The current study had demonstrated for the first time that total rupture of Achilles tendon evokes microglial activation and inflammatory response in the L5 spinal cord. Although it is widely described that total rupture of Achilles tendon promotes a painful recovery in injured patients, there is little data in the literature describing the effect of tendon injury on the central nervous system21,30,31. As previously described, rupture of the Achilles tendon triggers an in loco activation of inflammatory response which is characterized by hypercellularity, intense enervation and local angiogenesis[31, 32, 33].
The L5 spinal cord segment is a region of the CNS responsible for receiving input from the Achilles tendon-muscle complex34–36. The remarkable time-dependent astrogliosis and microglial activation observed in this region suggest that Achilles tendon rupture elicits activation of a neuroinflammatory response in the spinal cord at L5 segment. In addition, it was demonstrated in the behavioral test significant decrease in PWT values for ipsilateral paw which was sustained until o 14th dpt. The hallmarkers of neuroinflammation are the activation and infiltration of leukocytes, activation of glial cells, and increased production of inflammatory mediators37,38. The interactions between inflammation and pain are bidirectional, nociceptive sensory neurons not only respond to immune signals, but also directly modulate inflammation38.
Numerous non-neuronal cell types influence pain sensation, including immune, epithelial, mesenchymal and glial cells37. It is well documented that microglial activation represents an important response to neuronal injuries39,40. In accordance with our findings, previous studies already have described activation of microglial cells in damaged spinal cord of humans and animal models of spinal nerve injury25,41.
Several studies about peripheral nerve injury and spinal cord injury hypothesized that astrocytic reactivity occurs secondary to the microglial reactivity. Our results show evidence that astrocytes are involved in the maintenance, but not in the development of pain, being this function attributed to microglia. In a comparative study between two injury models, Romero-Sandoval, et al (paw incision and L5 nerve injury), showed that glial expression pattern is differentiated in each condition and divergent in the temporal course, suggesting that the onset and intensity of IBA-1 and GFAP expression are related to the cause of the primary lesion. Our data along with these findings show the dynamic and plastic nature of glial cells under pathological conditions, and that glial reactivity may demonstrate distinct temporal patterns of expression, depending on the lesion42.
GFAP overexpression and inflammatory response are events correlated to the impairment in spinal cord normal physiology as well as to the onset of hyperalgesia 22,43,44. The immunostaining sites on the spinal cord are relevant since the posterior region (dorsal horns) of the spinal cord are accountable for conduction of sensory stimuli. Beyond that, increased glial activation on the ipsilateral side of the dorsal horn, as well as decreased mechanical sensitivity in the post-rupture time course supports that longer hyperalgesic events associated with tendon rupture could be related with inflammatory activation in the spinal cord. The response of astrocytes may be triggered by progressive nerve degeneration and can be responsible for initiation of acute pain while microglial activation could mediate inflammatory response. However, posterior experiments need to be performed to ratify this hypothesis.
Inflammatory response in the L5 spinal cord induced by Achilles tendon rupture was confirmed by in loco expression of COX-2 and NOS-2 on the 7th and 14th day post tendon injury. Immunostaining results were also supported by biochemical findings that have demonstrated significant elevation of nitrite levels in the lumbar spinal cord of animals submitted to Achilles tendon rupture. It was widely described in the literature that COX-2 and NOS-2 expressions are intimately related with overproduction of prostaglandin E2 (PGE2) and nitric oxide (NO) which are important inflammatory mediators45–48. PGE2 and NO production are also evidenced in different kinds of spinal cord and nerve injuries and previous studies point to a close correlation between these mediators and injury in motor performance49–51. As presented in Fig. 9, our hypothesis is that Achilles tendon triggers glial activation and production of inflammatory mediators in spinothalamic tract of L5 spinal cord which favors the generation of acute and neuropathic pain. However, it is important to highlight that we do not discard that additional studies on Neuro-glial interactions are essentials to understand the exact mechanism involved in acute and chronic pain evoked by Achilles tendon rupture since neurons are also able to express NOS-2 and COX-2.
Taken together our findings demonstrate that total rupture of Achilles tendon directly affects the CNS on the L5 spinal cord level.