ABSTRACT
Spinal cord injury (SCI) is a devastating condition that has physical and psychological consequences for patients. SCI is accompanied by scar formation and systemic inflammatory response leading to an intense degree of functional loss. The catechin, epigallocatechin gallate (EGCG), an active compound found in green tea, holds neuroprotective features and is known for its anti-inflammatory potential. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that exists in two functionally distinct complexes termed mTOR complex 1 and 2 (mTORC1; mTORC2). Inhibition of mTORC1 by rapamycin causes neuroprotection, leading to partial recovery from SCI. In this study the effects of EGCG, PP242 (an inhibitor of both complexes of mTOR), and a combination of EGCG and PP242 in SCI have been examined. It has been found that both EGCG and PP242 significantly improved sensory/motor functions following SCI. However, EGCG appeared to be more effective (BBB motor test, from 2 to 8 weeks after SCI, p = 0.019, p = 0.007, p = 0.006, p = 0.006, p = 0.05, p = 0.006, and p = 0.003, respectively). The only exception was the Von Frey test, where EGCG was ineffective, while mTOR inhibition by PP242, as well as PP242 in combination with EGCG, significantly reduced withdrawal latency starting from week three (combinatorial therapy (EGCG + PP242) vs. control at 3, 5, and 7 weeks, p = 0.011, p = 0.007, and p = 0.05, respectively). It has been found that EGCG was as effective as PP242 in suppressing mTOR signaling pathways, as evidenced by a reduction in phosphorylated S6 expression (PP242 (t-test, p < 0.0001) or EGCG (t-test, p = 0.0002)). These results demonstrate that EGCG and PP242 effectively suppress mTOR pathways, resulting in recovery from SCI in rats, and that EGCG acts via suppressing mTOR pathways.
ABSTRACT
Traumatic spinal cord injury (SCI) is untreatable and remains the leading cause of disability. Neuroprotection and recovery after SCI can be partially achieved by rapamycin (RAPA) treatment, an inhibitor of mTORC1, complex 1 of the mammalian target of rapamycin (mTOR) pathway. However, mechanisms regulated by the mTOR pathway are not only controlled by mTORC1, but also by a second mTOR complex (mTORC2). Second-generation inhibitor, pp242, inhibits both mTORC1 and mtORC2, which led us to explore its therapeutic potential after SCI and compare it to RAPA treatment. In a rat balloon-compression model of SCI, the effect of daily RAPA (5 mg/kg; IP) and pp242 (5 mg/kg; IP) treatment on inflammatory responses and autophagy was observed. We demonstrated inhibition of the mTOR pathway after SCI through analysis of p-S6, p-Akt, and p-4E-BP1 levels. Several proinflammatory cytokines were elevated in pp242-treated rats, while RAPA treatment led to a decrease in proinflammatory cytokines. Both RAPA and pp242 treatments caused an upregulation of LC3B and led to improved functional and structural recovery in acute SCI compared to the controls, however, a greater axonal sprouting was seen following RAPA treatment. These results suggest that dual mTOR inhibition by pp242 after SCI induces distinct mechanisms and leads to recovery somewhat inferior to that following RAPA treatment.
ABSTRACT
Despite the variety of experimental models of spinal cord injury (SCI) currently used, the model of the ventral compression cord injury, which is commonly seen in humans, is very limited. Ventral balloon compression injury reflects the common anatomical mechanism of a human lesion and has the advantage of grading the injury severity by controlling the inflated volume of the balloon. In this study, ventral compression of the SCI was performed by the anterior epidural placement of the balloon of a 2F Fogarty's catheter, via laminectomy, at the level of T10. The balloon was rapidly inflated with 10 or 15 µL of saline and rested in situ for 5 min. The severity of the lesion was assessed by behavioral and immunohistochemical tests. Compression with the volume of 15 µL resulted in severe motor and sensory deficits represented by the complete inability to move across a horizontal ladder, a final Basso, Beattie and Bresnahan (BBB) score of 7.4 and a decreased withdrawal time in the plantar test (11.6 s). Histology and immunohistochemistry revealed a significant loss of white and gray matter with a loss of motoneuron, and an increased size of astrogliosis. An inflation volume of 10 µL resulted in a mild transient deficit. There are no other balloon compression models of ventral spinal cord injury. This study provided and validated a novel, easily replicable model of the ventral compression SCI, introduced by an inflated balloon of Fogarty´s catheter. For a severe incomplete deficit, an inflated volume should be maintained at 15 µL.
