Remote Limb Preconditioning Generates a Neuroprotective Effect by Modulating the Extrinsic Apoptotic Pathway and TRAIL-Receptors Expression

Abstract

As remote limb preconditioning (RPC) ameliorates brain damage after ischemic cerebral stroke (ICS), the purpose of the present study was to explore the molecular mechanisms in the course of RPC. Results of TUNEL staining and cleaved caspase-3 expression showed that ischemia-induced neuronal apoptosis was inhibited by RPC. The expression changes in cleaved caspase-8, cFLIP, Bid itself, and its truncated form represented that RPC suppressed the activation of extrinsic apoptotic pathway during ICS. Then, the level of cytoplasmic cytochrome c was also decreased by RPC. In addition, RPC might partially suppress TNF-related apoptosis-inducing ligand (TRAIL)-induced extrinsic apoptosis through downregulation of TRAIL death receptors and upregulation of TRAIL decoy receptors. As a counterproof, immunoneutralization of TRAIL in dMCAO rats resulted in significant restraint of tissue damage and in a marked functional recovery. Our data complemented the knowledge of RPC neuroprotective mechanism and provided new evidence for its clinical application.

Keywords: Ischemic cerebral stoke; Neuronal apoptosis; Remote limb preconditioning; TRAIL-receptors.

Fig. 1

Schematic diagram of animal modeling and infarct area measurement. a Schematic diagram of animal modeling in ICS groups. b Schematic diagram of animal modeling in RPC groups. (ICS ischemic cerebral stroke, RPC remote limb preconditioning, CCAsO bilateral common carotid arteries occlusion, dMCAO distal middle cerebral artery occlusion, FAO femoral artery occlusion, FAR femoral artery reperfusion.) c TTC staining of brain coronal sections of ICS 48 h and RPC 48 h groups. d Histogram represented infarct volume of ICS 48 h and RPC 48 h groups. (n = 6; ^&& P < 0.01, ICS 48 h group vs RPC 48 h group.) e Schematic diagram represented TTC-stained sections of ICS 48 h and RPC 48 h groups. (The orange area represented infarct area of RPC 48 h group; the light magenta area was defined as salvage area, which represented the difference of infarct area between ICS 48 h and RPC 48 h group; the apple green area represented the infarct area both observed in ICS 48 h and RPC 48 h group, which was defined as core area) (Color figure online)

Fig. 2

Neuronal apoptosis evaluated via TUNEL staining. a TUNEL staining (red) combined with NeuN immunofluorescent staining (green) in core area. White arrows represented TUNEL and NeuN double-positive (TUNEL⁺/NeuN⁺) cells; scale bar 50 μm. b Schematic diagram of brain coronal section, rectangle represented microscope examining fields is core area. c Histogram represented percentage of TUNEL-positive neurons. (n = 6; *P < 0.05, **P < 0.01, compared with ICS sham group; ^# P < 0.05, ^## P < 0.01, compared with RPC sham group; ^& P < 0.05, ICS group vs RPC group at 1, 2, and 3 days.) d TUNEL staining (red) combined with NeuN immunofluorescent staining (green) in salvage area. White arrows represented TUNEL and NeuN double-positive (TUNEL⁺/NeuN⁺) cells. e Schematic diagram of brain coronal section, rectangle represented microscope examining fields is salvage area. f Histogram represented percentage of TUNEL-positive neurons. (n = 6; **P < 0.01, compared with ICS sham group; ^# P < 0.05, ^## P < 0.01, compared with RPC sham group; ^&& P < 0.01, ICS group vs RPC group at 1, 2, 3, and 5 days) (Color figure online)

Fig. 3

Expression profiles of cleaved caspase-3 in salvage area. a Western blot analysis of cleaved caspase-3 and GAPDH in salvage area. b Histogram represented relative density of cleaved caspase-3 to GAPDH. (n = 6; **P < 0.01, compared with ICS sham group; ^## P < 0.01, compared with RPC sham group; ^&& P < 0.01, ICS group vs RPC group at 1, 2, and 3 days; ^& P < 0.05, ICS 5 days group vs RPC 5 days group.) c Immunofluorescent staining of cleaved caspase-3 (red), NeuN (green) and DAPI (blue); white arrows indicated cleaved caspase-3 and NeuN double-positive (cleaved caspase-3⁺/NeuN⁺) cells; scale bar 50 μm. d Histogram represented percentage of cleaved caspase-3 positive neurons (n = 6; ^&& P < 0.01, ICS group vs RPC group at 1, 2, and 3 days; ^& P < 0.05, ICS 5 days group vs RPC 5 days group.) e Schematic diagram of brain coronal section, rectangle represented microscope examining fields (Color figure online)

Fig. 4

Extrinsic apoptotic pathway in salvage area. a Western blot analysis of cleaved caspase-8 and GAPDH, cFLIP and GAPDH in salvage area. b Western blot of Bid, tBid, and GAPDH in salvage area. Western blot of cytosolic and mitochondrial cytochrome c in salvage area, internal references in cytosolic and mitochondrial protein were GAPDH and COX IV, respectively. c Histogram represented relative density of cleaved caspase-8 to GAPDH. (**P < 0.01, compared with ICS sham group; ^# P < 0.05, ^## P < 0.01, compared with RPC sham group; ^& P < 0.05, ICS group vs RPC group at 1, 3, and 5 days; ^&& P < 0.01, ICS 2 days group vs RPC 2 days group.) d Histogram represented relative density of cFLIP_L/S to GAPDH. (*P < 0.05, **P < 0.01, compared with ICS sham group; ^## P < 0.01, compared with RPC sham group; ^& P < 0.05, ICS group vs RPC group at 1, 2, 3, and 5 days.) e Histogram represented relative density of Bid to GAPDH. (**P < 0.01, compared with ICS sham group; ^# P < 0.05, ^## P < 0.01, compared with RPC sham group; ^& P < 0.05, ICS group vs RPC group at 1 and 3 days; ^&& P < 0.01, ICS 2 days group vs RPC 2 days group.) f Histogram represented relative density of tBid to GAPDH. (**P < 0.01, compared with ICS sham group; ^# P < 0.05, ^## P < 0.01, compared with RPC sham group; ^& P < 0.05, ICS group vs RPC group at 1 and 3 days; ^&& P < 0.01, ICS 2 days group vs RPC 2 days group.) g Histogram represented relative density of cytochrome c to GAPDH. (**P < 0.01, compared with ICS sham group; ^# P < 0.05, ^## P < 0.01, compared with RPC sham group; ^& P < 0.05, ICS group vs RPC group at 1 and 3 days; ^&& P < 0.01, ICS 2 days group vs RPC 2 days group.) h Schematic diagram of brain coronal section, rectangle represented microscope examining fields

Fig. 5

TRAIL-receptors expression in core area and salvage area. a, c Schematic diagram of brain coronal section, rectangle represented microscope examining fields is core area. b Western blot of DR4, DR5, DcR1,and DcR2 to GAPDH in ICS group. d Western blot of DR4, DR5, DcR1, and DcR2 to GAPDH in RPC group. e, g Schematic diagram of brain coronal section, rectangle represented microscope examining fields is salvage area. f Western blot of DR4, DR5, DcR1, and DcR2 to GAPDH in ICS group. h Western blot of DR4, DR5, DcR1, and DcR2 to GAPDH in RPC group. i Line graph represented relative density of DR4 to GAPDH. (^☆ P < 0.05, ^☆☆ P < 0.01, compared with ICS sham group in core area; **P < 0.01, compared with RPC sham group in core area; ^# P < 0.05, ^## P < 0.01, compared with ICS sham group in salvage area; ^&& P < 0.01, compared with RPC in salvage area; ^◇ P < 0.05, ICS 1 day group vs RPC 1 day group in core area; ^※※ P < 0.01, ICS group vs RPC group at 1 and 2 days in salvage area; ^※ P < 0.05, ICS 3 days group vs RPC 3 days group in salvage area.) j Line graph represented relative density of DR5 to GAPDH. (^☆ P < 0.05, ^☆☆ P < 0.01, compared with ICS sham group in core area; **P < 0.01, compared with RPC sham group in core area; ^# P < 0.05, ^## P < 0.01, compared with ICS sham group in salvage area; ^&& P < 0.01, compared with RPC in salvage area; ^◇ P < 0.05, ICS group vs RPC group at 1 and 2 days in core area; ^※※ P < 0.01, ICS 1 day group vs RPC 1 day group in salvage area; ^※ P < 0.05, ICS 2 days group vs RPC 2 days group in salvage area.) k Line graph represented relative density of DcR1 to GAPDH. (^☆ P < 0.05, compared with ICS sham group in core area; *P < 0.05, **P < 0.01, compared with RPC sham group in core area; ^# P < 0.05, ^## P < 0.01, compared with ICS sham group in salvage area; ^&& P < 0.01, compared with RPC in salvage area; ^◇◇ P < 0.01, ICS group vs RPC group at 1 and 3 days in core area; ^◇ P < 0.05, ICS 2 days group vs RPC 2 days group in core area; ^※ P < 0.05, ICS 1 day group vs RPC 1 day group in salvage area; ^※※ P < 0.01, ICS group vs RPC group at 2 and 3 days in salvage area.) l Line graph represented relative density of DcR2 to GAPDH. (^☆ P < 0.05, ^☆☆ P < 0.01 compared with ICS sham group in core area; *P < 0.05, **P < 0.01, compared with RPC sham group in core area; ^# P < 0.05, ^## P < 0.01, compared with ICS sham group in salvage area; ^& P < 0.05, ^&& P < 0.01, compared with RPC in salvage area; ^◇ P < 0.05, ICS group vs RPC group at 1 and 2 days in core area; ^※ P < 0.05, ICS group vs RPC group at 1 and 3 days in salvage area; ^※※ P < 0.01, ICS 2 days group vs RPC 2 days group in salvage area.)

Fig. 6

Double immunofluorescence staining of TRAIL-receptor and NeuN in core cortex. a Immunofluorescent staining of DR4 (red), NeuN (green), and DAPI (blue); white arrows indicated DR4 and NeuN double-positive (DR4⁺/DAPI⁺) cells; scale bar 50 μm. b Immunofluorescent staining of DR5 (red), NeuN (green), and DAPI (blue); white arrows indicated DR5 and NeuN double-positive (DR5⁺/DAPI⁺) cells. c Immunofluorescent staining of DcR1 (red), NeuN (green), and DAPI (blue); white arrows indicated DcR1 and NeuN double-positive (DcR1⁺/DAPI⁺) cells. d Immunofluorescent staining of DcR2 (red), NeuN (green) and DAPI (blue); white arrows indicated DcR2 and NeuN double-positive (DcR2⁺/DAPI⁺) cells. e Histogram represented percentage of DR4-positive neurons (n = 6; ^& P < 0.05, ICS 1 day group vs RPC 1 day group.) f Histogram represented percentage of DR5 positive neurons (n = 6; ^& P < 0.05, ICS group vs RPC group at 1 and 2 days.) g Histogram represented percentage of DcR1 positive neurons (n = 6; ^& P < 0.05, ICS group vs RPC group at 1 and 2 days; ^&& P < 0.01, ICS 3 days group vs RPC 3 days group.) h Histogram represented percentage of DcR2 positive neurons (n = 6; ^& P < 0.05, ICS group vs RPC group at 1, 2, and 3 days.) i Schematic diagram of brain coronal section, rectangle represented microscope examining fields (Color figure online)

Fig. 7

Double immunofluorescence staining of TRAIL-receptor and NeuN in salvage area. a Immunofluorescent staining of DR4 (red), NeuN (green), and DAPI (blue); white arrows indicated DR4 and NeuN double-positive (DR4⁺/DAPI⁺) cells; scale bar 50 μm. b Immunofluorescent staining of DR5 (red), NeuN (green), and DAPI (blue); white arrows indicated DR5 and NeuN double-positive (DR5⁺/DAPI⁺) cells. c Immunofluorescent staining of DcR1 (red), NeuN (green), and DAPI (blue); white arrows indicated DcR1 and NeuN double-positive (DcR1⁺/DAPI⁺) cells. d Immunofluorescent staining of DcR2 (red), NeuN (green), and DAPI (blue); white arrows indicated DcR2 and NeuN double-positive (DcR2⁺/DAPI⁺) cells. e Histogram represented percentage of DR4 positive neurons (n = 6; ^&& P < 0.01, ICS group vs RPC group at 1, 2, and 3 days.) f Histogram represented percentage of DR5-positive neurons (n = 6; ^&& P < 0.01, ICS 1 day group vs RPC 1 day group; ^& P < 0.05, ICS 2 days group vs RPC 2 days group.) g Histogram represented percentage of DcR1 positive neurons (n = 6; ^& P < 0.05, ICS group vs RPC group at 1 and 3 days; ^&& P < 0.01, ICS 2 days group vs RPC 2 days group.) h Histogram represented percentage of DcR2-positive neurons (n = 6; ^& P < 0.05, ICS group vs RPC group at 1 and 3 days; ^&& P < 0.01, ICS 2 days group vs RPC 2 days group.) i Schematic diagram of brain coronal section, rectangle represented microscope examining fields (Color figure online)

Fig. 8

Effect of anti-TRAIL administration on ischemic injury. a Infarct volume in rats subjected to ICS, ICS+ vehicle, ICS+ anti-TRAIL. (n = 6; ^& P < 0.05 vs ICS group and ICS+ vehicle group.) b Vibrissa test. Administration of anti-TRAIL antibody in ischemic rats ameliorated the deficit. (n = 6; *P < 0.05 vs ICS 24 h group and ICS+ vehicle 24 h group; **P < 0.01 vs ICS 48 h group and ICS+ vehicle 48 h group.) c Home cage test. Administration of anti-TRAIL antibody in ischemic rats decreased the biased usage of the ipsilateral forepaw. (n = 6; *P < 0.05 vs ICS 48 h group and ICS+ vehicle 48 h group)