Nerve Wrap for Local Delivery of FK506/Tacrolimus Accelerates Nerve Regeneration

Abstract

Peripheral nerve injuries (PNIs) occur frequently and can lead to devastating and permanent sensory and motor function disabilities. Systemic tacrolimus (FK506) administration has been shown to hasten recovery and improve functional outcomes after PNI repair. Unfortunately, high systemic levels of FK506 can result in adverse side effects. The localized administration of FK506 could provide the neuroregenerative benefits of FK506 while avoiding systemic, off-target side effects. This study investigates the utility of a novel FK506-impregnated polyester urethane urea (PEUU) nerve wrap to treat PNI in a previously validated rat infraorbital nerve (ION) transection and repair model. ION function was assessed by microelectrode recordings of trigeminal ganglion cells responding to controlled vibrissae deflections in ION-transected and -repaired animals, with and without the nerve wrap. Peristimulus time histograms (PSTHs) having 1 ms bins were constructed from spike times of individual single units. Responses to stimulus onsets (ON responses) were calculated during a 20 ms period beginning 1 ms after deflection onset; this epoch captures the initial, transient phase of the whisker-evoked response. Compared to no-wrap controls, rats with PEUU-FK506 wraps functionally recovered earlier, displaying larger response magnitudes. With nerve wrap treatment, FK506 blood levels up to six weeks were measured nearly at the limit of quantification (LOQ ≥ 2.0 ng/mL); whereas the drug concentrations within the ION and muscle were much higher, demonstrating the local delivery of FK506 to treat PNI. An immunohistological assessment of ION showed increased myelin expression for animals assigned to neurorrhaphy with PEUU-FK506 treatment compared to untreated or systemic-FK506-treated animals, suggesting that improved PNI outcomes using PEUU-FK506 is mediated by the modulation of Schwann cell activity.

Keywords: biopolymer; immunology; infraorbital nerve; neuroregeneration; peripheral nerve injury; tacrolimus.

Figure 1

(A) FK506 concentration within PEUU-FK506 wrap over six weeks shows sustained release of drug. FK506 concentration represented as a log₁₀ transformation of mean ± SEM. (B) Blood serum levels of FK506 are lower for PEUU-FK506-treated animals versus IP-FK506-treated animals. FK506 blood concentration for animals (n = 3–6/group/time point) treated with PEUU-FK506 (black line) and animals treated with IP-FK506 (dotted line) over six weeks. Significant difference in mean FK506 blood serum concentration between groups denoted as * with p < 0.05 using Mann–Whitney test. Beyond two weeks, IP-treated animals reached steady state FK506 blood concentrations represented as mean concentration (dotted arrow) and standard error of means (bracket). Blood concentration values represented as mean ± SEM. (C) PEUU-FK506 delivers higher drug concentrations to the infraorbital nerve when compared to IP-FK506 treatment at two weeks postoperatively. Comparative FK506 concentrations within the infraorbital nerve, medial rectus muscle, blood serum, and PEUU-FK506 wrap among animals (n = 3–6/group/time point) treated with IP-FK506 2 mg/kg/day or PEUU-FK506 infraorbital nerve wrap for two weeks. FK506 concentration represented as a log₁₀ transformation of mean ± SEM. (D) PEUU-FK506 delivers drug in higher concentrations locally versus in distal tissues. FK506 concentrations in the infraorbital nerve and medial rectus muscle over time among PEUU-FK506-treated animals (n = 3–6/group/time point). FK506 concentration represented as a log₁₀ transformation of mean ± SEM.

Figure 2

Response magnitude is lower for 4-week recovery cases without PEUU-FK506 treatment. Representative peristimulus time histograms of trigeminal ganglion cell to whisker deflection among treatment groups (A–D). Each peri-stimulus time histogram consists of n = 300 (0.1 ms) bins and is constructed from responses of all neurons within each experimental group (10 deflections × 8 directions × number of neurons; n = number of cells); histograms are scaled to the total number of stimuli. Stimulus waveform for movement onsets is shown in panel (E).

Figure 3

PEUU-FK506 treatment accelerates recovery but does not lead to improved long-term outcomes. Trigeminal ganglion cell to whisker deflection response magnitudes (A–C). ON responses are calculated as average spikes per stimulus evoked during the first 20 ms of the response following deflection onset. (A) ON responses evoked by movement at each cell’s maximally effective deflection angle. (B) ON responses averaged over all 8 deflection angles. (C) Maximal angle plateau responses calculated over 100 ms. Data for individual neurons within the 4 experimental groups are represented as discrete points. Population mean per group are represented as filled square blocks. Number of cells per group are reported in Table 1.

Figure 4

Myelin staining intensity is greater in PEUU-FK506-treated infraorbital nerves versus IP-FK506 treatment. (A–F) Representative confocal images of infraorbital nerves stained for myelin (red/TRITC), neurofilament medium chain (green/FITC), and nucleus (blue/DAPI). Confocal images acquired for each sample were modified using NIS-Elements software version 4.4.0. Scale bar = 50 microns. (A) Naїve; (B) cut-and-repair; (C) IP-FK506 4-wk; (D) IP-FK506 6-wk; (E) PEUU-FK506 wrap 4-wk; and (F) PEUU-FK506 wrap 6-wk. (G) Measured neurofilament medium chain (FITC) and myelin (TRITC) signal within each study cohort (n = 2–4 per group). (H) Comparison of myelin (TRITC) signal within each study cohort (n = 2–4 per group). (I) Proportion of infraorbital nerve FITC per TRITC signal within each study cohort (n = 2–4 per group). Signal intensity represented as mean ± SEM with significance denoted as p < 0.05.

Figure 5

Flowchart of cohorts assigned to FK506 concentration measurements in the blood serum, ION, medial rectus muscle, and PEUU-FK506 at designated timepoints. (A) Animal cohorts assigned to PEUU-FK506 treatment. (B) Animal cohorts assigned to IP-FK506 treatment. (C) Representative diagram of whisker/trigeminal system model for recording trigeminal nerve nuclei signal. (D) Spatial representation of anatomical structures involved in measuring mechanoreceptive afferent signal. (E) Naïve rat with intact ION (no surgery). (F) ION transection and repair with PEUU-FK506 treatment.