Exendin-4, a glucagon-like peptide-1 receptor agonist, provides neuroprotection in mice transient focal cerebral ischemia

Abstract

Glucagon-like peptide-1 (GLP-1) is an incretin hormone known to stimulate glucose-dependent insulin secretion. The GLP-1 receptor agonist, exendin-4, has similar properties to GLP-1 and is currently in clinical use for type 2 diabetes mellitus. As GLP-1 and exendin-4 confer cardioprotection after myocardial infarction, this study was designed to assess the neuroprotective effects of exendin-4 against cerebral ischemia-reperfusion injury. Mice received a transvenous injection of exendin-4, after a 60-minute focal cerebral ischemia. Exendin-4-treated vehicle and sham groups were evaluated for infarct volume, neurologic deficit score, various physiologic parameters, and immunohistochemical analyses at several time points after ischemia. Exendin-4 treatment significantly reduced infarct volume and improved functional deficit. It also significantly suppressed oxidative stress, inflammatory response, and cell death after reperfusion. Furthermore, intracellular cyclic AMP (cAMP) levels were slightly higher in the exendin-4 group than in the vehicle group. No serial changes were noted in insulin and glucose levels in both groups. This study suggested that exendin-4 provides neuroprotection against ischemic injury and that this action is probably mediated through increased intracellular cAMP levels. Exendin-4 is potentially useful in the treatment of acute ischemic stroke.

Figure 1

Neuroprotective effects of exendin-4 (Ex-4) against ischemia–reperfusion injury. (A) The trail of various doses (a) and schedules (b) of exendin-4 for determination of exendin-4 treatment protocol to be used in this study. (B) Typical infarct area in the vehicle (a) and exendin-4 (b) groups at 24 hours after reperfusion. Bar=2 mm. (C) Infarct volume in the vehicle and exendin-4 groups. (D) Neurologic deficit score in the vehicle and exendin-4 groups. Data are mean±s.e.m. of five mice (panels A, C, and D) in each group. ^*P<0.05, ^**P<0.001, compared with the vehicle group.

Figure 2

Physiologic parameters. (A) Changes in serum insulin (a) and plasma glucose (b) levels in the vehicle and exendin-4 groups until 24 hours after reperfusion. Base, nonoperation state; post, after middle cerebral artery occlusion (MCAO). (B) Temporal changes in rCBF. Pre, before MCAO; during, during MCAO. Data are mean±s.e.m. of four mice (panel A) and five mice (panel B) in each group. Ex-4, exendin-4; rCBF, regional cerebral blood flow.

Figure 3

Expression of GLP-1R in the brain. (A) Photomicrograph of GLP-1R in the mouse brain (normal, untreated). Arrowheads, positive cells. Bar=50 μm. (B) Double immunofluorescence staining for GLP-1R (green (a, d)), NeuN (red, b) and CD31 (red, e). Arrowheads, merged cells. Bar=20 μm. GLP-1R, glucagon-like peptide-1 receptor; NeuN, neuronal nuclei.

Figure 4

Effects of exendin-4 on oxidative stress. (A) Photomicrographs of 8-OHdG (a, c) and HHE (b, d) in the vehicle (a, b) and exendin-4 (c, d) groups at 72 hours after reperfusion. Bar=50 μm. (e) Number of 8-OHdG- and HHE-positive cells in the ischemic boundary zone. (B) (a) Immunoblot analysis of HHE. Equal protein loading was confirmed by measuring α-tubulin. Veh, vehicle group. (b) Densitometric analysis of HHE-modified protein. Data are mean±s.e.m. of five mice (panel A) and three mice (panel B) in each group. ^*P<0.001 compared with the vehicle group. 8-OHdG, 8-hydroxy deoxyguanosine; Ex-4, exendin-4; HHE, 4-hydroxy 2-hexenal.

Figure 5

Effect of exendin-4 on the inflammatory response. (A) Photomicrographs of Iba-1 in the vehicle (a) and exendin-4 (b) groups at 72 hours after reperfusion. Bar=50 μm. (c) Number of Iba-1-positive cells in the ischemic boundary zone. (B) Double immunofluorescence of iNOS (green, a) and Iba-1 (red, b) in the ischemic boundary zone at 24 hours after reperfusion, and that of iNOS (green, d) and Iba-1 (red, e) in the same zone at 72 hours. Bar=50 μm. (C) (a) Immunoblot analysis of iNOS. Equal protein loading was confirmed by measuring α-tubulin. Veh, vehicle group. (b) Densitometric analysis of iNOS protein. Data are mean±s.e.m. of five mice (panel A) and three mice (panel C) in each group. ^*P<0.001, compared with the vehicle group. iNOS, inducible nitric oxide.

Figure 6

Effect of exendin-4 on cell death. (A) TUNEL staining in the vehicle (a) and exendin-4 (b) groups at 72 hours after reperfusion. Bar=50 μm. (B) Number of TUNEL-positive cells in the ischemic boundary zone. Data are mean±s.e.m. of five mice in each group. ^*P<0.001 compared with the vehicle group. Ex-4, exendin-4; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling.

Figure 7

Induction of cAMP upregulation and CREB activation by exendin-4. (A) cAMP levels in the vehicle and exendin-4 groups. (B) Photomicrographs of pCREB in the vehicle (a) and exendin-4 (b) groups at 24 hours after reperfusion. Bar=50 μm. (c) Number of pCREB-positive cells in the ischemic boundary zone. (C) (a) Immunoblot analysis of pCREB and CREB. Equal protein loading was confirmed by measuring α-tubulin. Veh, vehicle group. (b) Densitometric analysis of pCREB protein. Data are mean±s.e.m. of three mice (Panels A and C) and five mice (panel B) in each group. ^*P<0.05, ^**P<0.001, compared with the vehicle group. cAMP, cyclic AMP; Ex-4, exendin-4; pCREB, phosphorylated cyclic AMP (cAMP) response element-binding protein.