Figure 2
Adeno-associated viral IL-10 blocks development of chronic sciatic inflammatory neuropathy (SIN) induced mechanical allodynia. After baseline (BL) assessment on the von Frey test, all rats received intrathecal AAV2-GFP (Control, encoding green fluorescent protein) or AAV2-r-IL-10. Behavior was reassessed Day 3 after intrathecal AAV, confirming that neither AAV2-GFP (Control) nor AAV2-r-IL-10 affected behavior prior to peri-sciatic injections (F 7,88 = 0.686, p > 0.68). After this Day 3 assessment, unilateral peri-sciatic injections of 0 (vehicle control; Panels A, B), 4 ug zymosan (to induce ipsilateral allodynia; Panels C, D), or 160 ug zymosan (to induce bilateral allodynia; Panels E, F) were delivered, with repeated re-administration across days to induce a chronic neuropathic state. Repeated measures ANOVA revealed reliable main effects of peri-sciatic zymosan dose (F 1,40 = 12.093, p < 0.002), IL-10 (F 1,40 = 69.829, p < 0.0001), and laterality (F 1,40 = 22.315, p < 0.0001), and interactions between zymosan dose and IL-10 (F 1,40 = 6.161, p < 0.02) and between IL-10 and laterality (F 1,40 = 15.412, p < 0.001). The construct pTR2-CB-r-IL-10 employed in an AAV vector for behavioral testing induced the production and release of rat IL-10 from transfected IB3 cells in culture. Increases in rat IL-10 protein were detected in supernatants of transfected cells versus untransfected vehicle control cultures (Panel A Inset). Neither AAV2-GFP nor AAV2-r-IL-10 affected the behavioral responses of rats receiving chronic peri-sciatic vehicle, as illustrated by data obtained from the hindpaws ipsilateral (Panel A) or contralateral (Panel B) to the peri-sciatic injections. Allodynia was induced in the ipsilateral hindpaw of intrathecal AAV2-GFP rats receiving 4 ug peri-sciatic zymosan (Panel C). This allodynia was largely blocked by AAV2-r-IL-10 (p > 0.045 through Day 11 compared to BL) with allodynia reappearing on Day 13 after AAV; that is, 10 days after initiation of chronic zymosan. Again, neither AAV2-GFP nor AAV2-r-IL-10 affected behaviors obtained from the contralateral, non-allodynic hindpaws (Panel D). Allodynia was induced in both the ipsilateral (Panel E) and contralateral (Panel F) hindpaws of intrathecal AAV2-GFP rats receiving 160 ug peri-sciatic zymosan. These ipsilateral and contralateral allodynias were largely blocked by AAV2-r-IL-10 (p > 0.15 through Day 11 compared to BL), until allodynia reappeared on Day 13 after AAV; that is, 10 days after initiation of chronic zymosan.