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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 1, 2002 as doi:10.1096/fj.02-0183fje. |
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Michael E. Debakey Department of Surgery, Division of Plastic Surgery, and
* Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, Texas, USA
2Correspondence: Michael E. DeBakey Department of Surgery, Division of Plastic Surgery, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. E-mail: ericr{at}bcm.tmc.edu
SPECIFIC AIMS
There is no known medical treatment that hastens the repair of damaged nerve and muscle. Using IGF-1 transgenic mice expressing the human recombinant IGF-1 in skeletal muscle, the aim of this study was to test the hypothesis that targeted gene expression of IGF-1 in skeletal muscle enhances motor nerve and muscle regeneration after a peripheral nerve injury.
PRINCIPAL FINDINGS
1. The IGF-1 transgene increases satellite cell proliferation after nerve injury
Transgenic homozygous female mice expressing the human IGF-1 transgene were produced, using a skeletal alpha actin promoter. After a sciatic nerve crush injury, gastrocnemius muscle was analyzed for muscle and nerve regeneration. Before nerve injury, satellite cell proliferation, identified by cyclin D1 expression, is not detected in IGF-1 or wild-type muscle using a chemiluminescence-based ELISA (Fig. 1
). Expression of cyclin D1 in IGF-1 muscle peaks on day 1 postinjury, increasing fourfold, then gradually declines through days 3 and 5 postinjury. In contrast, expression of cyclin D1 in wild-type muscle is not detected at day 1, but increases 1.5-fold at day 3 before declining to basal levels at day 5 postinjury. Immunohistochemical analysis shows cyclin D1-positive cells are satellite cells and IGF-1 muscle has significantly more proliferating satellite cells than wild-type muscle after a nerve injury. Therefore, satellite cell proliferation occurs earlier and to a greater extent in IGF-1 transgenic muscle than in wild-types.
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2. The IGF-1 transgene increases multipotent stem cell populations in muscle
Stem cell antigen (Sca-1)-positive cells in mouse skeletal muscle represent a population of multipotent stem cells derived from bone marrow. Sca-1 expression is detected in noninjured IGF-1 and wild-type muscle by CELISA, but basal Sca-1 expression is nearly 50% higher in IGF-1 transgenic muscle. At day 1 postinjury, Sca-1 expression increases sevenfold in IGF-1 transgenic muscle, and then gradually declines. In contrast, Sca-1 expression in wild-type muscle is first detected after day 3, increasing only twofold before declining to near basal levels at day 5. Immunohistochemical analysis reveals that SCA-1 is expressed in satellite cells. The identical time course of Sca-1 and cyclin D1 expression suggests that IGF-1 activates muscle stem cell precursors to proliferate.
3. The IGF-1 transgene increases muscle differentiation pathways after nerve injury
MyoD and myogenin are important in the initiation of muscle repair in the adult. In noninjured muscle, low levels of MyoD and myogenin are expressed in transgenic IGF-1 muscle but not in wild-type muscle. After 1 day postinjury, MyoD and myogenin expression increases seven- and fivefold, respectively, in IGF-1 transgenic muscle, before gradually declining. In contrast, wild-type mice first exhibits detectable MyoD and myogenin expression at 3 and 5 days postinjury, respectively, but expression levels are significantly lower vs. IGF-1 transgenic muscle. Immunohistochemical analysis detects MyoD and myogenin in satellite cells of noninjured IGF-1 transgenic muscle but not in noninjured wild-type muscle. After nerve injury, levels of MyoD and myogenin in satellite cells of IGF-1 transgenic muscle are significantly higher than wild-type muscle. Therefore, IGF-1 intensifies muscle regeneration after nerve injury by accelerating the myogenic differentiation pathway.
4. IGF-1 transgene enhances peripheral nerve regeneration
Neurofilaments are a major cytoskeleton protein of axons. Using neurofilament 150 kDa expression as a marker for muscle innervation, we analyzed the effects of locally expressed IGF-1 on axon growth after nerve injury by CELISA and immunohistochemistry. Neurofilament expression is increased 
twofold in IGF-1 transgenic muscle at 2 wk postinjury compared with injured wild-type muscle and remains elevated through 8 wk of recovery. Immunohistochemical analysis shows increased innervation in IGF-1 transgenic muscle after 3 wk postinjury compared with injured wild-type muscle. Therefore, the IGF-1 transgene accelerates reinnervation of muscle after a nerve injury.
In adults, muscle differentiation factors and nicotinic acetylcholine receptor subunits are known to be up-regulated by chemical or mechanical denervation of muscle, but return to baseline levels on functional innervation or after electrical stimulation. In our studies, myogenin and 
AchR mRNA are present in IGF-1 transgenic muscle and wild-type muscle 2 wk postinjury. Within 3 wk postinjury, however, myogenin and AchR mRNA levels return to uninjured levels in IGF-1 transgenic muscle whereas myogenin and AchR mRNA remains elevated in wild-types. The hastened restoration of myogenin and AchR mRNA to control levels strongly suggests that muscles of IGF-1 transgenic mice are reinnervated rapidly after injury.
Saltatory nerve conduction can only occur in myelinated, intact nerve muscle synapses and is therefore an excellent marker for functional recovery of movement (Fig. 2
). Nerve conduction velocity (NCV) is not evident 2 wk after injury in IGF-1 transgenic and wild-type mice, indicating that functional nerve/muscle synapses are not yet reestablished. In IGF-1 transgenic mice, NCV is first detectable 3 wk after injury, but is still not undetectable in wild-type mice. By 8 wk, nerve injured IGF-1 transgenic and wild-type controls recover 76% and 55%, respectively, of their function. These results show that the IGF-1 transgene enhanced the rate and degree of functional nerve recovery in the muscle after nerve injury.
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The return of gastrocnemius muscle mass after nerve injury was assessed in IGF-1 transgenic mice and wild-type controls. Muscle mass in wild-type mice declines to 50% of normal weight 2 wk postinjury. In contrast, muscle mass in IGF-1 transgenic mice decline to only 75% of normal weight. After 8 wk of recovery, IGF-1 transgenic mice regained 96% of normal weight, whereas wild-type mice regained only 83%. These results show that the IGF-1 transgene reduces the degree of atrophy and hastens recovery of normal muscle mass after nerve injury.
CONCLUSIONS
Although the peripheral nervous system has the ability to regenerate in response to an acute or chronic nerve injury, complete restoration of function is rare in many instances, even with patients undergoing physical rehabilitation. Aside from surgical intervention, there is no known treatment to enhance the repair of injured nerves. Growth factors, such as IGF-I, that affect motor neuron survival and regeneration have potential for enhancing nerve repair. Using a mouse transgenic line expressing the human IGF-1 in skeletal muscle, we show that the local expression of IGF-1 is capable of accelerating the regeneration of peripheral nerves and muscle after a nerve injury by enhancing muscle myogenesis and peripheral nerve growth and function.
IGF-1 has a role in inducing the initial phases of muscle differentiation in response to a peripheral nerve injury by causing satellite cell proliferation, as demonstrated by increased cyclin D1 expression. In noninjured IGF-1 and wild-type muscle, SCA-1-positive cells are shown to populate the muscle and are quiescent. With injury, parallel increases of SCA-1- and cyclin D1-positive cells in IGF-1 muscle suggest that IGF-1 induces the expansion of the satellite stem cell compartment after injury.
In wild-type muscle, the time course expression of MyoD parallels that of cyclin D expression. This is consistent with in vitro studies showing that MyoD is expressed in proliferating satellite cells. MyoD’s differentiating function remains in an inactive state due to the growth-promoting activity of cyclin D1, but becomes active when growth-promoting signals decline. Expression of myogenin is expressed later in the myogenic pathway. Thus, a sequential pattern of myogenesis exists after a nerve injury where MyoD (a myogenic determination factor) is expressed previous to myogenin (a terminal differentiation factor).
In contrast, induction of the myogenic program is accelerated in IGF-1 transgenic mice. The rise of these factors in IGF-1 transgenic muscle are rapid and simultaneous, occurring only after 1 day postinjury. This suggests that IGF-1 is inducing simultaneous activation of proliferation and differentiation. It is probable that distinct subpopulations of precursor cells are either committed satellite cells ready for immediate differentiation or precursor stem cells destined for proliferation.
The role of IGF-1 in inducing nerve regeneration is demonstrated by 1) increased number of neurofilament staining axons in muscle, 2) the hastened return of muscle differentiation factors to denervation levels, 3) the accelerated return of nerve conduction velocity to denervated levels, and 4) diminished muscle atrophy and acceleration of long-term recovery of muscle mass. IGF-1 could be signaling the motor nerve cell body by either direct retrograde transport or indirectly via receptor activation on the axon. IGF-1’s effects on axonal growth are also supported by studies showing IGF-1-mediated up-regulation of neurofilament and tubulin and increased myelination. Exogenously administered IGF-1 protein can accumulate at the neuromuscular junction. Therefore, enhanced nerve growth in IGF-1 transgenic mice could be a result of multiple targeting to motor neurons, Schwann cells, and the neuromuscular junction. Taken together, we show that IGF-1 acts at diverse stages of nerve and muscle regeneration process (Fig. 3
). These findings strengthen the concept that IGF-1 can be used as a muscle-based gene therapy to enhance the functional innervation and regeneration of skeletal muscle after an acute nerve injury.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0183fje; to cite this article, use FASEB J. (November 1, 2002) 10.1096/fj.02-0183fje 
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