Normalisation of glucose metabolism by exendin-4 in the chronic phase after stroke promotes functional recovery in male diabetic mice

Abstract

Background and purpose: Glucagon-like peptide-1 (GLP-1) receptor activation decreases stroke risk in people with Type 2 diabetes (T2D), while animal studies have shown the efficacy of this strategy to counteract stroke-induced acute brain damage. However, whether GLP-1 receptor activation also improves recovery in the chronic phase after stroke is unknown. We investigated whether post-acute, chronic administration of the GLP-1 receptor agonist, exendin-4, improves post-stroke recovery and examined possible underlying mechanisms in T2D and non-T2D mice.

Experimental approach: We induced stroke via transient middle cerebral artery occlusion (tMCAO) in T2D/obese mice (8 months of high-fat diet) and age-matched controls. Exendin-4 was administered for 8 weeks from Day 3 post-tMCAO. We assessed functional recovery by weekly upper-limb grip strength tests. Insulin sensitivity and glycaemia were evaluated at 4 and 8 weeks post-tMCAO. Neuronal survival, stroke-induced neurogenesis, neuroinflammation, atrophy of GABAergic parvalbumin+ interneurons, post-stroke vascular remodelling and fibrotic scar formation were investigated by immunohistochemistry.

Key results: Exendin-4 normalised T2D-induced impairment of forepaw grip strength recovery in correlation with normalised glycaemia and insulin sensitivity. Moreover, exendin-4 counteracted T2D-induced atrophy of parvalbumin+ interneurons and decreased microglia activation. Finally, exendin-4 normalised density and pericyte coverage of micro-vessels and restored fibrotic scar formation in T2D mice. In non-T2D mice, the exendin-4-mediated recovery was minor.

Conclusion and implications: Chronic GLP-1 receptor activation mediates post-stroke functional recovery in T2D mice by normalising glucose metabolism and improving neuroplasticity and vascular remodelling in the recovery phase. The results warrant clinical trial of GLP-1 receptor agonists for rehabilitation after stroke in T2D.

Linked articles: This article is part of a themed issue on GLP1 receptor ligands (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.4/issuetoc.

Keywords: GLP-1R agonist; T2D; fibrotic scar; neurological recovery; stroke; vascular remodelling.

FIGURE 1

The experimental design. Experimental design of (a) diabetic study and (b) non‐diabetic study

FIGURE 2

The effect of T2D and exendin‐4 treatment on neurological recovery, metabolic parameters and ischaemic volume after tMCAO. (a) Grip strength recovery and ANOVA table. Dashed line in (a) indicates mean of pre‐stroke grip strength. (b) Body weight. (c) Fasting glucose. (d) Insulin sensitivity (insulin tolerance test, ITT). (e) Ischaemic volume. (f) Representative images of NeuN staining. The dotted lines on images on (f) panel indicate stroke area. Data in (a), (b) and (e) are presented as mean ± SD. Box plots on (c) and (d) show min–max values. Group sizes for (b)–(d): Non‐T2D n = 9, T2D‐Ve n = 9, T2D‐Ex‐4 n = 7. For (a), one statistical outlier was excluded from the T2D‐Ve group, resulting in the following group sizes: Non‐T2D n = 9, T2D‐Ve n = 8, T2D‐Ex‐4 n = 7. For (e), one statistical outlier was excluded from the non‐T2D group, resulting in the following group sizes: Non‐T2D n = 8, T2D‐Ve n = 9, T2D‐Ex‐4 n = 7. Two‐way repeated measures ANOVA with Geisser–Greenhouse's correction followed by Sidak's test was used to analyse grip strength, body weight, fasting glucose and insulin sensitivity (a–d). Kruskal–Wallis with uncorrected Dunn's test was used to analyse ischaemic volume (e). *^{, #} P < 0.05, significantly different from T2D‐Ve; ^$ P < 0.05, Non‐T2D significantly different from T2D‐Ex‐4

FIGURE 3

Exendin‐4 counteracts the diet‐induced atrophy of PV+ cells. (a) Number of PV+ cells in the contralateral (represented by dotted line) versus ipsilateral striatum. (b) The average soma volume of PV+ cells. Data presented as mean ± SD. Group sizes for all figures: Non‐T2D n = 9, T2D‐Ve n = 9, T2D‐Ex‐4 n = 7. One‐way ANOVA with Tukey's post hoc test was used to analyse the number of PV+ cells. Two‐way ANOVA followed by two‐stage linear step‐up procedure of Benjamini, Krieger and Yekutieli was used to compare the volume of PV+ cells between non‐T2D and T2D‐Ve and T2D‐Ex‐4. * P < 0.05, significantly different as indicated; ^# P < 0.05, contralateral significantly different from ipsilateral, in the same group. (c) Representative images of PV+ interneurons in contralateral and ipsilateral striatum, scale bars = 200 and 25 μm for inserts (c1) and (c2)

FIGURE 4

Exendin‐4 decreases neuroinflammation after stroke. (a) Density of Iba‐1+ cells at 8 weeks after stroke. Representative image of Iba‐1+ cells in contralateral (a1) and ipsilateral striatum (a2). (b) Quantification of Iba‐1+ cells in an infarct‐adjacent region of interest (ROI). (b1) Photomicrograph of Iba1 staining, ROI is outlined by dashed line. (c) Number of CD68+ cells in the ipsilateral striatum. (c1) Representative image of CD68+ cells. Data are presented as mean ± SD. Group sizes for all figures: Non‐T2D n = 9, T2D‐Ve n = 9, T2D‐Ex‐4 n = 7. Two‐way ANOVA followed by two‐stage linear step‐up procedure of Benjamini, Krieger and Yekutieli was used to compare the number of Iba‐1+ cells in the contralateral and ipsilateral striatum (in a); and in contralateral versus ROI (in b) between non‐T2D and T2D‐Ve and T2D‐Ex‐4. Kruskal–Wallis with uncorrected Dunn's test was used to compare the number of CD68+ cells between non‐T2D and T2D‐Ve and T2D‐Ex‐4. * P < 0.05, significantly different as indicated; ^# P < 0.05, contralateral significantly different from ipsilateral, in the same group. Scale bars in (a1), (a2), and (c1) = 50 μm

FIGURE 5

Exendin‐4 restores the density and maturity of vessels in T2D mice after stroke. (a) Representative images of CD31+ (vessels) and CD13+ (pericytes) staining in the ipsilateral striatum. Representative images of the contralateral striatum are included in Supporting Information Figure S2. Scale bar: 100 μm for the left panel (CD31); 50 μm for the two centre and right panels (higher magnification). (b) Density of CD31+ vessels at 8 weeks after stroke. (c) Coverage of vessels by pericytes. (d) Total density of CD13+ pericytes, including both perivascular and parenchymal pericytes. (e) Density of parenchymal CD13+ pericytes. Data presented as mean ± SD. Group sizes for (a)–(d): Non‐T2D n = 9, T2D‐Ve n = 9, T2D‐Ex‐4 n = 7. For (e), one statistical outlier was excluded from the T2D‐Ex‐4 group, resulting in the following group sizes: Non‐T2D n = 9, T2D‐Ve n = 9, T2D‐Ex‐4 n = 6. Two‐way ANOVA followed by two‐stage linear step‐up procedure of Benjamini, Krieger and Yekutieli was used to compare the density of CD31+ vessels, the density of CD13+ pericytes, and coverage of CD31+ vessels by CD13+ pericytes, between non‐T2D and T2D‐Ve and T2D‐Ex‐4, in the contralateral and ipsilateral striatum. One‐way ANOVA with Tukey's post hoc test was used to analyse the density of parenchymal CD13+ pericytes, in the ipsilateral striatum. * P < 0.05, significantly different as indicated; ^# P < 0.05, contralateral significantly different from ipsilateral, in the same group

FIGURE 6

The effect of Exendin‐4 treatment after tMCAO in non‐diabetic mice. (a) Recovery of grip strength and ANOVA table. (b) Ischaemic volume. (c) Average volume of PV+ interneurons. (d) Number of Iba‐1+ cells. (e) Number of CD68+ cells. (f) Density of CD31+ vessels. (g) Coverage of vessels by pericytes. (h) Total density of CD13+ pericytes (including both perivascular and parenchymal pericytes). (i) Serum insulin levels. Data are presented as mean ± SD. Group sizes for (a)–(e): Non‐T2D‐Ve n = 6, non‐T2D‐Ex‐4 n = 8. For (f)–(h), one statistical outlier was excluded from the non‐T2D‐Ex‐4 group, resulting in the following group sizes: Non‐T2D‐Ve n = 6, non‐T2D‐Ex‐4 n = 7. For (i), insulin levels were only measured in n = 6 mice for both groups, resulting in the following group sizes: Non‐T2D‐Ve n = 6, non‐T2D‐Ex‐4 n = 6. Two‐way repeated measures ANOVA with Geisser–Greenhouse's correction followed by Sidak's test was used to compare grip strength between non‐T2D‐Ve and non‐T2D‐Ex‐4. Two‐way ANOVA followed by two‐stage linear step‐up procedure of Benjamini, Krieger and Yekutieli was used to compare the volume of PV+ cells, the number of Iba‐1+ cells, the density of CD31+ vessels, the density of CD13+ pericytes, and coverage of CD31+ vessels by CD13+ pericytes. Unpaired t test was used to analyse ischaemic volume, CD68+ cells and insulin levels. (* P < 0.05, significantly different as indicated; ^# P < 0.05, contralateral significantly different from ipsilateral, in the same group