I G F - I and I G F - I I ) possess neurotrophic activities 2-5'1°'11. In response to I G F - I I cultured n e u r o b l a s t o m a cells extend neurites and exhibit characteristics of the differentiated neuron 1°'11 and I G F - I has been shown to p r o m o t e the survival of cultured CNS neurons 3. Hansson et al. 4 r e p o r t e d that I G F - I accumulated in the rat sciatic nerve after transection and suggested that I G F - I could be involved in peripheral nerve regeneration. We have d e v e l o p e d a m e t h o d which allows local superfusion of the regenerating rat sciatic nerve with drugs or growth factors for several days 6. This m e t h o d was used to test the effects of I G F - I and antibodies to 1GF-I on regeneration of the rat sciatic nerve after a crush lesion. H e r e we r e p o r t that I G F - I superfusion stimulates nerve regeneration and that endogenous extracellular I G F - I appears to be involved in regeneration of sensory nerve fibers. S p r a g u e - D a w ley ( A l a b , Sweden) rats weighing approximately 200 g were used throughout the experiments. Rats were anaesthetized with an intraperitoneal injection of 0.3 ml of a mixture of p e n t o b a r b i t a l (60 mg/ml), diazepam (5 mg/ml) and 0.9% NaC1 in 1:2:1 volume
proportions. The sciatic nerve was e x p o s e d along the thigh and the nerve was transected at the level of the knee. The nerve was then crushed a p p r o x i m a t e l y 10 mm proximal to the transection with specially designed pliers. The site of the lesion was labelled by attaching a 9:0 epineural suture. The crush lesion and the distal segment of the proximal stump were then enclosed in a t h r e e - p o r t silicone tube c h a m b e r (STC) as previously described 6. T h e STC was filled with Ringer solution with or without growth factors and/or antibodies. The STC was attached via a catheter to a miniosmotic p u m p , filled with Ringer solution with or without growth factors or antibodies. The p u m p was i m p l a n t e d subcutaneously on the a b d o m e n of the rat. The wounds were then closed by sutures. R e g e n e r a t i o n was evaluated 3 o r 4 days later. The animals were reanesthetized and consecutive segments (0.5 m m apart) of the sciatic nerve were pinched, starting from the distal end. W h e n the regenerated sensory axons were crushed they elicited a reflex response noted as a contraction of muscles on the back of the rat. The distance b e t w e e n the site of the first reflex response and that of the original crush was m e a s u r e d with calipers under a
Correspondence: M. Kanje, Department of Zoophysiology, University of Lund, Helgonav/igen 3 B, S-223 62 Lund, Sweden. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
397 TABLE I
Effects of lGF-I on regeneration of the rat sciatic nerve Concentration as m e a s u r e d in the miniosmotic p u m p . Values are m e a n + S.E.M. with the n u m b e r given in parentheses. R e g e n e r a t i o n was m e a s u r e d 3 days after crushing the sciatic nerve.
Treatment
Concentration (l~g/ml)
Rdgeneration distance (mm )
Control IGF-I
100
4.7 + 0.5 (6) 7.0 + 0.1" (3)
* Student's t-test, P < 0.05.
dissection microscope and regarded as the regeneration distance. Regeneration distances obtained with this method are similar to those obtained when regeneration is estimated by other methods including axonal transport of radiolabelled proteins or staining for neurofilaments in the regenerating fibers 12. In some experiments both sciatic nerves were crushed and IGF-I was administered, unilaterally, to the dorsal root ganglia via osmotic pumps. Recombinant IGF-I produced in yeast was used. The batches were 2104 and 2111 with specific activities of 14,900 U/mg and 13,600 U/mg, respectively, according to radio receptor assay. Antibodies to IGF-I were raised in rabbits. Antibody K624 was made against human serum derived IGF-I and kindly provided by Johnny Brandt. Antibody 863/5 was raised against recombinant IGF-I and kindly provided by Dr. P. Gluckman, Auckland, New Zealand. Antibodies to insulin was obtained from Milab, Maim6, Sweden, Nerve growth factor (2.5 S) was purchased from Sigma. Superfusion of the nerve with IGF-I at a pump concentration of 100/tg/ml for 3 days increased the regeneration distance by 49% as compared to nerve perfused with Ringer solution alone (Table I). Superfusion with lower concentrations did not affect the regeneration distance (Table III). A stimulatory effect of IGF-I was also observed when the dorsal root ganglia were perfused (Table II). Superfusion of the STC with NGF (2.5 S) for 4 days had no effect (not shown). To test the specificity of the IGF-I response and to study if endogenous IGF-I was important for regeneration the STC surrounded nerves were perfused with antibodies to IGF-I (Table III). Superfusion with either of two different
antisera K624 and 863/5 significantly inhibited regeneration during a 3-day superfusion period. This inhibition was neutralized by addition of IGF-I at 5 or 25 /zg/ml to the miniosmotic pump. At these concentrations IGF-I alone does not stimulate regeneration. In contrast, superfusion with non-immune serum diluted 1/25 or antibodies to insulin failed to affect the regeneration distances. The present results showed that local administration of IGF-I to the regenerating rat sciatic nerve stimulated regeneration. Superfusion with specific antibodies to IGF-I inhibited regeneration, suggesting that extracellular IGF-I plays a role during regeneration of peripheral sensory fibers. We assumed that the antibodies acted by reducing the level of extracellular IGF-I since they, due to their size, are unlikely to gain access to the intracellular compartments. A role for IGF-I during peripheral nerve regeneration is also supported by immunohistochemical data which shows that IGF-I accumulate in Schwann cells in the regenerating rat sciatic nerve 4. Our observation 7 that human growth hormone which partly regulates the synthesis of IGF-I, stimulated regeneration of the rat sciatic nerve, when given systemically, lends further support to this idea. The IGF-I response appeared to be specific since neither NGF nor antibodies to insulin affected regeneration. We do not know if IGF-I acts via its own receptors, or via cross-reaction with other receptors. However, we have found that the sciatic
T A B L E II
Effects of ganglionic application of lGF-I Values are m e a n S.D. R e g e n e r a t i o n was m e a s u r e d 4 days after crushing of the sciatic nerve.
Rat I Rat 2 Rat 3
IGF-I contralateral IGF-I contralateral IGF-I contralateral
Student's t test, P < 0.05.
Regeneration (mm)
Ratio IGF-1/ contralateral
8.9 7.6 8.6 7.8 9.8 7.9 Mean
1.17 1.10 1.24 1.17 + 0.07
398 TABLE III Effects of antibodies to 1GF-I on regeneration
Values are mean + S.D. with the number given in parentheses. Dunnett's test was used for test of significance. The antibodies were diluted to give the same binding capacity. Treatment
Dilution
Regeneration distance (ram) a 3 Days
4 Days
Preimmune serum Preimmune serum + IGF-I Preimmune serum + IGF-I
1:25 1:25 + 5/~g/ml 1:25 + 25/~g/ml
3.8 + 0.4 (6) 4.0 + 0.2 (4) -
8.0_+ 0.6 (5) 7.0+1.6 (6) 8.2_+0.3 (3)
Antibody K624 Antibody K624 + IGF-I Antibody 863/5 Anti-insulin
1:25 1:25 + 5/~g/ml 1:1000 1:25
2.3 + 1.0" (5) 1.8 + 0.6* (4) -
4.8 _+2.2* (4) 7.9 _+0.4 (3) 2.1 (2) 8.3 (2)
P < 0.05.
nerve possesses receptors for I G F - I as measured by specific binding of 125I-IGF-I (unpublished results). The present result showed that I G F - I acted at the site of the crush lesion but also at the level of the dorsal root ganglia. It is possible that locally synthesized IGF-I, analogous to N G F 1'8'9'13, is picked up by the regenerating fibers, retrogradely transported t o t h e cell body where it exerts its action. The finding that I G F - I is axonally transported 5 lends some support to this idea. It is therefore tempting to
1 Abrahamson, I.K., Bridges, D. and Rush, R.A., Transport of endogenous nerve growth factor in the proximal stump of sectioned nerves, J. Neurocytol., 16 (1987) 417-422. 2 Burgess, S.K., Jacobs, S., Cuatrecasas, P. and Sahyoun, N., Characterization of a neuronal subtype of insulin-like growth factor I receptor, J. Biol. Chem., 262 (1987) 1618-1622. 3 Fellows, R., AI-Hadar, A. and Kadle, R., IGF-I supports survival and differentiation of fetal rat brain neurons in serum-free hormone free defined medium, Soc. Neurosci. Abstr., 13 (1987) 1615. 4 Hansson, H.-A., Dahlin, L.B., Danielsen, N., Fryklund, L., Nachemson, A.K., Polleryd, P., Rozell, B., Skottner, A., Stemme, S. and Lundborg, G., Evidence indicating trophic importance of IGF-I in regenerating peripheral nerve, Acta Physiol. Scand., 126 (1986) 609-614. 5 Hansson, H.-A., Rozell, B. and Skottner, A., Rapid axoplasmic transport of insulin-like growth factor, Cell Tissue Res., 247 (1987) 241-247. 6 Kanje, M., Lundborg, G. and Edstr6m, A., A new method for studies of the effects of locally applied drugs on peripheral nerve regeneration in vivo, Brain Research, 439 (1988) 116-121. 7 Kanje, M., Skottner, A. and Lundborg, G., Effects of
suggest that locally produced and secreted I G F - I could be taken up by the growth cones and reach the cell body by retrograde axonal transport where it could trigger or increase regenerative changes. The present study was supported by grants from Swedish Natural Research Council and K a b i V i t r u m AB. Thanks are due to Marie A d l e r Maihofer, Eva L6fberg and Vibeke Arrhenius-Nyberg for expert technical assistance.
8 9
10
11
12 13
growth hormone treatment on the regeneration of rat sciatic nerve, Brain Research, 475 (1988) 254-258. Levi-Montalcini, R., The nerve growth factor 35 years later, Science, 237 (1987) 1154-1162. Paravicini, U., St6ckel, K. and Thoenen, H., Biological importance of retrograde axonal transport of nerve growth factor in adrenergic neurons, Brain Research, 84 (1975) 279-291. Recio-Pinto, E., Lang, EE and Ishii, D.N., Insulin and insulin-like growth factor II permit nerve growth factor binding and the neurite formation response in cultured human neuroblastoma cells, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 2562-2566. Recio-Pinto, E. and Ishii, D.N., Effects of insulin, insulinlike growth factor II and nerve growth factor on neurite outgrowth in cultured human neuroblastoma cells, Brain Research, 302 (1984) 323-334. Sj6berg, J., Kanje, M. and Edstr6m, E., Influence of non-neuronalcells on regeneration of the rat sciatic nerve, Brain Reseach, 453 (1988) 221-226. St6ckel, K., Paravicini, U. and Thoenen, H., Specificity of the retrograde axonal transport of nerve growth factor, Brain Research, 76 (1974) 413-421.