Diffusion MRI Biomarker Validation through 3D Histology: Updates and Developing Strategies

Introduction: Neurotrauma kills millions of people worldwide annually and disproportionately affects younger individuals [1, 2]. Peripheral nerve injury in particular can have devastating consequences, leaving one disabled for life if not treated in a timely manner [3]. Unfortunately, current clinical tools are often of limited value early after injury/surgery, resulting in delayed decision-making and suboptimal outcomes. In contrast, advanced MRI modalities (e.g., diffusion tensor imaging, DTI) may predict outcomes earlier. However, these novel approaches need to be validated against pathology prior to clinical usage. Conventional pathology uses thin 2D sections, which creates a mismatch with 3D MRI images. The objective of this study is to develop 3D histological methods for peripheral nerves to overcome this limitation and facilitate validation against 3D DTI.

Methods: Three cohorts (sham, crush, cut-repair) of Sprague-Dawley rats were studied at 2-, 4-, and 12-weeks after surgery (n=3 per time/surgery cohort, total=21). Weekly behavior was collected on each animal. Following euthanasia, the sciatic nerve was extracted, ex-vivo MRI was performed (7T Bruker), and nerves were processed for immunohistochemistry. Nerves were cleared, and either immunolabeled and whole-mounted, or sectioned at 120 microns prior to immunolabeling. Full sample confocal images were obtained at 10X magnification, and single z-stacks were acquired at 40X magnification at key regions along the nerve.

Results: Behavioral data showed recovery in crush animals around 4-weeks and continued deficits with greater variation within the cut-repair groups (similar to [4]). DTI showed a decrease in fractional anisotropy that was more pronounced with injury severity, which was consistent with previous studies [4].

Conclusion : 3D immunolabeling showed well-organized tracts proximal to lesion sites, with disorganized axons within lesions. Future work will focus on establishing correlations between 3D histology and DTI to evaluate DTI as a monitoring and/or prognostic biomarker of recovery following trauma and surgical repair.