Enhanced Repair Effect of Toll-Like Receptor 4 Activation on Neurotmesis: Assessment Using MR Neurography

Animals and Surgery

All interventions and animal care procedures were performed in accordance with the Guidelines and Policies for Animal Surgery provided by our university and were approved by the Institutional Animal Use and Care Committee. All animals were obtained from the Animal Experiment Center of our university and were housed in a standard animal facility with 12-hour on/off light conditions, and allowed free access to standard food and water. Forty-eight adult Sprague-Dawley rats weighing 250 ± 20 g were used to establish peripheral nerve transection injury models. Animals were anesthetized with an intraperitoneal injection of sodium pentobarbital at a dosage of 30 mg per kilogram of body weight (Sigma-Aldrich, St Louis, Missouri). After anesthesia, the left hind limb of each animal was selected to establish sciatic nerve transection injury. In brief, the sciatic nerve was exposed and transected at the midportion of the nerve trunk. Immediately after transection, the nerve was repaired in an end-to-end coaptation with the manner of epineural suturing. The wound was then closed and the animal was returned and housed separately. The right hind limb served as the control, on which sham operations (incision and exploration of the nerve only) were performed.

After surgery, animals were randomly assigned to 2 groups by means of a random number table. The first group was subdivided into groups A (n = 6) and B (n = 6), and the second group was subdivided into groups C (n = 18) and D (n = 18). Groups A and C received daily intraperitoneal injection of LPS (Sigma-Aldrich) at a dosage of 0.32 mg per kilogram of body weight for 7 days postoperatively, as previously reported,⁸ whereas groups B and D received intraperitoneal injection of the same volume of phosphate buffered saline (PBS) as controls. Animals of the first group (subgroups A and B) underwent serial MR imaging before surgery (baseline), at 3 days, and 1, 2, 3, 4, 6, and 8 weeks after the surgery. In the second group (subgroups C and D), 6 animals each were randomly selected and sacrificed for histologic evaluation at 1, 3, and 6 weeks after MR imaging.

MR Imaging

MR imaging was performed on a 1.5T scanner (Intera; Philips Medical Systems, Best, the Netherlands) with use of a 5-cm linearly polarized birdcage radiofrequency mouse coil (Chenguang Medical Technologies, Shanghai, China). After anesthesia, animals were placed in the prone position. Coronal images including T2WI and T2 measurements and gadofluorine-enhanced T1WI were obtained in a longitudinal plane parallel to the course of the sciatic nerve. T2WI was acquired by using a 3D FSE sequence (TR/TE = 2500/138 ms, flip angle = 90°, echo-train length = 25, section thickness = 1 mm, section gap = 0 mm); T2 relaxation data were obtained by using a single-section multi-spin-echo (TR/TE = 2000/20–160 ms; echo spacing, 20 ms) sequence as previously described.¹¹ Gadofluorine M (250 mmol Gd/L; Bayer Schering Pharma, Berlin, Germany) enhanced imaging was acquired by using fat-suppressed 2D FSE T1WI (TR/TE = 300/17 ms, flip angle = 120°, section thickness = 1 mm, section gap = 0 mm) 24 hours after intravenous injection of gadofluorine M at a dose of 0.1 mmol/L/kg body as previously described.¹³ Other parameters of all sequences were: FOV = 60 × 60 mm, matrix size = 512 × 512, and NEX = 2.

The nerve signal abnormalities were observed in a blinded manner by 2 authors in consensus (J.S., with more than 10 years of experience with musculoskeletal MR imaging, and F.Z., with 5 years of experience with musculoskeletal MR imaging). Nerve signal intensities (SIs) and T2 relaxation times were measured by these 2 authors independently and in a blinded manner. The average values from the 2 datasets were used for analysis. SIs of the distal stumps of the injured nerves and the sham-operated nerves were measured on gadofluorine M–enhanced T1WI. Nerve enhancement rate was calculated as the ratio of SI of the injured nerve to SI of contralateral sham-operated nerves. T2 relaxation times of the distal stumps were measured by using the region of interest technique. For each measurement, a rectangular region of interest with a minimal size of 85 pixels covering a 10-mm proximal segment of the distal stump was placed within the nerve and along the course of the nerve. The average value generated from all analyzed pixels of each region of interest was adopted and an effort was made to avoid including fatty tissue, edema, and muscle in the measured volume, as previously described.¹³

Functional Assessment

Sciatic nerve functional recovery was assessed in subgroups A and B by using walking track analysis, which was performed at each time point before MR imaging by 2 authors (X.Z. and X.H.W., both with 3 years of experience with sciatic nerve functional assessment) in a blinded manner by consensus. The sciatic functional index (SFI) was calculated by the formula of Bain et al¹⁴ as an indicator of nerve locomotor dysfunction.

Histologic Examination

Animals in the second group (subgroups C and D) were sacrificed after MR imaging at specific time points by transcardial perfusion with PBS followed by 4% paraformaldehyde in 0.1 mol/L PBS (pH = 7.4). The distal stumps of the injury nerves were harvested and postfixed in 4% paraformaldehyde for 1 hour and then cryoprotected in 20% sucrose solution until examination. In addition, a 5-mm segment distal to the injured site was prepared from each distal nerve stump. Contiguous 6-μm cross-sections were obtained from the distal end of the tissue specimens and processed for Luxol fast blue (LFB) (Sigma-Aldrich) and oil red (Sigma-Aldrich) staining as described⁷ to detect myelin load and myelin debris. Contiguous 15-μm thickness longitudinal sections were obtained in the proximal end of the tissue specimens and processed for multiple immunohistochemical staining to assess macrophage infiltration, TLR4 expression, Schwann cell proliferation, and axonal regeneration. Briefly, sections were blocked with donkey serum (10:100 vol/vol) for 30 minutes, followed by incubation overnight at 4°C with primary antibodies against cluster of differentiation 68 (CD68) (1:200 vol/vol; Abcam, Cambridge, United Kingdom) to detect activated macrophages, S-100 (1:200 vol/vol; Abcam) to detect Schwann cells, TLR4 (1:200 vol/vol; Abcam) to detect TLR4 expression, or small proline-rich protein 1A (SPRR1A) to detect regenerating axons (1:200 vol/vol; Abcam). After being rinsed 3 times with PBS, sections were allowed to incubate with the corresponding Alexa Fluor 594- or 488-conjugated secondary antibody (1:200 vol/vol; Invotrogen, Carlsbad, California) at room temperature for 30 minutes and subsequently with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining. The immunoreactive signals were then observed by a confocal laser scanning microscope (LSM710; Carl Zeiss, Jena, Germany).

The quantification of LFB myelin staining, the number of activated macrophages, and the number of regenerating axons were measured per nerve segment. For the quantification of LFB myelin staining, the proportional area of tissue occupied by labeling within the entire cross-sectional area was measured by using a microscope (Axio Observer; Carl Zeiss) equipped with a digital imaging analysis system, as described previously.⁷ For the quantification of activated macrophages and regenerating axons, the number of labeled cells on CD68-stained longitudinal sections or axons on SPRR1A-stained longitudinal sections per nerve segment was estimated by the optical fractionator method⁷ using the digital image analysis system equipped with a confocal laser scanning microscope (LSM710).

Statistical Analysis

All data are presented as mean ± standard deviation. In groups A and B, gadofluorine enhancement rates, T2 values, and SFIs obtained at specific acquisition points were compared by using a repeated-measures 1-way analysis of variance, followed by the Student-Neuman-Keuls post hoc test for multiple pair-wise comparisons among different times. In groups C and D, the quantification of myelin LFB staining, the number of activated macrophages, and regenerating axons were compared using an independent t test. A 2-sided P value less than .05 was considered to indicate a significant difference. All statistical tests were performed by using SPSS 13.0 software (IBM, Armonk, New York).