Toll-like receptors 2 and 4 influence neuronal survival and glial reactions following ventral root crush injury in mice

Peripheral nerve injuries are followed by an increase in the expression of immune molecules, such as the major histocompatibility complex class I (MHC-I) and Toll-like receptors (TLR 2 and 4), which are mainly related to synaptic stability or elimination. The present study aimed to evaluate the putative role of TLR2 and 4 in neuronal survival, glial reaction and synaptic coverage after ventral root crush (VRC). To this end, 8-week-old male C57BL6/JUnib, TLR2 knockout, and TLR4 knockout strains were used. The animals underwent surgery for the crushing of L4, L5, and L6 ventral roots, and C57BL/6JUnib mice were further subdivided into control groups and groups of animals previously stimulated with TLR2 and TLR4 agonists (Pam3CSK4 and LPS, respectively; 1 h before surgery). Seven- and fourteen-days post-injury, spinal cords were collected for motoneuron survival analysis, immunohistochemistry (anti-Iba1, anti-GFAP, anti-synaptophysin), and synaptic coverage quantification. A subset of mice was also processed at 3 dpi for RT-qPCR and Western blotting. The results showed that TLR4 knockout mice exhibited greater susceptibility to motoneuron loss and decreased synaptic coverage after VRC, accompanied by increased NF-κB expression. In contrast, TLR2 knockout mice showed altered microglial responses, with reduced expression of Il10 and a distinct glial activation profile compared to wild-type mice and TLR4-deficient animals. Additionally, TLR2 stimulation with Pam3CSK4 led to a more intense microglial reaction surrounding motoneurons, along with increased Il10 gene expression, suggesting an anti-inflammatory phenotype. In the absence of TLR4, at 7 dpi, a reduction in amoeboid microglia was observed, which, coupled with the increased susceptibility of motoneurons and decreased synaptic coverage, reinforces the importance of immunomodulation in the early stages after injury. Together, these findings highlight distinct but complementary roles of TLR2 and TLR4 in shaping the neuroimmune response following VRC, with implications for understanding how innate immune signaling influences motoneuron survival and repair mechanisms.