Peripheral denervation participates in heterotopic ossification in a spinal cord injury model

Abstract

We previously reported the development of a new acquired neurogenic HO (NHO) mouse model, combining spinal cord transection (SCI) and chemical muscle injury. Pathological mechanisms responsible for ectopic osteogenesis after central neurological damage are still to be elucidated. In this study, we first hypothesized that peripheral nervous system (PNS) might convey pathological signals from injured spinal cord to muscles in NHO mouse model. Secondly, we sought to determine whether SCI could lead to intramuscular modifications of BMP2 signaling pathways. Twenty one C57Bl6 mice were included in this protocol. Bilateral cardiotoxin (CTX) injection in hamstring muscles was associated with a two-stage surgical procedure, combining thoracic SCI with unilateral peripheral denervation. Volumes of HO (Bone Volume, BV) were measured 28 days after surgery using micro-computed tomography imaging techniques and histological analyses were made to confirm intramuscular osteogenesis. Volume comparisons were conducted between right and left hind limb of each animal, using a Wilcoxon signed rank test. Quantitative polymerase chain reaction (qPCR) was performed to explore intra muscular expression of BMP2, Alk3 and Id1. Nineteen mice survive the complete SCI and peripheral denervation procedure. When CTX injections were done right after surgery (n = 7), bilateral HO were detected in all animals after 28 days. Micro-CT measurements showed significantly increased BV in denervated paws (1.47 mm3 +/- 0.5) compared to contralateral sides (0.56 mm3 +/-0.4), p = 0.03. When peripheral denervation and CTX injections were performed after sham SCI surgery (n = 6), bilateral HO were present in three mice at day 28. Quantitative PCR analyses showed no changes in intra muscular BMP2 expression after SCI as compared to control mice (shamSCI). Peripheral denervation can be reliably added to spinal cord transection in NHO mouse model. This new experimental design confirms that neuro inflammatory mechanisms induced by central or peripheral nervous system injury plays a key role in triggering ectopic osteogenesis.

Fig 1. Sciatic nerve excision procedure by posterior approach.

A: skin incision over posterior surface of proximal hind limb exposes superficial muscular layers [gluteus maximus (1) and biceps femoralis (2)]. B: Biceps femoralis retraction allows sciatic nerve identification. C: Pelvic insertion of gluteus maximus muscle is freed to expose deep muscular layers [gluteus medius and gluteus minimus (3)]. D & E: Greater trochanter (4) exposure after deep muscles retraction. Sciatic nerve is followed throughout its course towards the sciatic notch. E: Proximal cut of sciatic nerve. F: Divisional branches of the sciatic nerve to hamstring muscles.

Fig 2. Anterior approach of femoral nerve.

A: skin incision through inguinal fold. B: femoral neuro-vascular bundle exposure. C: proximal part of femoral nerve laying over psoas muscle.

Fig 3. Experimental design and bone volume measurements.

Fig 3A. SCI: spinal cord injury; PNS: peripheral nervous system; CTX: cardiotoxin. T0: CTX was injected during surgery. D10: CTX was injected 10 days after surgery BV: HO Bone Volume measured with CT scan in right hind limb (RHL) versus left hind limb (LHL). Fig 3B. CT scan of harvested hind limbs showed bilateral intramuscular HO in one mouse from group 1 and 3 and no HO in mouse from group 2. Fig 3C. HO volumes in hind limbs of 6 mice from group 1 showing significant increased BV after peripheral denervation. Fig 3D. Amyotrophic right hind limb after peripheral denervation 28 days after surgery.

Fig 4. Muscular samples after SCI and CTX injection.

HES staining showed bone matrix (1), osteocytes (white arrow) mineralized nodules (2), regenerated muscular fibers (3) infiltration of inflammatory cells (4).

Fig 5. QPCR analyses of BMP signaling pathway in muscular samples of C57bl6 mice, 18 hours after spinal cord injury (SCI) or sham surgery (sham SCI).

Studied genes were BMP2, Alk3 (encoding BMP type 1 receptor) and ID1 (encoding inhibitor Of DNA Binding 1, HLH Protein). For each gene, presented results are the means of qPCR triplicate results, reported to housekeeping genes 18S and GADPH. p values were 0.08 and 0.76 respectively for BMP2/18S and BMP2/GADPH; 0.05 and 0.9 respectively for AlK3/18S and AlK3/GADPH; 0.8 and 0.15 respectively for ID1/18S and ID1/GADPH.