The chronic protective effects of limb remote preconditioning and the underlying mechanisms involved in inflammatory factors in rat stroke

Abstract

We recently demonstrated that limb remote preconditioning (LRP) protects against focal ischemia measured 2 days post-stroke. Here, we studied whether LRP provides long-term protection and improves neurological function. We also investigated whether LRP transmits its protective signaling via the afferent nerve pathways from the preconditioned limb to the ischemic brain and whether inflammatory factors are involved in LRP, including the novel galectin-9/Tim-3 inflammatory cell signaling pathway, which induces cell death in lymphocytes. LRP in the left hind femoral artery was performed immediately before stroke. LRP reduced brain injury size both at 2 days and 60 days post-stroke and improved behavioral outcomes for up to 2 months. The sensory nerve inhibitors capsaicin and hexamethonium, a ganglion blocker, abolished the protective effects of LRP. In addition, LRP inhibited edema formation and blood-brain barrier (BBB) permeability measured 2 days post-stroke. Western blot and immunostaining analysis showed that LRP inhibited protein expression of both galectin-9 and T-cell immunoglobulin domain and mucin domain 3 (Tim-3), which were increased after stroke. In addition, LRP decreased iNOS and nitrotyrosine protein expression after stroke. In conclusion, LRP executes long-term protective effects against stroke and may block brain injury by inhibiting activities of the galectin-9/Tim-3 pathway, iNOS, and nitrotyrosine.

Figure 1. LRP reduced brain injury after focal ischemia.

A. Diagram of the LRP protocol. In the LRP group, 3 cycles of 15 min occlusion/reperfusion of the left femoral artery was induced before stroke onset. In the control group, 90 min of isoflurane was applied before ischemia, as a vehicle control for LRP. B. Top: Representative brain sections of TTC staining from rats receiving focal ischemia with and without LRP. Bottom: Bar graph showing the quantitation of infarct sizes in the ischemic cortex measured at each level and normalized to the non-ischemic contralateral cortex, and expressed as percentage. C. Top: Representative staining of cresyl/violet from rat brains 60 d post-stroke. The lost and damaged tissues are traced with dashed lines. Bottom: Bar graph showing the average value from 4 levels of brain sections. Control, control ischemia. LRP, limb remote preconditioning. N = 6–7/group. **, ***, P<0.01, 0.001, respectively, vs. control.

Figure 2. LRP attenuated behavioral deficits for up to 2 months post-ischemia.

Three standard tests were performed. A. Vibrissae-elicited forelimb placement test. All sham rats showed normal forelimb placing. Control ischemic rats exhibited unsuccessful placing of the contralateral forelimb (right) after stroke. The reflex was tested 10 times on each side per trial, and 2 trials occurred per test session. The percentage of vibrissa stimulations in which a paw placement occurred was calculated. LRP attenuated the overall deficit from 2 to 60 d after stroke. B. Postural reflex test. Scores were increased in control rats at 1, 2, 7, 10, and 21 d after stroke; LRP reduced scores at 10 and 14 d after stroke compared with control stroke. C. Home cage forelimb use test. The number of times the animal used its forelimbs to brace itself against the wall of the cage was counted, with separate counting for the ipsilateral, contralateral, or both forelimbs until 20 contacts were reached. The percentage of times out of 20 that the ipsilateral forelimb (left) was used was computed. The ratio of left-limb-use was increased at 1, 2 and 7 d compared to control ischemia; LRP blocked this increase at 2 d. *, ** vs. control ischemia, and #, ##, vs. sham, P<0.05, 0.01 at the corresponding time points, respectively. N = 6–8/group.

Figure 3. LRP attenuated edema induced by stroke.

A. Diagram of the dissected regions of the ischemic penumbra and core for BBB leakage measurement. The penumbra (I) is defined as the ischemic region spared by LRP, while the core (II) is the ischemic part that developed into the infarction. The same regions were also dissected for Western blotting. B. LRP inhibited BBB leakage. Evans blue was injected 2 h before the rat was euthanized. The ischemic penumbra and core, as well as the corresponding non-ischemic hemisphere were dissected for Evans blue detection. LRP reduced BBB leakage at 48 h after stroke in the penumbra but not in the core (n = 6/group). * vs. control ischemia, P<0.05. C. LRP mitigated edema after stroke. The ischemic and non-ischemic hemispheres from each rat brain were separated, weighed for wet weight, baked at 90±2°C for 1 wk, and weighed again for dry weight. Water contained in the brain tissues was calculated and is presented in the bar graph (n = 6–7/group). *** vs. contralateral hemisphere, P<0.001; ### vs. sham and control ischemia, P<0.001.

Figure 4. Blocking nerve pathways enlarged infarct size in rats receiving LRP. A. Systemic injection of capsaicin enlarged infarction in rats receiving LRP.

Capsaicin was subcutaneously injected into rats for 4 consecutive days. LRP and focal ischemia were conducted 2 wks after capsaicin injection, and infarct sizes were measured 2 d after stroke. The bar graphs represent the mean values of infarct size in 4 groups: 1) ischemia, control ischemia; 2) LRP, animals receiving LRP plus ischemia; 3) capsaicin+LRP, animals receiving capsaicin injection, LRP and ischemia; 4) capsaicin+ischemia, animals receiving capsaicin and control ischemia. B. Local application of capsaicin onto the thigh nerve in the hind limb abolished the protective effects of LRP. The nerve was soaked with a capsaicin solution for 30 min. Four days later LRP and focal ischemia were conducted. The bar graphs show average infarct sizes. C. The ganglion blocker hexamethonium blocked the protective effects of LRP. Hexamethonium was intravenously injected into rats 30 min before LRP induction. Infarct sizes were measured at 2 days after stroke. The bar graphs show the average values of infarct size of 4 groups. 1) Ischemia, control ischemia without LRP; 2) LRP, animals receiving ischemia and LRP; 3) Hex+LRP, animals receiving hexamethonium, LRP and ischemia; 4) Hex+ischemia, animals receiving hexamethonium and ischemia without LRP. N = 7/group. *, *** vs. ischemia, P<0.05, 0.001, respectively. #, ##, vs. LRP.

Figure 5. LRP inhibited galectin-9 expression 24 h after stroke.

A and B. Representative protein bands of galectin-9 from Western blots for control ischemia and LRP with ischemia, respectively. C and D. The bar graphs show protein band quantitation results corresponding to A and B, respectively. Brain tissue from ischemic penumbras were dissected for Western blotting, as indicated in Fig. 3A. *, vs. sham, P<0.05, ***, vs. sham, P<0.001. n = 6/group. E. Results from confocal microscopy indicate that galectin-9 was increased in the ischemic penumbra 24 h after stroke, and such expression was inhibited by LRP. Scale bar, 50 µM.

Figure 6. Tim-3 expression was increased after stroke and inhibited by LRP.

A and B. Western blots showing representative protein bands of Tim-3 and β-actin in the ischemic penumbra for rats receiving control ischemia alone and ischemia plus LRP, respectively. C. Bar graphs indicating that Tim-3 was slightly increased as early as 1 h and peaked at 24 h after stroke. D. This was inhibited by LRP. ***, vs. sham, P<0.001. n = 6/group. E. The results were further confirmed using immunofluorescent confocal microscopy in control ischemia and LRP 24 h after stroke. An ischemic brain was collected from a surviving rat 24 h after stroke, fixed for 24 h with 4% PFA, stained, and examined with confocal microscopy. F. Double staining of MAP-2 and Tim-3 suggests that Tim-3 was expressed in neurons. Scale bar, 50 µM.

Figure 7. LRP blocked iNOS expression and nitrotyrosine after stroke.

A and C. Representative protein bands of iNOS and quantitative results in the penumbra are shown for control ischemia. B and D. Representative protein bands of iNOS in the penumbra and quantitative results for the animals receiving control ischemia and LRP. iNOS was increased from 1 to 24 h after stroke in rats receiving control ischemia. LRP inhibited iNOS expression. *, **, *** vs. sham, P<0.05, 0.01. 0.001, respectively. n = 6/group. E. LRP blocked nitrotyrosine expression. Nitrotyrosine is a product of iNOS activities. Immunostaining suggested that nitrotyrosine expression was increased 24 h after stroke in control ischemic rats, and this was attenuated by LRP. Scale bar, 50 µM.