Development of polarization-sensitive optical coherence tomography imaging platform and metrics to quantify electrostimulation-induced peripheral nerve injury in vivo in a small animal model

Abstract

Significance: Neuromodulation devices are rapidly evolving for the treatment of neurological diseases and conditions. Injury from implantation or long-term use without obvious functional losses is often only detectable through terminal histology. New technologies are needed that assess the peripheral nervous system (PNS) under normal and diseased or injured conditions.

Aim: We aim to demonstrate an imaging and stimulation platform that can elucidate the biological mechanisms and impacts of neurostimulation in the PNS and apply it to the sciatic nerve to extract imaging metrics indicating electrical overstimulation.

Approach: A sciatic nerve injury model in a 15-rat cohort was observed using a newly developed imaging and stimulation platform that can detect electrical overstimulation effects with polarization-sensitive optical coherence tomography. The sciatic nerve was electrically stimulated using a custom-developed nerve holder with embedded electrodes for 1 h, followed by a 1-h recovery period, delivered at above-threshold Shannon model $k$ -values in experimental groups: sham control (SC, $n=5$ , $0.0\mathrm{mA}/0\mathrm{Hz}$ ), stimulation level 1 (SL1, $n=5$ , $3.4\mathrm{mA}/50\mathrm{Hz}$ , and $k=2.57$ ), and stimulation level 2 (SL2, $n=5$ , $6.8\mathrm{mA}/100\mathrm{Hz}$ , and $k=3.17$ ).

Results: The stimulation and imaging system successfully captured study data across the cohort. When compared to a SC after a 1-week recovery, the fascicle closest to the stimulation lead showed an average change of $+4\%/-309\%$ (SL1/SL2) in phase retardation and $-79\%/-148\%$ in optical attenuation relative to SC. Analysis of immunohistochemistry (IHC) shows a $+1\%/-36\%$ difference in myelin pixel counts and $-13\%/+29\%$ difference in axon pixel counts, and an overall increase in cell nuclei pixel count of $+20\%/+35\%$ . These metrics were consistent with IHC and hematoxylin/eosin tissue section analysis.

Conclusions: The poststimulation changes observed in our study are manifestations of nerve injury and repair, specifically degeneration and angiogenesis. Optical imaging metrics quantify these processes and may help evaluate the safety and efficacy of neuromodulation devices.

Keywords: nerve stimulation; neuromodulation; optical metrics; peripheral nerve injury; polarization-sensitive optical coherence tomography; rat; sciatic nerve.

Fig. 1

Optical coherence tomography (OCT) imaging: full system layout. This custom-built swept-source polarization-sensitive OCT system was designed to capture volumetric scans of the sciatic nerve during electrical stimulation. This system noninvasively extracts tissue features (structure, birefringence, and perfusion) without stains or dyes. (a) Nerve stabilizer 3D-model and (b) stabilizer with sciatic nerve in situ. Deinsulated portions of the electrodes are noted with yellow bars, nearest to the rightmost fascicle.

Fig. 2

Platform output: processed PSOCT data, animal #2: SC, D7. Representative data from a rat sciatic nerve as observed in this study of the six data channels captured. En face representations and cross-sectional scans for each channel are shown. BwOA, birefringence-weighted optic axis. Scale bar: $500\mu \mathrm{m}$.

Fig. 3

Surgical and imaging experimental protocol. Day 1 began with a baseline assessment of nerve function, followed by sciatic nerve exposure surgery. Afterward, the animal was placed into the imaging setup for a 30-min acclimation period (PREP). A baseline (B) OCT scan of the nerve was captured before stimulation. Stimulation was active for 1 h (ON) followed by a 1-h recovery (OFF). Simultaneously, OCT scans of the stimulation region were collected every 30 min (during stimulation: S1, S2; and recovery: R1, R2). On day 7, nerve assessment tests were repeated, and the left sciatic nerve re-exposed for imaging. OCT scans were collected using a tiled collection strategy to ensure capture of the stimulation region from day 1. Nerve exposure and tiled imaging was repeated on the contralateral (right) leg to serve as an internal control. Finally, the sciatic nerves from both legs were harvested for histological analysis.

Fig. 4

Data extraction methods. (a) Data channels were organized and co-registered across each imaging timepoint for analysis (from top to bottom: intensity, phase retardation, DOPU, and optic axis). (b) A 3D representation of nerve volume is plotted using BwOA channel. ROI selection: five analysis regions within the stimulation region were selected to extract representative frames. At each location, 25 frames were averaged to generate a resultant frame. An arithmetic or coherent mean (CM) was used to ensure data integrity during processing. For each of the five resultant averaged frames, ROIs were manually segmented using histology and PSOCT image morphology to select only the fascicle (teal) and not the epi- or perineurium (red). From each ROI, a mean value for each fascicle can be extracted and group statistics computed. (c) To analyze angiography data, the full en face dataset from each timepoint was processed to extract both vessel diameter and density metrics. Changes in vessel diameter (top) were calculated by manually selecting at least 30 segments per timepoint, extracting the mean FWHM. To compute vessel fraction (bottom), a coverage mask was computed. A ratio (%) of vessel to total (vessel + background) pixels is calculated for each frame.

Fig. 5

Representative imaging results, animal #4: SL1. (a) (left) OCT scans capture temporal dynamics of the left sciatic nerve during stimulation and recovery. Polarization phantoms (*, red/purple) shown in (a, left series), cropped in all other figures. (a, center): Overlapping scans are co-registered using cues from the structural organization of fascicles and perfusion maps. Tile #3 (purple, solid outline) corresponds best to day 1 scans (teal, dotted outline). BwOA colormap [(a), right] relates color to the physical orientation of tissue subunits. (b) Day 1 baseline, including en face projections of the structure (left), birefringent-weighted optic axis (BwOA, center), perfusion map (right), and cross-sectional scans (below) from all data channels. (c) Day 7 tile #3, co-registered data showing changes after a 7-day recovery. Note: yellow lines mark electrodes and stimulation region (day 1). Orange circles demonstrate one perfusion map visual cue for co-registration. White-dotted lines denote matching locations in cross-sectional data. Cross-sectional scans, from corresponding en face projections: (row 1) structural, BwOA, and angiography/perfusion and (row 2) phase retardation, optic axis, and degree of polarization uniformity. Distal and proximal labels indicate sciatic nerve orientation. Scale bars (white, solid): $500\mu \mathrm{m}$.

Fig. 6

OCT metrics: changes after 1 week. (a) Phase retardation, D7 to D1. The rightmost fascicle nearest the stimulation electrode has the greatest effect, with the SL2 group with a significant reduction in phase retardation average value (SL2, ***$p<0.001$) after recovery. (b) Optical attenuation, D7 to D1. Results show a decrease in mean attenuation in the center and right fascicles in both SL1 and SL2. Left/center/right (L/C/R) denotes fascicle position, with R closest to the deinsulated portion of the electrode. (c) Blood vessel diameter (whole nerve), all timepoints across D1 and D7. Mean vessel diameter results demonstrate the stimulation group values increase in diameter under stimulation as expected and slowly decrease during the recovery period. Notably, a small increase over baseline values persists at 1 week for SL1 and SL2. (d) Blood vessel fraction (coverage, whole nerve), all timepoints across D1 and D7. Vessel fraction results show no appreciable change in the number of vessels over the course of day 1 as expected, though an increase in the total amount of vessels is seen at day 7 (SL1 **$p<0.005$ and SL2 *$p<0.05$). Error bars: ±SD. $\mathrm{\Delta }\text{Metric}={\mathrm{avg}}_{\text{stim}\mathrm{grp}}(\mathrm{D}{7}_{2\mathrm{D}\text{fasc}}-\mathrm{D}{1}_{2\mathrm{D}\text{fasc}}$).

Fig. 7

Histology co-registration and comparison. Representative IHC and H&E-stained sections from each stimulation group. Insets are expanded views of the fascicle nearest to the stimulation leads (right side). Sections were co-registered with PSOCT data and evaluated for signs of injury. IHC and H&E scale bars represent $125\mu \mathrm{m}$ ($35\mu \mathrm{m}$ inset), acquired at $10\times$.

Fig. 8

IHC image analysis results. Relative structural components are quantified in fluorescent IHC images by summing the fluorescent pixels within a fascicle ROI (D7 to D1) and comparing between stimulation groups. (a) Red pixels (myelin, SL2 **$p<0.005$), (b) green pixels (axons), (c) blue pixels (cell nuclei/DAPI), and (d) the ratio of myelin/axon pixel counts (SL2 *$p<0.05$). Left/center/right (L/C/R) denotes the fascicle position, with R closest to the deinsulated portion of the electrode. Error bars: ±SD. $\mathrm{\Delta }\text{Metric}={\mathrm{avg}}_{\text{stim}\mathrm{grp}}(\mathrm{D}{7}_{2\mathrm{D}\text{fasc}}-\mathrm{D}{1}_{2\mathrm{D}\text{fasc}}$).