Expression profile of nerve growth factor after muscle incision in the rat

Abstract

Background: Previous studies have demonstrated that nerve growth factor (NGF) is an important mediator of pathologic pain. Many studies have focused on cutaneous mechanisms for NGF-induced hyperalgesia; few have examined its contribution in deeper tissues like muscle. This study examined pain behaviors and the expression of NGF in incised hind paw flexor digitorum brevis muscle.

Methods: Adult Sprague-Dawley rats were pretreated with anti-NGF peptibody and underwent skin or skin plus deep fascia and muscle incision. Guarding pain behaviors were measured. Muscle NGF messenger RNA (mRNA) was measured by reverse-transcriptase polymerase chain reaction. Changes in NGF protein expression were measured using Western blot, enzyme-linked immunosorbent assay, and immunohistochemistry. In situ hybridization for NGF mRNA was also performed.

Results: Pretreatment with anti-NGF peptibody (100 mg/kg) decreased the guarding behavior caused by deep fascia and muscle incision. Muscle NGF mRNA increased abruptly 2 h after incision and was the same as control by postoperative day 1. NGF protein increased from 4 h after incision and was sustained for several days. NGF was localized in many calcitonin gene-related peptide-positive axons, few N52-positive axons, but not isolectin B4-positive axons in incised muscle. The sources of NGF mRNA included keratinocytes in epidermis and fibroblasts in deeper tissues.

Conclusion: Fibroblasts adjacent to the injury are sources of NGF in incised muscle. NGF is upregulated by incision of muscle and contributes to guarding pain behavior.

Fig. 1

Effect of anti-nerve growth factor (NGF) peptibody on guarding pain ehavior caused by plantar incision. A: Cumulative pain score after anti-NGF treatment in rats that underwent only skin incision. B: Cumulative pain score after anti-NGF treatment in rats that underwent skin, fascia and muscle incisions. t, indicates P<0.05 vs baseline. Asterisks indicate *P<0.05 and **P<0.01 vs vehicle, respectively.

Fig. 2

Nerve growth factor (NGF) expression in incised or sham operated plantar flexor digitorum brevis muscle. A: Changes in NGF mRNA relative to β-actin mRNA examined by real time polymerase chain reaction. Data are expressed as mean ± SEM of ratio of incised flexor muscle to sham controls. B: NGF protein determined by enzyme-linked immunosorbent assay. Data are shown as mean ± SEM of nanogram of NGF protein per milligram protein. C: Western blots for NGF and actin immunoreactivity. Top: Example of western blot showing immunoreactive NGF and actin control bands in detailed time course after plantar incision. Bottom: Summary of relative changes in NGF protein expression after plantar incision. Data are expressed as mean ± SEM of ratio of incised flexor muscle to sham controls. Non, contralateral non-incised muscle. PO, post operative. POD, post operative day. * P<0.05, **P<0.01 vs sham. N = 6 per group.

Fig. 3

3’3-diaminobenzidine immunohistochemistry for nerve growth factor (NGF) in plantar flexor digitorum brevis muscle. A: NGF immunohistochemistry in sham operated flexor muscle. B: NGF in nerve-like structures from incised muscle on POD (post operative day) 1. C: A higher magnification of the rectangular area C in Fig. B, showing immunoreactivity for NGF within a transverse sectioned nerve-like structure. D: A higher magnification of the rectangular area D in B, showing immunoreactivity for NGF within a longitudinal sectioned nerve-like structure. E: NGF immunohistochemistry in skin, fascia and muscle after preabsorption with blocking peptide. The arrow indicates the incision.

Fig. 4

Confocal image of nerve fibers in muscle tissue for nerve growth factor (NGF) and protein gene product 9.5 (PGP 9.5), calcitonin gene related peptide (CGRP), isolectin B4 (IB4) fluorescent immunohistochemistry. A, B and C: NGF fluorescent immunohistochemistry (red) within incised flexor muscle on POD (post operative day) 1. A1: PGP 9.5 immunohistochemistry for nerve axons (green) in incised flexor muscle. A2: Merged image shows NGF colocalizes with PGP9.5 immunoreactive axons (yellow). B1: CGRP immunohistochemistry for axons (green) within incised flexor muscle. B2: Merged image shows NGF colocalizes with CGRP immunoreactive axons (yellow). C1: IB4 labeling (green) in incised flexor muscle. C2: Merged image shows NGF does not colocalize with IB4 axons (yellow).

Fig. 5

Confocal image for nerve growth factor (NGF) and calcitonin gene related peptide (CGRP), isolectin B4 (IB4), neurofilament 200 (N52) fluorescent double labeling immunohistochemistry in large nerve in deep tissue. A, B and C: NGF fluorescent immunohistochemistry one day after plantar incision. A1: CGRP immunohistochemistry for axons (green) in large nerve. A2: Merged image shows NGF colocalizes with CGRP immunoreactive axons (yellow). B1: IB4 labelling for axons (green) in large nerve. B2: Merged image shows NGF does not colocalize with IB4 axons (yellow). C1: N52 labelling for axons (green) in large nerve. C2: Merged image shows NGF colocalizes with some N52 axons (yellow).

Fig. 6

In situ hybridization for nerve growth factor (NGF) mRNA in cortex of adult rat brain. Sections are counter stained with fast red for nuclei. A: In situ hybridization (ISH) for NGF mRNA with antisense probe. B: A higher magnification of the rectangular area in A, showing NGF expression (blue) in neuron cytoplasm. C: ISH for NGF mRNA with sense probe. D: A higher magnification of the rectangular area in C.

Fig. 7

In situ hybridization (ISH) for nerve growth factor (NGF) mRNA in sham operated plantar skin and flexor digitorum brevis muscle. Sections are counter stained with fast red for nuclei. A: ISH for NGF mRNA with antisense probe. B–F: Higher magnification photographs show NGF mRNA expression (blue) within epidermis and dermis (B), dense connective tissue in subcutaneous tissue (C), perimysium which separates muscle fibers (D), peri-vascular connective tissue (E) and large nerve bundle (F) in deep layer which innervates plantar skin and flexor muscle.

Fig. 8

In situ hybridization (ISH) for nerve growth factor (NGF) mRNA in incised plantar skin and flexor digitorum brevis muscle. Sections are counter stained with fast red for nuclei. A: ISH for NGF mRNA with the antisense probe 2 hours after incision. B–H: Higher magnification photographs show NGF mRNA expression within epidermis and dermis (B), dense connective tissue in subcutaneous region (C), perimysium (D), peri-vascular connective tissue (E) and large nerve bundle (F) in deep tissue layer. ISH in subcutaneous region adjacent to skin incision (G), and flexor muscle fibers adjacent to muscle incision (H). I: ISH for NGF mRNA with sense probe at 2 hours after incision showing no positive staining. The arrows indicate the incision. PO – post operative.