Effect of locally delivered IGF-1 on nerve regeneration during aging: an experimental study in rats

Abstract

Age is an important predictor of neuromuscular recovery after peripheral nerve injury. Insulin-like growth factor 1 (IGF-1) is a potent neurotrophic factor that is known to decline with increasing age. The purpose of this study was to determine if locally delivered IGF-1 would improve nerve regeneration and neuromuscular recovery in aged animals. Young and aged rats underwent nerve transection and repair with either saline or IGF-1 continuously delivered to the site of the nerve repair. After 3 months, nerve regeneration and neuromuscular junction morphology were assessed. In both young and aged animals, IGF-1 significantly improved axon number, diameter, and density. IGF-1 also significantly increased myelination and Schwann cell activity and preserved the morphology of the postsynaptic neuromuscular junction (NMJ). These results show that aged regenerating nerve is sensitive to IGF-1 treatment.

FIGURE 1

Diagram of experimental setup. The tibial nerve was transected 1 cm from the insertion into the gastrocnemius and allowed to relax to create a 7-mm gap. A custom-made T-tube device was interposed between the nerve ends and secured with a 9-0 nylon suture. The middle arm of the T-tube was connected to an osmotic pump, which delivered either IGF-1 or normal saline at 0.25 µl/h.

FIGURE 2

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.]

FIGURE 3

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.]

FIGURE 4

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.]

FIGURE 5

Response of the neuromuscular junction to saline or IGF-1 treatment. (A) Young saline, (B) aged saline, (C) young IGF-1, and (D) aged IGF-1. The postsynaptic NMJ was stained with fluorescent-labeled α-bungarotoxin and imaged under a confocal microscope. In saline-treated young animals (A), postsynaptic NMJ was highly contiguous, complex, and had deep gutters. In aged saline-treated animals (B), there was a loss of complexity, with flattening of the gutters and a reduction in total postsynaptic area. This morphologic appearance is consistent with chronic denervation. In young IGF-1–treated animals (C), there were no differences when compared with saline-treated animals. In aged IGF-1–treated animals (D), there was an increase in the complexity and gutter depth, similar to the morphology seen in young animals. However, there was increased fragmentation seen in the aged IGF-1 group compared with the young IGF-1 or young saline groups. Scale bar = 20 µm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]