What Is IGF-1 Lr3 – The Jack of All Traits
Cartilage Repair – An Insulin-like Growth Factor-I Study
The role of IGF-I in articular cartilage metabolism has been extensively investigated in both health and disease . When added exogenously to monolayer or explant cultures of normal articular cartilage from a variety of species, IGF-I induces a plethora of anabolic effects and decreases catabolic responses . Chondrogenic differentiation of MSCs is induced by IGF-I but is enhanced when IGF-I and TGF-β1 are used in combination
The premise that IGF-I is required to maintain articular cartilage integrity is supported by an in vivo study in rats in which chronic IGF-I deficiency led to the development of articular cartilage lesions In animal models, IGF-I has led to enhanced repair of extensive cartilage defects and protection of the synovial membrane from chronic inflammation . However, there is a decreased capacity of chondrocytes to respond to IGF-I with age and in OA Evidence suggests an uncoupling of IGF-I responsiveness in OA with IGF-I having the ability to robustly simulate matrix synthesis with the inability to decrease matrix catabolism
Despite the diminished ability of IGF-I to decrease catabolism in aged and OA cartilage, studies suggest that a combination of IGF-I and BMP-7 results in greater repair potential than either growth factor alone These studies demonstrated that in general, BMP-7 was more potent than IGF-I in stimulating matrix synthesis in aged and OA cells, but the greatest increase in matrix synthesis was observed after combination treatment with BMP-7 and IGF-I.
EFFECT OF LOCALLY DELIVERED IGF-1 ON NERVE REGENERATION DURING AGING
IGF-1 Increases Axon Number, Diameter, and Density in Regenerated Nerve of both Young and Aged Animals
Examination of the peripheral nerve revealed that, in aged saline-treated nerves, there were fewer total axons, fewer axons per mm2, and less myelination than in young saline-treated nerves. IGF-1 had a marked effect on both young and aged animals, increasing axon number, diameter, and density, in addition to increasing myelination (Fig. 2). Quantitative data are presented in Figure 3. For the number of axons per nerve, IGF-1 increased total axon number in both young and aged animals; however, this was only statistically significant for aged animals. For axon density, IGF-1 increased axon density in both young and aged animals; however, this was only statistically significant for aged animals. For average axonal diameter, there was a significant increase in average axonal diameter in both young and aged animals when they were treated with IGF-1. There was no difference between the young IGF-1 group and aged IGF-1 group for number of axons per nerve, axon density, and average axonal diameter.
Cross-section of regenerated nerve segment. After 3 months, the regenerated nerve segment was removed, sectioned to 1 µm, stained with toluidine blue, and viewed at 1000×. (A) Young saline, (B) aged saline, (C) young IGF-1, and (D) aged IGF-1. Asterisk indicates representative myelinated axons. Note that each axon is surrounded by myelin of varying thickness. A myelinating Schwann cell (SC) can be seen in (D). In the saline-treated group, the young nerve had a larger number of axons and a greater axon density. With IGF-1 treatment, both groups had increases in axon number, diameter, and density (axons per mm2). In addition, IGF-1 increased myelin thickness and myelin/fiber ratio (g-ratio). In the saline-treated groups, there are fewer myelinated axons, and the myelin is thinner (A, B). By histomorphometric analysis, however, the young saline-treated nerves (A) show a greater total number of axons and greater axon density (axons per mm2). With IGF-1 treatment, both groups had increases in axon number, diameter, and density (axons per mm2). In addition, IGF-1 increased myelin thickness and the myelin/fiber ration (g-ratio). Scale bar = 10 µm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Effect of age and IGF-1 on axon number, density, and diameter. Quantification of histologic images reveals a significant effect of IGF-1 on (A) axons per nerve, (B) axon density (axons per mm2), and (C) average axonal diameter. Although there were differences due to age for axons per nerve and axon density, these were not statistically significant. Data are expressed as mean ± standard error *P < 0.05. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
IGF-1 Increases Myelination and Schwann Cell Activity in Regenerated Nerve of both Young and Aged Animals
Three parameters of Schwann cell activity and myelination were examined: (1) myelin thickness; (2) axon/fiber ratio (g-ratio); and (3) GAP43 mRNA expression, a marker of Schwann cell activity. 30,31 We found that IGF-1 significantly affected all measured aspects of Schwann cell activity and myelination (Fig. 4). For myelin thickness and g-ratio, this effect was not age-dependent. However, for GAP43 expression, aged animals had a greater response to IGF-1 than young animals (Fig. 4C).
Effect of age and IGF-1 on measures of myelination. Quantification of histologic images revealed a significant effect of IGF-1 on multiple facets of myelination and Schwann cell function. (A) Myelin thickness was significantly increased in IGF-1–treated nerves compared with saline-treated nerves. (B) Myelin thickness is known to increase with increases in axon diameter, and thus the g-ratio (axon:fiber diameter) was calculated. The significantly lower g-ratio in the IGF-1–treated groups indicates that the increases in myelination were independent of the increases in axon diameter. (C) GAP43 is a marker of Schwann cell activity and myelination. GAP43 is a marker of Schwann cell activity and myelination. mRNA for GAP43 was assayed by RT-PCR. GAP43 expression in young and aged animals was not significantly different when both were treated with saline. GAP43 expression levels were found to be significantly increased by IGF-1 treatment in both young and aged animals compared with saline treatment. The GAP43 expression in aged IGF-1–treated animals was significantly higher than in young IGF-1 animals. Data are expressed as mean ± standard error. *P < 0.05 [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
[highlight color=”orange”]In this study we sought to determine whether IGF-1 would improve neuromuscular recovery after nerve injury during aging. We found that IGF-1 improved nerve regeneration by acting on the axons and Schwann cells, and secondarily on the NMJs. These results suggest that there is no loss of sensitivity to IGF-1 with age and that IGF-1 can improve regeneration after nerve injury during aging.
Previous studies have examined the effect of systemically administered IGF-1 during neuromuscular recovery in neonatal rats. Vergani et al. found that systemic IGF-1 promoted neuronal survival following crush injury and improved muscle reinnervation.34 Kanje et al. found that locally delivered IGF-1 following crush injury in juveniles led to improved sensory function.35 Tiancgo et al. found that IGF-1 locally delivered to an end-to-side repair improved muscle function in young rats.36 To the authors’ knowledge, the current study is the first to examine the effect of IGF-1 during nerve regeneration in aged animals.
Axonal outgrowth is central to nerve regeneration, and IGF-1 is a well-documented promoter of motor neuron survival,37 axonal growth,38 and axonal branching.18 In the regenerating nerve, IGF-1 is a key promoter of initial sprouting and subsequent elongation of axons39 (for review, see Rabinovsky2). Although there has been extensive investigation of the effect of IGF-1 on neuron outgrowth and survival, nearly all of the studies were performed in vitro or in embryonic animals. Thus, it is not known how increased age affects the neuronal response to IGF-1. In this study we found that, for metrics of neuronal trophism, aged animals had a robust response to IGF-1 treatment, increasing axonal number, diameter, and density in the regenerated nerve segment. Thus, the neuronal response to IGF-1 of the regenerating axon does not appear to be limited due to age.
The Schwann cell is a key factor in promotion of nerve regeneration. The effect of IGF-1 on Schwann cells has also been studied. IGF-1 is known to promote Schwann cell survival,40 motility,41 proliferation, 42 and myelination.43 However, the physiology of Schwann cells from aged animals is largely unknown. This study has addressed some of the parameters of Schwann cell function in vivo, particularly myelination and GAP43 expression. GAP43 is upregulated in nearly all myelinating Schwann cells of the distal stump following axotomy.31 Expression of GAP43 is postulated to play a role in the interaction between the regenerating axon and Schwann cell.30 In this study, we found that GAP43 expression was significantly upregulated in both young and aged animals with IGF-1 treatment. In addition, GAP43 expression was higher in aged animals treated with IGF-1 than in similarly treated young animals, suggesting that age increases sensitivity to IGF-1 for this aspect of Schwann cell function. However, due to pooling of the whole muscle mRNA, in this study we could differentiate between an increase in Schwann cell number and an increase in activity. Furthermore, because the sample was from muscle, the increase in GAP43 expression was due to intramuscular neurons, terminal Schwann cells, or both. GAP43 expression in the regenerated segment itself was not assayed. In addition to GAP43 activity, IGF-1 increased myelination in the regenerated nerve, independent of the increase in axon diameter. Thus, at the Schwann cell level, age does not impair the response to IGF-1 and results in an increased regenerative response.
Failure to achieve reinnervation of the muscle is thought to contribute to impaired neuromuscular recovery after nerve injury.44 In this study we found by histologic examination that the motor endplate regained an innervated, pre-injury morphology in all but the aged saline group. This failure to reinnervate may have been due to delayed nerve regeneration or instability of the postsynaptic NMJ, both of which have been shown to occur with aging.9 In this study, treatment with IGF-1 may have accelerated axonal regeneration, leading to earlier reinnervation of the muscle, and thus preservational of the postsynaptic NMJ.
One limitation of our study is the lack of functional and electrical assessments. Functional recovery is the gold standard for clinical outcomes in human subjects, but, historically, functional recovery in rodent models has been difficult to assess.45 In rats, there is a poor correlation between functional, electrophysiologic, and histologic outcomes. 46 In this study, electrical measures of function were performed and showed a trend toward improvement with IGF-1 treatment, but this was not statistically significant (data not shown). Nonetheless, we were able to demonstrate that IGF-1 improved nerve regeneration in aged animals.
An important strength of our study is the in vivo comparison between young adult and aged rats after a complete nerve transection. Previous studies of IGF-1 on nerve regeneration utilized in vitro, neonatal, or juvenile animal models. In addition, many previous studies utilized crush injury models. Crush injuries in rodents typically have a near 100% rate of recovery and do not accurately model clinical scenarios wherein neuromuscular recovery is often very poor. Although previous studies have helped to answer many questions about the effects of IGF-1, this study is the first to examine in vivo the effects of age and IGF-1 on neuromuscular recovery following nerve transection.[/highlight]
[highlight color=”red”]Overall, our study has provided substantial evidence that locally delivered IGF-1 improves neuromuscular recovery after nerve injury in aged animals. We showed that, when local IGF-1 levels were equally supplemented in young and aged animals, nerve regeneration and NMJ preservation were similar, suggesting that IGF-1 may be a contributing factor in age-related impairment of neuromuscular recovery. However, our study has not conclusively demonstrated that IGF-1 deficiency is the immediate cause of age-related impairments in neuromuscular recovery after injury.[/highlight]
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