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Beneficial effects of insulin-like growth factor-I on wobbler mouse motoneuron disease
JOURNAL OF THE
NEUROLOGICAL SCIENCES ELSEVIER
Journal of the Neurological Sciences 129 (Suppl.) (1995) 122-126
Beneficial effects of insulin-like growth factor-I on wobbler mouse motoneuron disease D. Hanta'i a, * M. Akaaboune a, C. Lagord a,b M. Murawsky a L.J. Houenou c, B.W. Festoff d, J.L. Vaught e, F. Rieger a, B. Blondet a,b INSERM U. 153 and CNRS ERS 64, 17 rue du Fer h Moulin, 75005 Paris, France b Universitd Paris XII, Cr~teil, France c Department of Neurobiology and Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC, USA a Veterans Affairs Medical Center, Kansas City, MO and KS, USA e Cephalon Inc., West Chester, PA, USA
Accepted 21 February 1995
Abstract Recombinant human insulin-like growth factor-I (IGF-I) is being considered as a possible therapeutic agent for the treatment of motoneuron diseases like amyotrophic lateral sclerosis. The neurological mutant mouse wobbler, carries an autosomal recessive gene (wr) and has been characterized as a model of lower motoneuron disorders with associated muscle atrophy, denervation and reinnervation. The purpose of the present study was to determine the possible beneficial effect of IGF-I administration in this mouse model. Upon diagnosis at 4 weeks of age, affected mice and their control normal littermates received human recombinant IGF-I (1 mg/kg) or vehicle solution, once a day, for 6 weeks. Body weight and grip strength were evaluated periodically during the treatment period. Mean muscle fiber diameter on biceps brachii sections, choline acetyltransferase activity in muscle extracts, and motoneuron numbers in spinal cord sections were determined. IGF-I treated wobbler mice showed a marked weight increase from 3 to 6 weeks of treatment in comparison with placebo treated mutant mice. At the end of the treatment, grip strength, estimated by dynamometer resistance, was 40% higher in IGF-I treated versus placebo treated animals. Mean muscle fiber diameter which is smaller in wobbler mice than in normal mice was increased in IGF-I treated mutants. However, in this study the muscle choline acetyltransferase activity and the number of spinal cord motoneurons were unchanged. Thus, IGF-I administration mainly results in a significant effect on the behavioral and skeletal muscle histochemical parameters of the wobbler mouse mutant. Keywords: Trophic factors; Mutant mouse; Motoneuron disease; Human recombinant insulin-like growth factor-l; hrIgF-1;
Wobbler mouse (wr/wr); Dynamometry; Grip strength; Choline acetyltransferase (CHAT); Morphometry; Motoneuron counts; Muscle fiber diameter
1. Introduction A b e t t e r u n d e r s t a n d i n g of t h e factors which influence t h e d i f f e r e n t i a t i o n , survival or d e a t h of m o t o n e u rons is of c o n s i d e r a b l e physiological a n d clinical interest. H u m a n m o t o n e u r o n d i s e a s e s i n c l u d e several familial a n d s p o r a d i c c o n d i t i o n s involving t h e d e g e n e r a t i o n o r d e a t h o f m o t o n e u r o n s in the spinal c o r d a n d brain. T h e y p r e s e n t an i n t e r e s t i n g o p p o r t u n i t y for t h e r a p e u t i c
* Corresponding author. Tel.: (+33-1) 43 36 46 31; Fax: (+33-1) 43 37 85 22. E-mail: [email protected]. 0022-510X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0022-5 10X(95)00081-X
i n t e r v e n t i o n with n e u r o t r o p h i c proteins. I G F - I p r o m o t e s n e u r i t i c growth (both in vivo a n d in vitro), influe n c e s t h e m a i n t e n a n c e a n d r e g u l a t i o n o f n e u r o n a l dev e l o p m e n t a n d r e g e n e r a t i o n ( R e c i o - P i n t o a n d Ishii, 1988; K a n j e et al., 1989; C o n t r e r a s et al., 1993) a n d has n e u r o p r o t e c t i v e p r o p e r t i e s . I G F - I p r o m o t e s the survival of n e u r o n s in c u l t u r e ( A i z e n m a n a n d de Vellis, 1987; T o r r e s - A l e m a n et al., 1990; A n g et al., 1992; N e f f et al., 1993) a n d in d e v e l o p i n g spinal c o r d in vivo ( N e f f et al., 1993). I G F - I also functions in the r e g u l a t i o n o f o l i g o d e n d r o c y t e a n d myelin m e t a b o l i s m in vivo ( K o m o l y et al., 1992). F o r t h e s e reasons, I G F - I is likely to b e involved, directly or indirectly, in t h e a e t i o l o g y of a
D. Hanta'tet al. /Journal of the Neurological Sciences 129 (Suppl.) (1995) 122-126 variety of neurodegenerative disorders and, therefore, might be of therapeutic value for these diseases. To assess the therapeutic effect of IGF-I, we administered this factor to the neurological mutant mouse wobbler. Wobbler has been proposed as an animal model of human motoneuron diseases, such as the fatal amyotrophic lateral sclerosis, and spinal muscular atrophies (Mitsumoto and Gambetti, 1986; Melki et al., 1991). This spontaneous mutation is inherited as an autosomal recessive trait and displays a muscular atrophy associated with motoneuron degeneration in early post-natal development (Duchen et al., 1966). Clinical abnormalities are apparent at 3 - 4 weeks of age when the mutants become smaller than their littermates and exhibit general muscle weakness and wasting. At this stage, several authors have observed progressive partial denervation of skeletal muscle (Duchen et al., 1966; Duchen and Strich, 1968; Harris and Ward, 1974). On the other hand, it has been established that significant axonal regeneration and attempted reinnervation coincides with axonal degeneration (Mitsumoto, 1985; Melki et al., 1991; Blondet et al., 1992), all p h e n o m e n a where IGF-I administration might be of interest. Here, we show that treatment with I G F - I from 4 - 1 0 weeks of age, significantly improve behavioral and muscle histochemical parameters in this mutant wobbler mouse.
2. M a t e r i a l s
and methods
Mutant mice, homozygous ( w r / w r ) for the gene wr and littermates of normal phenotype ( + / . 9 ) were obtained from our mouse colony (C57BL/6J; Blondet et al., 1992). The phenotypically normal littermates were used as controls. Their weight and grip strength (measured with a dynamometer) were determined periodically. Recombinant human I G F - I (rhIGF-I; 1 m g / k g ) or vehicle solution was administered once a day during 6 weeks, from 4 to 10 weeks postnatally. One wobbler mouse and one control littermate were placed in the same cage and both of them were treated blind with either r h I G F - I or placebo. A total of 6 wobbler mice and 6 non affected littermates was used. To determine muscle C h A T activity, biceps brachii, triceps and gastrocnemius muscles were dissected and homogenized with a Polytron PT1200 homogenizer in 10% ( w / v ) medium containing 100 mM Tris-HC1, 2 mM E D T A , p H 7.6, all at 4° C. Homogenates were centrifuged at 20 000 × g for 30 min at 4° C. The supernatant was used without dilution. ChAT activity was determined by the method of Rand and Johnson (1981). A modification we made was that the enzymatic incubation was carried out at 37°C for 2 h. Motoneuron cell count in the lateral motor column of the spinal
123
cord was performed as described previously (Oppenheim, 1986; Li et al., 1994). Muscle fiber diameter distribution was determined by computer assisted morphometric analysis (SAMBA, TITN-Alcatel, Grenoble, France) on biceps brachii transverse cryostat sections. Protein determination was estimated by using the bicinchoninic acid (BCA protein assay, Pierce, Rockford, IL) protein assay. Statistical analysis was performed using the two-tailed Student's t-test.
3. R e s u l t s
El:olution of the weight of the animals From day 1 to day 21 of treatment, we did not observe any significant difference between I G F - I treated versus placebo treated animals, either wobbler mice or normal littermates. However, beginning on day 21 of treatment, the I G F - I treated wobbler mice showed a striking ( × 4) weight increase, compared to placebo treated wobbler mice (Fig. 1). For the I G F - I treated versus placebo treated normal littermates, we also noticed an increase but to a lesser extent ( × 2; Fig. 1). Grip strength Similar to total body weight, we did not detect any significant difference in grip strength in I G F - I treated versus vehicle treated animals before 3 weeks of treatment. After 25 days of treatment, the difference in grip strength between I G F - I treated versus placebo treated wobbler mice became apparent (Fig. 2). Note that, before 25 days of treatment, the placebo and I G F - I treated wobbler mice never exceeded 0.25 on the scale of the dynamometer. However, after 25 days the strength of the placebo treated wobbler mice started to
-F
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, , . , . , , , , . , , , , . , - , < .
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IGF
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Fig. 1. Weight increase between 21 and 39 days of treatment. The difference between IGF-I treated versus placebo treated wobbler mice is statistically significant (2.2+0.16 vs 0.63+0.26 g; values are means + SEM; comparison of means p < 0.01; n = 3 for each group).
D. Hanta't'et al, /Journal of the Neurological Sciences 129 (Suppl.) (1995) 122-126
124
wobbler
decline. At the same time, the grip strength of IGF-I treated mutant mice increased, reaching 0.35 on the scale of the dynamometer at day 40, i.e. a 40% increase in comparison with placebo treated wobbler mice. All controls, either placebo or IGF-1 treated, gave scores superior to 1.0 (the upper limit of the scale of the dynamometer) and therefore are not reported here.
mice
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0,0
I
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Fig. 2. Increase in grip strength as measured by dynamometry. Note that starting at day 30 there is a statistically significant increase in grip strength between IGF-I treated versus placebo treated wobbler mice ( + 4 0 % over control at day 40; values are means_+SEM: p < 0.01; n = 3 for each group).
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fiber
diameter measurements
Using morphometric analysis of biceps transverse cryostat sections, we found, as expected, that the wobbler mice have smaller muscle fiber diameters than the controls (Fig. 3). When wobbler mutants were treated with IGF-I a shift of the fiber diameter distribution to the right (i.e. towards the higher diameters) was observed when compared with placebo treated mutants. The muscle fiber diameter distribution in normal littermates did not significantly change following treatment with IGF-I (Fig. 3).
diameter
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Fig, 3. Plot of the percentage distribution of muscle fiber diameters. A total of 1000 muscle fibers were examined for each group.
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Fig. 4. Choline acetyltransferase (CHAT) activity in biceps, triceps and gastrocnemius muscle expressed as radioactive acetylcholine formed per muscle. Values are means_+ SEM; n = 3 for each muscle except for biceps (pool of 3 muscles).
D. Hanta~"el al. /Journal of the Neurological Sciences 129 (Suppl.) (1995) 122-126 Muscle choline acetyltransferase (CHAT) actiuity per muscle In all muscle studied (gastrocnemius, triceps, biceps; Fig. 4), C h A T activity per muscle in wobbler mice represented only 3 0 - 4 0 % of the activity in corresponding littermates. No statistically significant difference in C h A T activity per muscle between I G F - I versus placebo treated wobbler mutants and control mice was observed in any of the specific muscles studied (gastrocnemius, triceps, biceps; Fig. 4). Motoneuron cells count in the spinal cord Using an established technique ( O p p e n h e i m , 1986; Li et al., 1994), no statistically different n u m b e r of m o t o n e u r o n s in the spinal cord between I G F - I and placebo treated wobbler mice was observed (data shown). However m o t o n e u r o n survival and differentiation in wobbler mice is currently being reexamined in order to determine m o t o n e u r o n survival and to what extent this mutation affects m o t o n e u r o n survival.
4. Discussion T h e results from this study show that daily subcutaneous administration of r h I G F - I (1 m g / k g ) to wobbler mice beginning at the time the phenotypic diagnosis is made (i.e., at 4 weeks of age) and for a further 6 weeks resulted in a marked increase in (1) body weight; (2) grip strength; (3) mean skeletal muscle fiber diameters. T h e increase in body weight, likely reflects the increase in skeletal muscle mass since the latter represents mor,: than 80% of total body weight. This increase is expected since IGF-I exerts known nutrient effects in skeletal muscle by binding to I G F or insulin receptors (Froesch et al., 1985). The shift to a more " n o r m a l " distribution of the muscle fiber diameter also confirms the myotrophic effect of IGF-I. It is also more informative since it means that there are less atrophic muscle fibers of denervated type and more large muscle fibers. This may reflect a u g m e n t e d regeneration and reinnervation in wobbler mice due to I G F - I administration. O f particular interest is the increase in grip strength since it implies that there is also an action on m o t o r nerves, in addition to muscle. Such an increase in grip strength was also observed in wobbler animals by Mitsumoto et al. (1994) after c o t r e a t m e n t with B D N F and C N T F , but not with B D N F or C N T F alone. A m o n g the known neurotrophic factors, I G F - I has been proposed as the e n d o g e n o u s nerve sprouting factor as both m R N A and protein increase markedly in the distal stump after nerve injury (Caroni and Grandes, 1990), as they do in Botulinum toxin A paralyzed muscle (Caroni and Schneider, 1994). Additionally, I G F - I also has a wide range of action in muscle devel-
125
o p m e n t and growth (Florini et al., 1991) where I G F binding proteins might have regulatory roles (Caroni and Schneider, 1994). However, a unique direct action on the m o t o r nerve is less likely since we did not find any changes in choline acetyltransferase (CHAT) activity in muscle of I G F - I treated wobbler mice versus placebo-treated animals, particularly because C h A T activity reflects the degree of muscle innervation (Blondet et al., 1992). O u r failure to find an increased n u m b e r of m o t o n e u tons in the spinal cord using established techniques ( O p p e n h e i m , 1986; Li et al., 1994) argues also against the direct action of I G F - I on the m o t o r nerve. In conclusion, despite the absence of detection of an effect on the nerve side with the parameters that we have tested, we f o u n d an indisputable beneficial effect of I G F - I on the strength and muscle histochemical parameters of the neurological m u t a n t m o u s e wobbler. W h e t h e r these results are applicable to the t r e a t m e n t of h u m a n m o t o n e u r o n pathology, such as amyotrophic lateral sclerosis, remains to be determined.
Acknowledgements Support for these studies was provided, in part, by the Association Fran~aise contre les Myopathies (AFM), Cephalon, Inc., and the Muscular Dystrophy Association (MDA).
References Aizenman, Y. and de Vellis, J. (1987) Brain neurons develop in a serum and glial free environment: effects of transferrin, insulin, insulin-like growth factor-I and thyroid hormone on neuronal survival, growth and differentiation. Brain Res., 406: 32-42. Aug, L., Bhaumick, B., Munaz, D., Sass, J. and Juurlink, B. (1992) Effects of astrocytes, insulin and insulin-like growth factor I on the survival of motoneurons in vitro. J. Neurol. Sci., 109: 396-398. Blondet, B., Barlovatz-Meimon, G., Festoff, B.W., Soria, C., Soria, J., Rieger, F. and Hanta'i, D. (1992) Plasminogen activators in the neuromuscular system of the wobbler mutant mouse. Brain Res., 580: 303-310. Caroni, P. and Grandes, P. (1990) Nerve sprouting in innervated adult skeletal muscle induced by exposure to elevated levels of insulin-like growth factors. J. Cell Biol., 110: 1307-1317. Caroni, P. and Schneider, C. (1994) Signaling by insulin-like growth factors in paralyzed skeletal muscle: rapid induction of IGF 1 expression in muscle fibers and prevention of interstitial cell proliferation by IGF-BP5 and IGF-BP4. J. Neurosci., 14: 33783388. Contreras, P.C., Steffier, C. and Vaught, J.L. (1993) rhIGF-I enhances functional recovery from sciatic crush. Time-course and dose-response study. Ann. N. Y. Acad. Sci., 27: 314-316. Duchen, L.W. and Strich, S.J. (1968) An hereditary motor neuron disease with progressive denervation of muscle in the mouse: the mutant "wobbler". J. Neurol. Neurosurg. Psychiat., 31: 535-542. Duchen, L.W., Falconen, D.S. and Strich, S.J. (1966) Hereditary
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D. Hanta~et al. /Journal of the Neurological Sciences 129 (Suppl.) (1995) 122-126
progressive neurogenic muscular atrophy in the mouse, J. Physiol., 183: 53-55. Florini, J.R., Ewton, D.Z. and Magri, K.A. (1991) Hormones, growth factors, and myogenic differentiation. Annu. Rev. Physiol., 53: 201-216. Froesch, E.R., Schmid, Ch., Schwander, J. and Zapf, J. (1985) Actions of insulin-like growth factors. Annu. Rev. Physiol., 47: 443-467. Harris, J.B. and Ward, M.R. (1974) A comparative study of "denervation" in muscles from mice with inherited progressive neuromuscular disorders. Exp. Neurol., 42: 169-180. Kanje, M., Skottner, A., Sjoberg, J. and Lundborg, G. (1989) Insulin-like growth factor I (IGF-I) stimulates regeneration of the rat sciatic nerve. Brain Res., 486: 396-398. Komoly, S., Hudson, L.D., Webster, H. and Bondy, C. (1992) Insulin-like growth factor I gene expression is induced in astrocytes during experimental demyelination. Proc. Natl. Acad. Sci. USA, 89: 1894-1898. Li, L., Oppenheim, R.W., Lei, M. and Houenou, L. (1994) Neurotrophic agents prevent motoneuron death following sciatic nerve section in neonatal mouse. J. Neurobiol., 25: 759-766. Melki, J., Blondet, B., Pin~on-Raymond, M., Dreyfus, P. and Rieger, F. (1991) Generalized molecular defects of the neuromuscular junction in skeletal muscle of the wobbler mutant mouse. Neurochem. Int., 18: 425-433. Mitsumoto, H. (1985) Axonal regeneration in wobbler motor neuron
disease: quantitative histologic and axonal transport studies, Muscle Nerve, 8: 44-51. Mitsumoto, H. and Gambetti, P. (1986) Impaired slow axonal transport in wobbler mouse motor neuron disease. Ann. Neurol., 19: 36-43. Mitsumoto, H., lkeda, K., Klinkosz, B., Cedarbaum, J.M., Wong, V. and Lindsay, R.M. (1994) Arrest of motor neuron disease in wobbler mice cotreated with CNTF and BDNF. Science, 265: 1107-1110. Neff, N.T., Prevette, D., Houenou, L.J., Lewis, M.E., Glicksman, M.A., Yin Q.-W. and Oppenheim, R.W. (1993) Insulin-like growth factors: putative muscle-derived trophic agents that promote motoneuron survival. J. Neurobiol., 24: 1578-1588. Oppenheim, R.W. (1986) The absence of significant postnatal motoneuron death in the brachial and lumbar spinal cord of the rat. J. Comp. Neurol., 246: 281-286. Rand, J.B. and Johnson, C.D. (1981) A single-vial biphasic extraction assay for choline acetyltransferase using [3H]choline. Anal. Biochem., 116: 361-371. Recio-Pinto, E. and Ishii, D. (1988) Insulin and related growth factors: effects on the nervous system and mechanism for neurite growth and regeneration. Neurochem. Int., 12: 397-414. Torres-Aleman, I., Naftolin, F., and Robbins, R.J. (1990) Trophic effects of insulin-like growth factor-I on fetal rat hypothalamic cells in culture. Neuroscience, 35: 601-608.
NEUROLOGICAL SCIENCES ELSEVIER
Journal of the Neurological Sciences 129 (Suppl.) (1995) 122-126
Beneficial effects of insulin-like growth factor-I on wobbler mouse motoneuron disease D. Hanta'i a, * M. Akaaboune a, C. Lagord a,b M. Murawsky a L.J. Houenou c, B.W. Festoff d, J.L. Vaught e, F. Rieger a, B. Blondet a,b INSERM U. 153 and CNRS ERS 64, 17 rue du Fer h Moulin, 75005 Paris, France b Universitd Paris XII, Cr~teil, France c Department of Neurobiology and Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC, USA a Veterans Affairs Medical Center, Kansas City, MO and KS, USA e Cephalon Inc., West Chester, PA, USA
Accepted 21 February 1995
Abstract Recombinant human insulin-like growth factor-I (IGF-I) is being considered as a possible therapeutic agent for the treatment of motoneuron diseases like amyotrophic lateral sclerosis. The neurological mutant mouse wobbler, carries an autosomal recessive gene (wr) and has been characterized as a model of lower motoneuron disorders with associated muscle atrophy, denervation and reinnervation. The purpose of the present study was to determine the possible beneficial effect of IGF-I administration in this mouse model. Upon diagnosis at 4 weeks of age, affected mice and their control normal littermates received human recombinant IGF-I (1 mg/kg) or vehicle solution, once a day, for 6 weeks. Body weight and grip strength were evaluated periodically during the treatment period. Mean muscle fiber diameter on biceps brachii sections, choline acetyltransferase activity in muscle extracts, and motoneuron numbers in spinal cord sections were determined. IGF-I treated wobbler mice showed a marked weight increase from 3 to 6 weeks of treatment in comparison with placebo treated mutant mice. At the end of the treatment, grip strength, estimated by dynamometer resistance, was 40% higher in IGF-I treated versus placebo treated animals. Mean muscle fiber diameter which is smaller in wobbler mice than in normal mice was increased in IGF-I treated mutants. However, in this study the muscle choline acetyltransferase activity and the number of spinal cord motoneurons were unchanged. Thus, IGF-I administration mainly results in a significant effect on the behavioral and skeletal muscle histochemical parameters of the wobbler mouse mutant. Keywords: Trophic factors; Mutant mouse; Motoneuron disease; Human recombinant insulin-like growth factor-l; hrIgF-1;
Wobbler mouse (wr/wr); Dynamometry; Grip strength; Choline acetyltransferase (CHAT); Morphometry; Motoneuron counts; Muscle fiber diameter
1. Introduction A b e t t e r u n d e r s t a n d i n g of t h e factors which influence t h e d i f f e r e n t i a t i o n , survival or d e a t h of m o t o n e u rons is of c o n s i d e r a b l e physiological a n d clinical interest. H u m a n m o t o n e u r o n d i s e a s e s i n c l u d e several familial a n d s p o r a d i c c o n d i t i o n s involving t h e d e g e n e r a t i o n o r d e a t h o f m o t o n e u r o n s in the spinal c o r d a n d brain. T h e y p r e s e n t an i n t e r e s t i n g o p p o r t u n i t y for t h e r a p e u t i c
* Corresponding author. Tel.: (+33-1) 43 36 46 31; Fax: (+33-1) 43 37 85 22. E-mail: [email protected]. 0022-510X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0022-5 10X(95)00081-X
i n t e r v e n t i o n with n e u r o t r o p h i c proteins. I G F - I p r o m o t e s n e u r i t i c growth (both in vivo a n d in vitro), influe n c e s t h e m a i n t e n a n c e a n d r e g u l a t i o n o f n e u r o n a l dev e l o p m e n t a n d r e g e n e r a t i o n ( R e c i o - P i n t o a n d Ishii, 1988; K a n j e et al., 1989; C o n t r e r a s et al., 1993) a n d has n e u r o p r o t e c t i v e p r o p e r t i e s . I G F - I p r o m o t e s the survival of n e u r o n s in c u l t u r e ( A i z e n m a n a n d de Vellis, 1987; T o r r e s - A l e m a n et al., 1990; A n g et al., 1992; N e f f et al., 1993) a n d in d e v e l o p i n g spinal c o r d in vivo ( N e f f et al., 1993). I G F - I also functions in the r e g u l a t i o n o f o l i g o d e n d r o c y t e a n d myelin m e t a b o l i s m in vivo ( K o m o l y et al., 1992). F o r t h e s e reasons, I G F - I is likely to b e involved, directly or indirectly, in t h e a e t i o l o g y of a
D. Hanta'tet al. /Journal of the Neurological Sciences 129 (Suppl.) (1995) 122-126 variety of neurodegenerative disorders and, therefore, might be of therapeutic value for these diseases. To assess the therapeutic effect of IGF-I, we administered this factor to the neurological mutant mouse wobbler. Wobbler has been proposed as an animal model of human motoneuron diseases, such as the fatal amyotrophic lateral sclerosis, and spinal muscular atrophies (Mitsumoto and Gambetti, 1986; Melki et al., 1991). This spontaneous mutation is inherited as an autosomal recessive trait and displays a muscular atrophy associated with motoneuron degeneration in early post-natal development (Duchen et al., 1966). Clinical abnormalities are apparent at 3 - 4 weeks of age when the mutants become smaller than their littermates and exhibit general muscle weakness and wasting. At this stage, several authors have observed progressive partial denervation of skeletal muscle (Duchen et al., 1966; Duchen and Strich, 1968; Harris and Ward, 1974). On the other hand, it has been established that significant axonal regeneration and attempted reinnervation coincides with axonal degeneration (Mitsumoto, 1985; Melki et al., 1991; Blondet et al., 1992), all p h e n o m e n a where IGF-I administration might be of interest. Here, we show that treatment with I G F - I from 4 - 1 0 weeks of age, significantly improve behavioral and muscle histochemical parameters in this mutant wobbler mouse.
2. M a t e r i a l s
and methods
Mutant mice, homozygous ( w r / w r ) for the gene wr and littermates of normal phenotype ( + / . 9 ) were obtained from our mouse colony (C57BL/6J; Blondet et al., 1992). The phenotypically normal littermates were used as controls. Their weight and grip strength (measured with a dynamometer) were determined periodically. Recombinant human I G F - I (rhIGF-I; 1 m g / k g ) or vehicle solution was administered once a day during 6 weeks, from 4 to 10 weeks postnatally. One wobbler mouse and one control littermate were placed in the same cage and both of them were treated blind with either r h I G F - I or placebo. A total of 6 wobbler mice and 6 non affected littermates was used. To determine muscle C h A T activity, biceps brachii, triceps and gastrocnemius muscles were dissected and homogenized with a Polytron PT1200 homogenizer in 10% ( w / v ) medium containing 100 mM Tris-HC1, 2 mM E D T A , p H 7.6, all at 4° C. Homogenates were centrifuged at 20 000 × g for 30 min at 4° C. The supernatant was used without dilution. ChAT activity was determined by the method of Rand and Johnson (1981). A modification we made was that the enzymatic incubation was carried out at 37°C for 2 h. Motoneuron cell count in the lateral motor column of the spinal
123
cord was performed as described previously (Oppenheim, 1986; Li et al., 1994). Muscle fiber diameter distribution was determined by computer assisted morphometric analysis (SAMBA, TITN-Alcatel, Grenoble, France) on biceps brachii transverse cryostat sections. Protein determination was estimated by using the bicinchoninic acid (BCA protein assay, Pierce, Rockford, IL) protein assay. Statistical analysis was performed using the two-tailed Student's t-test.
3. R e s u l t s
El:olution of the weight of the animals From day 1 to day 21 of treatment, we did not observe any significant difference between I G F - I treated versus placebo treated animals, either wobbler mice or normal littermates. However, beginning on day 21 of treatment, the I G F - I treated wobbler mice showed a striking ( × 4) weight increase, compared to placebo treated wobbler mice (Fig. 1). For the I G F - I treated versus placebo treated normal littermates, we also noticed an increase but to a lesser extent ( × 2; Fig. 1). Grip strength Similar to total body weight, we did not detect any significant difference in grip strength in I G F - I treated versus vehicle treated animals before 3 weeks of treatment. After 25 days of treatment, the difference in grip strength between I G F - I treated versus placebo treated wobbler mice became apparent (Fig. 2). Note that, before 25 days of treatment, the placebo and I G F - I treated wobbler mice never exceeded 0.25 on the scale of the dynamometer. However, after 25 days the strength of the placebo treated wobbler mice started to
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Fig. 1. Weight increase between 21 and 39 days of treatment. The difference between IGF-I treated versus placebo treated wobbler mice is statistically significant (2.2+0.16 vs 0.63+0.26 g; values are means + SEM; comparison of means p < 0.01; n = 3 for each group).
D. Hanta't'et al, /Journal of the Neurological Sciences 129 (Suppl.) (1995) 122-126
124
wobbler
decline. At the same time, the grip strength of IGF-I treated mutant mice increased, reaching 0.35 on the scale of the dynamometer at day 40, i.e. a 40% increase in comparison with placebo treated wobbler mice. All controls, either placebo or IGF-1 treated, gave scores superior to 1.0 (the upper limit of the scale of the dynamometer) and therefore are not reported here.
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Fig. 2. Increase in grip strength as measured by dynamometry. Note that starting at day 30 there is a statistically significant increase in grip strength between IGF-I treated versus placebo treated wobbler mice ( + 4 0 % over control at day 40; values are means_+SEM: p < 0.01; n = 3 for each group).
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Using morphometric analysis of biceps transverse cryostat sections, we found, as expected, that the wobbler mice have smaller muscle fiber diameters than the controls (Fig. 3). When wobbler mutants were treated with IGF-I a shift of the fiber diameter distribution to the right (i.e. towards the higher diameters) was observed when compared with placebo treated mutants. The muscle fiber diameter distribution in normal littermates did not significantly change following treatment with IGF-I (Fig. 3).
diameter
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Fig. 4. Choline acetyltransferase (CHAT) activity in biceps, triceps and gastrocnemius muscle expressed as radioactive acetylcholine formed per muscle. Values are means_+ SEM; n = 3 for each muscle except for biceps (pool of 3 muscles).
D. Hanta~"el al. /Journal of the Neurological Sciences 129 (Suppl.) (1995) 122-126 Muscle choline acetyltransferase (CHAT) actiuity per muscle In all muscle studied (gastrocnemius, triceps, biceps; Fig. 4), C h A T activity per muscle in wobbler mice represented only 3 0 - 4 0 % of the activity in corresponding littermates. No statistically significant difference in C h A T activity per muscle between I G F - I versus placebo treated wobbler mutants and control mice was observed in any of the specific muscles studied (gastrocnemius, triceps, biceps; Fig. 4). Motoneuron cells count in the spinal cord Using an established technique ( O p p e n h e i m , 1986; Li et al., 1994), no statistically different n u m b e r of m o t o n e u r o n s in the spinal cord between I G F - I and placebo treated wobbler mice was observed (data shown). However m o t o n e u r o n survival and differentiation in wobbler mice is currently being reexamined in order to determine m o t o n e u r o n survival and to what extent this mutation affects m o t o n e u r o n survival.
4. Discussion T h e results from this study show that daily subcutaneous administration of r h I G F - I (1 m g / k g ) to wobbler mice beginning at the time the phenotypic diagnosis is made (i.e., at 4 weeks of age) and for a further 6 weeks resulted in a marked increase in (1) body weight; (2) grip strength; (3) mean skeletal muscle fiber diameters. T h e increase in body weight, likely reflects the increase in skeletal muscle mass since the latter represents mor,: than 80% of total body weight. This increase is expected since IGF-I exerts known nutrient effects in skeletal muscle by binding to I G F or insulin receptors (Froesch et al., 1985). The shift to a more " n o r m a l " distribution of the muscle fiber diameter also confirms the myotrophic effect of IGF-I. It is also more informative since it means that there are less atrophic muscle fibers of denervated type and more large muscle fibers. This may reflect a u g m e n t e d regeneration and reinnervation in wobbler mice due to I G F - I administration. O f particular interest is the increase in grip strength since it implies that there is also an action on m o t o r nerves, in addition to muscle. Such an increase in grip strength was also observed in wobbler animals by Mitsumoto et al. (1994) after c o t r e a t m e n t with B D N F and C N T F , but not with B D N F or C N T F alone. A m o n g the known neurotrophic factors, I G F - I has been proposed as the e n d o g e n o u s nerve sprouting factor as both m R N A and protein increase markedly in the distal stump after nerve injury (Caroni and Grandes, 1990), as they do in Botulinum toxin A paralyzed muscle (Caroni and Schneider, 1994). Additionally, I G F - I also has a wide range of action in muscle devel-
125
o p m e n t and growth (Florini et al., 1991) where I G F binding proteins might have regulatory roles (Caroni and Schneider, 1994). However, a unique direct action on the m o t o r nerve is less likely since we did not find any changes in choline acetyltransferase (CHAT) activity in muscle of I G F - I treated wobbler mice versus placebo-treated animals, particularly because C h A T activity reflects the degree of muscle innervation (Blondet et al., 1992). O u r failure to find an increased n u m b e r of m o t o n e u tons in the spinal cord using established techniques ( O p p e n h e i m , 1986; Li et al., 1994) argues also against the direct action of I G F - I on the m o t o r nerve. In conclusion, despite the absence of detection of an effect on the nerve side with the parameters that we have tested, we f o u n d an indisputable beneficial effect of I G F - I on the strength and muscle histochemical parameters of the neurological m u t a n t m o u s e wobbler. W h e t h e r these results are applicable to the t r e a t m e n t of h u m a n m o t o n e u r o n pathology, such as amyotrophic lateral sclerosis, remains to be determined.
Acknowledgements Support for these studies was provided, in part, by the Association Fran~aise contre les Myopathies (AFM), Cephalon, Inc., and the Muscular Dystrophy Association (MDA).
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