Improving stroke outcomes in hyperglycemic mice by modulating tPA/NMDAR signaling to reduce inflammation and hemorrhages

Abstract

The pharmacological intervention for ischemic stroke hinges on intravenous administration of the recombinant tissue-type plasminogen activator (rtPA, Alteplase/Actilyse) either as a standalone treatment or in conjunction with thrombectomy. However, despite its clinical significance, broader use of rtPA is constrained because of the risk of hemorrhagic transformations (HTs). Furthermore, the presence of diabetes or chronic hyperglycemia is associated with an elevated risk of HT subsequent to thrombolysis. This detrimental impact of tPA on the neurovascular unit in patients with hyperglycemia has been ascribed to its capacity to induce endothelial N-methyl-D-aspartate receptor (NMDAR) signaling, contributing to compromised blood-brain barrier integrity and neuroinflammatory processes. In a mouse model of thromboembolic stroke with chronic hyperglycemia, we assessed the effectiveness of rtPA and N-acetylcysteine (NAC) as thrombolytic agents. We also tested the effect of blocking tPA/NMDAR signaling using a monoclonal antibody, Glunomab. Magnetic resonance imaging, speckle contrast imaging, flow cytometry, and behavioral tasks were used to evaluate stroke outcomes. In hyperglycemic animals, treatment with rtPA resulted in lower recanalization rates and increased HTs. Conversely, NAC treatment reduced lesion sizes while mitigating HTs. After a single administration, either in standalone or combined with rtPA-induced thrombolysis, Glunomab reduced brain lesion volumes, HTs, and neuroinflammation after stroke, translating into improved neurological outcomes. Additionally, we demonstrated the therapeutic efficacy of Glunomab in combination with NAC or as a standalone strategy in chronic hyperglycemic animals. Counteracting tPA-dependent endothelial NMDAR signaling limits ischemic damages induced by both endogenous and exogenous tPA, including HTs and inflammatory processes after ischemic stroke in hyperglycemic animals.

Graphical abstract

Figure 1.

Chronic hyperglycemia induces rtPA resistance and increases HT in a mouse model of thromboembolic stroke. (A) Schematic representation of the experimental protocol. (B) Quantification of ischemic lesion volume, 24 hours after MCAo assessed by T2-weighted imaging (7T MRI) in saline or rtPA treated (10 mg/kg; Actilyse, 10% bolus, 90% perfusion during 40 minutes) on nonhyperglycemic mice (Ctrl) or on hyperglycemic mice (STZ). Individual values, means, and standard error of the mean (SEM) are plotted; 23.38 mm³ for Ctrl group (n = 13); 12.88 mm³ for Ctrl-rtPA group (n = 13); 36 mm³ for STZ group (n = 12); 39.58 mm³ for STZ-rtPA group (n = 12). Ordinary 1-way analysis of variance (ANOVA) (P < .01); Tukey test for multiple comparisons (∗∗P < .01; ∗∗∗∗P < .0001). (C) Representative T2-weighted 7T MRI brain images (left) and representation of the lesion distribution around bregma (right), 24 hours after MCAo in Ctrl, Ctrl-rtPA, STZ, and STZ-rtPA groups. (D) Percentage of angiographic scores 24 hours after MCAo assessed by FLASH TOF 2D imaging (7T MRI) in Ctrl (n = 13), Ctrl-rtPA (n = 12), STZ (n = 12), and STZ-rtPA (n = 12) groups. No recanalization = complete occlusion (orange); partial recanalization = incomplete filling of the distal bed (light green); and complete recanalization = complete filling of the distal bed (dark green). Kruskal-Wallis test (P < .01); Dunns test for multiple comparisons (∗∗P < .01). (E) Proportion of HT per groups, 24 hours after MCAo assessed by T2∗-weighted imaging (deoxyhemoglobin; 7T MRI) in Ctrl (n = 13), Ctrl-rtPA (n = 13), STZ (n = 12), and STZ-rtPA (n = 12) groups. Fisher exact tests between groups (∗P < .05). (F) Quantification of the specific left paw–strength deficit measured by grip-test ratio (strength of left/right paws) of Ctrl, Ctrl-rtPA, STZ, and STZ-rtPA groups (n = 12) before MCAo and on days 1, 3, and 7 after MCAo. Data were assessed in grams. Results are represented in mean ± SEM; 2-way ANOVA: time × group effect = 0.0211; Tukey test for multiple comparison (∗P < .05 between groups at each time; $P < .5 vs baseline for each group: impact of Stroke; #P < .5 vs day 1 for each group: recovery). (G) Quantification of the global strength deficit measured by grip-test of forepaws of Ctrl, Ctrl-rtPA, STZ, and STZ-rtPA groups (n = 12) before and on days 1, 3, and 7 after MCAo. Data were assessed in grams and converted in percentage normalized for each animal with the corresponding baseline value (before MCAo). Results are represented in mean ± SEM; 2-way ANOVA: time factor <0.0001, and group factor <0.001; Tukey test for multiple comparison (∗P < .05 between groups at each time; $P < .5 vs baseline for each group: impact of stroke; #P < .5 vs day 1 for each group: recovery).

Figure 2.

Chronic hyperglycemia induces rtPA resistance and increases HT independently of the therapeutic window. (A) Schematic representation of the experimental protocol. (B) Quantification of ischemic lesion volume, 24 hours after MCAo assessed by T2-weighted imaging (7T MRI) in hyperglycemic mice treated with saline (STZ group) or rtPA (10 mg/kg; Actilyse, 10% bolus, 90% perfusion during 40 minutes), in early 20 minutes after MCAo (STZ-rtPA-early) or in late 4 hours after MCAo (STZ-rtPA-late). Individual values, means, and SEM are plotted; 34.45 mm³ for STZ group (n = 10); 37.01 mm³ for STZ-rtPA-early group (n = 5); and 33.98 mm³ for STZ-rtPA-late group (n = 5). Ordinary 1-way ANOVA (P = .14). (C) Representative T2-weighted 7T MRI brain images (left) and representation of the lesion distribution around bregma (right) 24 hours after MCAo in STZ, STZ-rtPA-early and STZ-rtPA-late groups. (D) Percentage of angiographic scores, 24 hours after MCAo assessed by FLASH_TOF_2D imaging (7T MRI) in STZ (n = 10), STZ-rtPA-early (n = 5), and STZ-rtPA-late (n = 5) groups. No recanalization = complete occlusion (orange); partial recanalization = incomplete filling of the distal bed (light green); and complete recanalization = complete filling of the distal bed (dark green). Kruskal-Wallis test (P = .16). (E) Proportion of HT per group, 24 hours after MCAo assessed by T2∗-weighted imaging (deoxyhemoglobin; 7T MRI) in STZ (n = 10), STZ-rtPA-early (n = 5), and STZ-rtPA-late (n = 5) groups. Fisher exact tests between groups (P > .05).

Figure 3.

N-Acetyl-Cysteine induces recanalization after acute thrombosis in chronically hyperglycemic animals without risk of HT. (A) Schematic representation of the experimental protocol. (B) Quantification of ischemic lesion volume 24 hours after MCAo assessed by T2-weighted imaging (7T MRI) in hyperglycemic mice treated after stroke with saline (STZ) or rtPA (10mg/kg; Actilyse, 10% bolus, 90% perfusion during 40 minutes; STZ-rtPA group), NAC (400 mg/kg, slow bolus during 60 seconds; STZ-NAC group) or a combination of NAC and rtPA (STZ-NAC-rtPA group). 40.20 mm³ for STZ group (n = 14); 40.43 mm³ for STZ-rtPA group (n = 14); 26.92 mm³ for STZ-NAC group (n = 14); 21.59 mm³ for STZ-NAC-rtPA group (n = 14). Ordinary one-way ANOVA (P < .001); Tukey test for multiple comparisons (∗P < .05; ∗∗∗P < .001). (C) Representative T2-weighted 7T MRI brain images (left) and representation of the lesion distribution around bregma (right) 24 hours after MCAo in STZ, STZ-rtPA, STZ-NAC and STZ-NAC-rtPA groups. (D) Percentage of angiographic scores 24 hours after MCAo assessed by FLASH_TOF_2D imaging (7T MRI) in STZ, STZ-rtPA, STZ-NAC and STZ-NAC-rtPA groups (n = 13-14 per group). No recanalization = complete occlusion (orange); partial recanalization = incomplete filling of the distal bed (light green); complete recanalization = complete filling of the distal bed (dark green). Kruskal-Wallis test (P < .01); Dunn’s test for multiple comparisons (∗P < .05, ∗∗P < .01). (E) Proportion of HT per groups 24 hours after MCAo assessed by T2∗-weighted imaging (deoxyhemoglobin; 7T MRI) in STZ, STZ-rtPA, STZ-NAC and STZ-NAC-rtPA groups (n = 14 per group). Fisher exact tests between groups (∗P < .05). (F) Quantification of the specific left paw strength deficit measured by grip-test ratio (strength of left/right paws) on day before and on day 1, day 3, and day 7 after MCAo in STZ, STZ-rtPA, STZ-NAC and STZ-NAC-rtPA groups (n = 14 per group). Data were assessed in grams. Results are represented in mean ± SEM. 2-way ANOVA: Time factor <0.0001 and group factor <0.001; Tukey test for multiple comparison (∗P < .05 between groups at each time; $P < .5 vs baseline for each group: impact of Stroke; #P < .5 vs day 1 for each group: recovery). (G) Quantification of the global strength deficit measured by grip-test of forepaws before and on days 1, 3, and 7 after MCAo in STZ, STZ-rtPA, STZ-NAC and STZ-NAC-rtPA groups (n = 14 per group). Data were assessed in grams and converted in percentage normalized for each animal with the corresponding baseline value (before MCAo). Results are represented in mean ± SEM. 2-way ANOVA: Time factor <0.0001 and group factor <0.001; Tukey test for multiple comparison (∗P < .05 between groups at each time; $P < .5 vs baseline for each group: impact of Stroke; #P < .5 vs day 1 for each group: recovery).

Figure 4.

Targeting endothelial tPA-dependent NMDAR signaling with Glunomab increases the benefit of rtPA-mediated thrombolysis in nonhyperglycemic mice. (A) Schematic representation of the experimental protocol. (B) Quantification of ischemic lesion volume, 24 hours after MCAo assessed by T2-weighted imaging (7T MRI) in mice treated with saline (vehicle group), rtPA (10 mg/kg; Actilyse, 10% bolus, 90% perfusion during 40 minutes; rtPA group), Glunomab (300 μg, 100% bolus; Gluno group), or a combination of Glunomab-rtPA (Gluno-rtPA group). Individual values, means and SEM are plotted. 21.57 mm³ for vehicle group (n = 12); 12.07 mm³ for tPA group (n = 12); 10.26 mm³ for Gluno group (n = 12); and 11.04 mm³ for Gluno-rtPA group (n = 12). Ordinary 1-way ANOVA (P < .001); Tukey test for multiple comparisons (∗P < .05 and ∗∗P < .01). (C) Percentage of angiographic scores, 24 hours after MCAo assessed by FLASH_TOF_2D imaging (7T MRI) in vehicle, rtPA, Gluno, and Gluno-rtPA groups (n = 12 per group). No recanalization = complete occlusion (orange); partial recanalization = incomplete filling of the distal bed (light green); and complete recanalization = complete filling of the distal bed (dark green). Kruskal-Wallis test (P = .33). (D) Proportion of HT per group, 24 hours after MCAo assessed by T2∗-weighted imaging (deoxyhemoglobin; 7T MRI) in vehicle, rtPA, Gluno, and Gluno-rtPA groups (n = 12 per group). Fisher exact tests between groups (P > .05).

Figure 5.

Glunomab coupled with a thrombolytic agent improves stroke outcome and protects against HT in chronically hyperglycemic animals. (A) Schematic representation of the experimental protocol. (B) Quantification of ischemic lesion volume, 24 hours after MCAo assessed by T2-weighted imaging (7T MRI) in mice treated with saline (STZ group), rtPA (10mg/kg; Actilyse, 10% bolus, 90% perfusion over 40 minutes; and STZ-rtPA group), Glunomab (300 μg, 100% bolus; STZ-Gluno group), a combination of Glunomab and rtPA (STZ-Gluno-rtPA group), and a combination of Glunomab and NAC (400 mg/kg, slow bolus) on hyperglycemic mice. Individual values, means, and SEM are plotted; 28.80 mm³ for STZ group (n = 16); 27.66 mm³ for STZ-rtPA group (n = 15); 20.14 mm³ for STZ-Gluno group (n = 15); 15.64 mm³ for STZ-Gluno-rtPA group (n = 16); and 14.88 mm³ for STZ-NAC-Gluno group (n = 15). Ordinary 1-way ANOVA (P < .001); Tukey multiple comparisons (∗∗P < .01 and ∗∗∗P < .001). (C) Representative T2-weighted 7T MRI brain images (left) and representation of the lesion distribution around bregma (right), 24 hours after MCAo in STZ, STZ-rtPA, STZ-Gluno, STZ-Gluno-rtPA, and STZ-NAC-Gluno groups. (D) Percentage of angiographic scores, 24 hours after MCAo assessed by FLASH_TOF_2D imaging (7T MRI) in STZ (n = 15), STZ-rtPA (n = 14), STZ-Gluno (n = 15), STZ-Gluno-rtPA (n = 15), and STZ-Gluno-NAC (n = 14) groups. No recanalization = complete occlusion (orange); partial recanalization = incomplete filling of the distal bed (light green); and complete recanalization = complete filling of the distal bed (dark green). Kruskal-Wallis test (P < .05); Dunns test for multiple comparisons (∗P < .01 and ∗∗P < .01). (E) Proportion of HT per groups, 24 hours after MCAo assessed by T2∗-weighted imaging (deoxyhemoglobin; 7T MRI) in STZ (n = 16), STZ-rtPA (n = 15), STZ-Gluno (n = 15), STZ-Gluno-rtPA (n = 16), and STZ-Gluno-NAC (n = 15) groups. Fisher exact tests between groups (∗P < .05). (F) Quantification of the specific left paw–strength deficit measured by grip-test ratio (strength of left/right paws) of STZ (n = 16), STZ-rtPA (n = 15), STZ-Gluno (n = 15), STZ-Gluno-rtPA (n = 16), and STZ-Gluno-NAC (n = 15) groups, 1 day before, and on day 1, day 3, and day 7 after MCAo. Data were assessed in grams. Results are represented in mean ± SEM; 2-way ANOVA: time effect <0.0001 and group effect <0.01; Tukey test for multiple comparison (∗P < .05 between groups at each time; $P < .5 vs baseline for each group: impact of Stroke; #P < .5 vs day 1 for each group: recovery). (G) Quantification of the global strength deficit measured by grip-test of forepaws of STZ (n = 16), STZ-rtPA (n = 15), STZ-Gluno (n = 15), STZ-Gluno-rtPA (n = 16), and STZ-Gluno-NAC (n = 15) groups 1 day before and on day 1, day 3, and day 7 after MCAo. Data were assessed in grams and converted in percentage normalized for each animal with the corresponding baseline value (before MCAo). Results are represented in mean ± SEM; 2-way ANOVA: time factor <0.0001; Tukey test for multiple comparison (∗P < .05 between groups at each time; $P < .5 vs baseline for each group: impact of Stroke; #P < .5 vs day 1 for each group: recovery).

Figure 6.

Targeting endothelial tPA-dependent NMDAR signaling with Glunomab reduces invasion of the ischemic brain tissues by circulating inflammatory cells. (A) Schematic representation of the experimental protocol. (B) Representative flow cytometry dot plots and gating strategy used for quantification of CD11b⁺CD45^low microglia, CD11b⁺CD45^hi activated microglia or macrophages and CD11b⁺CD45⁺Ly6G⁺ neutrophils, 5 days after stroke in ipsilateral mice brain. (C) Flow cytometry quantification of microglia, activated microglia or macrophages, and neutrophils 5 days after stroke in ipsilateral mice brain in the Ctrl group (n = 6), STZ group (n = 5), STZ-rtPA group (n = 5), STZ-Gluno group (n = 5), and STZ-Gluno-rtPA group (n = 5); ∗P < .05; ordinary 1-way ANOVA; Tukey multiple comparisons. (D) Representative flow cytometry dot plots and gating strategy used for quantification of CD3⁺ T cells, CD3⁺CD8⁺ cytotoxic T cells, and CD3⁺CD4⁺ regulatory/helper T cells, 5 days after stroke in ipsilateral mice brain. (E) Flow cytometry quantification of CD3⁺ T cells, CD3⁺CD8⁺ cytotoxic T cells, and CD3⁺CD4⁺ regulatory/helper T cells, 5 days after stroke in ipsilateral mice brain in the Ctrl group (n = 6), STZ group (n = 5), STZ-rtPA group (n = 5), STZ-Gluno group (n = 5), and STZ-Gluno-rtPA group (n = 5); ∗P < .05; ordinary 1-way ANOVA; Tukey multiple comparisons.