Neurogenic heterotopic ossifications (NHOs) are pathological bone formations that develop in periarticular muscles after spinal-cord injury (SCI), traumatic brain injury (TBI), or stroke. As its pathophysiology is still obscure, there is a need to understand the factors that contribute to this pathology. We have established a mouse model for NHO by combining a complete spinal-cord transection together with an intramuscular injection of cardiotoxin into the hamstring muscles to cause muscle injury. Using this model, we have been able to identify numerous pathways that trigger NHO formation.
Retrospective studies have demonstrated that the prevalence of NHO is higher in patients with concomitant infections, particularly urinary tract infections and pneumonia. To demonstrate a direct link between infections and enhanced NHO formation, we employed purified pathogen-associated molecular patterns (PAMPs). C57BL/6 mice were subjected to a complete spinal-cord transection between T11-T13 vertebrae, followed by an intramuscular injection of cardiotoxin in the hamstring muscles. Mice additionally received intraperitoneal injections of purified PAMPs in different dosages, while control mice were injected with the vehicle. At 7- and 21-days post-surgery, NHO volumes were quantified using micro-computed tomography.
Bacterial PAMPs such as lipoteichoic acid (LTA) – TLR2 (Toll-like receptor-2) ligand, Pam2CSK4 - TLR2/6 ligand, GlcC14C18 – Mincle ligand, viral PAMPS such as Gardiquimod (TLR7/8 ligand), Poly(I:C) (TLR3, RIG1, MDA5, and PKR ligand), and a fungal PAMP Zymosan – Dectin1/2, TLR2/6 ligand significantly increased NHO volumes when compared to control mice. In support of these findings, receptors for the aforementioned PAMPs were expressed by sorted muscle macrophages and/or fibro-adipogenic progenitors.
These data illustrate that PAMPs exacerbate NHO development in mice. Our data are consistent with previous retrospective studies showing a higher prevalence of NHO in SCI patients with infections. This work highlights the necessity to increase microbe vigilance to reduce the incidence of NHO in SCI or TBI patients.