Quantification of Collagen Organization after Nerve Repair

Abstract

Background: Clinical outcomes after nerve injury and repair remain suboptimal. Patients may be plagued by poor functional recovery and painful neuroma at the repair site, characterized by disorganized collagen and sprouting axons. Collagen deposition during wound healing can be intrinsically imaged using second harmonic generation (SHG) microscopy. The purpose of this study was to develop a protocol for SHG imaging of nerves and to assess whether collagen alignment can be quantified after nerve repair.

Methods: Sciatic nerve transection and epineural repair was performed in male rats. The contralateral nerves were used as intra-animal controls. Ten-millimeter nerve segments were harvested and fixed onto slides. SHG images were collected using a 20× objective on a multiphoton microscope. Collagen fiber alignment was calculated using CurveAlign software. Alignment was calculated on a scale from 0 to 1, where 1 represents perfect alignment. Statistical analysis was performed using a linear mixed-effects model.

Results: Eight male rats underwent right sciatic nerve repair using 9-0 Nylon suture. There were gross variations in collagen fiber organization in the repaired nerves compared with the controls. Quantitatively, collagen fibers were more aligned in the control nerves (mean alignment 0.754, SE 0.055) than in the repairs (mean alignment 0.413, SE 0.047; P < 0.001).

Conclusions: SHG microscopy can be used to quantitate collagen after nerve repair via fiber alignment. Given that the development of neuroma likely reflects aberrant wound healing, ex vivo and/or in vivo SHG imaging may be useful for further investigation of the variables predisposing to neuroma.

Fig. 1.

Maximum intensity projection SHG images of nerve samples (fibrillar collagen = green, autofluorescence = red). A, Repaired nerve #1, epineural repair, 4 weeks after repair. The Nylon suture material is visible in red. B, Control #1, uninjured sciatic nerve from left hind limb. Scale bar 500 µm.

Fig. 2.

SHG images of repair and control nerve samples. A, Repaired nerve #2, epineural repair, 4 weeks after repair. Box inset is enlarged and depicted on the right. Scale bar 500 µm. B, Enlarged inset from repaired nerve. Perineural vasculature (with intravascular biconcave red blood cells) is depicted in red (autofluorescence). Scale bar 100 µm.

Fig. 3.

Qualitative comparison of nerve specimens imaged using SHG microscopy (far left) and traditional histologic preparations. The same nerve specimen is imaged using a variety of methodologies, depicted as examples here. A, SHG three-dimensional images of nerve specimens before histological processing. The collagen fibers are imaged intrinsically without sectioning or the application of stains or dyes for imaging. B, Appearance of the same nerve posthistological processing (fixation, embedding, sectioning, staining) using PSR, GTC, and H&E stains. Although each technique allows for qualitative visualization of collagen at the repair site, collagen is depicted in different colors, depending on the stain. The gross morphology of each of the images is the same though each imaging/processing technique allows for visualization of nuances in the tissue and cellular structures being imaged. Scale bar for all images 100 µm.