Intranasal insulin helps overcome brain insulin deficiency and improves survival and post-stroke cognitive impairment in male mice

Abstract

Obesity increases the risk for stroke and is associated with worse post-stroke outcomes; however, the mechanisms are poorly understood. Diet-induced obesity leads to insulin resistance and subsequently, brain insulin deficiency. The purpose of this study was to investigate the potential impact of brain insulin deficiency on post-stroke outcomes. To accomplish this, brain insulin levels were assessed in male C57BL/6J (B6) mice placed on either a standard diet or 54% kcal high-fat diet, a known model of insulin resistance. Mice were subjected to either a sham surgery (control) or 30-min middle cerebral artery occlusion to induce an ischemic stroke and administered either intranasal saline (0.9%) or intranasal insulin (1.75 U) twice daily for 5 days beginning on day 1 post-stroke. High-fat diet-induced brain insulin deficiency was associated with increased mortality, neurological and cognitive deficits. On the other hand, increasing brain insulin levels via intranasal insulin improved survival, neurological and cognitive function in high-fat diet mice. Our data suggests that brain insulin deficiency correlates with worse post-stroke outcomes in a diet-induced mouse model of insulin resistance and increasing brain insulin levels may be a therapeutic target to improve stroke recovery.

Keywords: RRID:IMSR_JAX:000664; RRID:SCR_000306; brain insulin deficiency; high-fat diet; insulin resistance; ischemic stroke; middle cerebral artery occlusion; neuroprotection.

Figure 1.. Middle cerebral artery occlusion significantly reduced blood flow during occlusion in male and female mice.

(a) Percentage (%) of blood flow measured using a Laser Doppler during occlusion (white bar) and reperfusion (black bar) normalized to baseline blood flow in (a) male mice (n=24 for occlusion and n=18 for reperfusion); ****P<0.0001 based on unpaired t-test; t=7.854, df=40 and (b) female mice (n=28 for occlusion and n=28 for reperfusion); ****P<0.0001 based on unpaired t-test; t=6.851, df=54. Data presented as mean ± SD.

Figure 2.. Study design.

Summary of experimental timeline in mice on either standard diet (STD) or a high-fat diet (HFD) for assessing insulin levels in the brain (Experiment 1) and stroke outcomes following the middle cerebral artery occlusion (MCAO), model of stroke (Experiment 2).

Figure 3.. HFD reduced brain insulin levels.

Hippocampal insulin levels in standard (STD) and high-fat (HFD) diet male mice after (a) 6 weeks of diet; n=8 (STD) and n=8 (HFD); **P<0.01 based on unpaired t-test (P=0.0017; t=3.856, df=14) and (b) 24 weeks of diet; n=10 (STD) and n=10 (HFD); *P<0.05 based unpaired t-test (P=0.0464; t=2.138, df=18). (c) Cerebral spinal fluid (CSF) insulin levels in male mice after 24 weeks of diet; n=8 (STD) and n=11 (HFD); **P<0.01 based on unpaired t-test (P=0.0020; t=3.635, df=17). Data presented as mean ± SD.

Figure 4.. Body weight reduced post-stroke.

The body weight in grams (g) in male mice on either a standard diet (STD) or high-fat diet (HFD) following middle cerebral artery occlusion (MCAO); n=10 (STD-S), n=20 (HFD-S), or n=14 (HFD treated with intranasal insulin, HFD-I) and n=6 (STD-I). A three-way ANOVA with mixed-effects modeling reveals a significant interaction with days Post-stroke (F(1.915, 62.05) = 72.54; P<0.0001), diet (F(1, 46) = 55.10; P<0.0001), and between days post-stroke and diet (F(5, 162) = 8.927; P<0.0001). Tukey’s post-hoc analysis reveals that STD-S mice experienced significant decrease in weight at 1 (P = 0.0327), 2 (P = 0.0048), and 3 (P = 0.0079) days post-stroke compared to baseline (0 day), STD-I experienced significant decrease in weight at 2 (P = 0.0004) and 3 (P = 0.0072) days post-stroke compared to baseline. The data is presented as mean ± SD.

Figure 5.. Intranasal insulin improves post-stroke survival and neurological deficits.

(a) Percent survival with following SHAM surgery or middle cerebral artery occlusion (MCAO), model of stroke in mice on either a standard diet (STD) or high-fat diet (HFD) treated with either intranasal saline (S) or insulin (I); A comparison of survival curves used Log-rank (Mantel-Cox) test (Chi square = 16.91, df=4, P=0.0020) and Log-rank test for trend (Chi square = 7.600, df=1, P = 0.0058). (b) Modified neurological severity score (mNSS) on a scale of 0 (normal) through 5 (moribund) at Day 1 and Day 2 post-stroke in STD-S (n=10), HFD-S (n=19), STD-I (n=6), and HFD-I (n=14) mice. Three-way ANOVA with mixed-effects modeling revealed a significant interaction with diet (F (1, 28)=6.262, P=0.0184) and treatment (F(1, 54)=12.68, P=0.0008). Tukey’s multiple comparisons test revealed a significant improvement in mNSS scores in HFD-I mice compared to HFD-S mice at 2 days post-stroke (P=0.0194). Data presented as mean ± SD.

Figure 6.. Intranasal Insulin improved post-stroke cognitive impairment.

(a) Baseline percentage (%) exploration of novel location in male mice on either a standard diet (STD) or high-fat diet (HFD). **P<0.01 based on a one sample t-test (STD: P=0.0085; HFD: P=0.0028); STD; n=10, HFD; n=14. (b) % Total Memory of exploring the novel location in male mice 14 days following middle cerebral artery occlusion (MCAO), model of stroke, in mice on either STD or HFD receiving either intranasal saline (0.9%) or insulin (1.75U) beginning Day 1 post-stroke. *P<0.05 in Controls (P=0.465), **P<0.01 in STD-I (P=0.0020) and HFD-I (P=0.0095) based on one-sample t-test; Controls (n=5), STD-I (n=6), HFD-S (n=5), and HFD-I (n=5). There are no significant differences between groups with Tukey’s multiple comparisons test. Data presented as mean ± SD.

Figure 7.. Body Weight homeostasis significantly impacted in HFD MCAO female mice immediately after MCAO.

The body weight in grams (g) in female mice on either a standard diet (STD) or high-fat diet (HFD) following middle cerebral artery occlusion (MCAO). (STD-S; n=6), high-fat diet (HFD-S; n=6), or HFD treated with intranasal insulin (HFD-I; n=6) and (STD-I; n=6). Based on a three-way ANOVA with Tukey’s multiple comparison test, STD-S mice experienced significant decrease in weight at 1 (P=0.0181) and 2 (P=0.0114) days post-stroke compared to baseline (0 day). STD-I experienced significant decrease in weight at 1 day (P=0.0025) post-stroke compared to baseline. HFD-S mice experienced significant decrease in weight at 1 (P=0.0257), 2 (P=0.0075), 3 (P=0.0065), and 4 (P=0.0214) days post-stroke compared to baseline (0 day). HFD-I mice experienced significant decrease in weight at 1 (P=0.0.0011), 2 (P=0.0095), 3 (P=0.0184), 4 (P=0.0302), and 5 (P=0.0145) days post-stroke compared to baseline (0 day). The data is presented as mean ± SD.

Figure 8.. Intranasal insulin improves post-stroke survival and neurological deficits in female mice.

(a) Percent survival with following SHAM surgery or middle cerebral artery occlusion (MCAO), model of stroke in mice on either a standard diet (STD) or high-fat diet (HFD) treated with either intranasal saline (S) or insulin (I). A comparison of survival curves used Log-rank (Mantel-Cox) test (Chi square=2.769, df=5, P=0.7355) and Log-rank test for trend (Chi square=0.1412, df=1, P = 0.7071). (b) Modified neurological severity score (mNSS) on a scale of 0 (normal) through 5 (moribund) at Day 1 and Day 2 post-stroke in STD-S (n=6), HFD-S (n=6), STD-I (n=6), and HFD-I (n=6) mice. Three-way ANOVA with mixed-effects modeling with Tukey’s multiple comparisons test revealed no significant interactions or differences between groups. Data presented as mean ± SD.

Figure 9.. Intranasal Insulin had no impact on post-stroke cognitive impairment in high-fat diet female mice.

(a) Baseline percentage (%) exploration of novel location in female mice on either a standard diet (STD) or high-fat diet (HFD). ####P<0.0001 based on one sample t-test (STD: t=12.25, df=13, P<0.0001; HFD: t=27.30, df=13, P<0.0001); ****P<0.0001 based on unpaired t-test (t=6.536, df=26); STD: n=14, HFD: n=14. (b) % Total Memory of exploring the novel location in female mice 14 days following middle cerebral artery occlusion (MCAO), model of stroke, in mice on either STD or HFD receiving either intranasal saline (0.9%) or insulin (1.75U) beginning Day 1 post-stroke. ****P<0.0001 in Controls, ns (not significant) in STD-I, **P=0.0023 in HFD-S, and *P=0.0180 in HFD-I based on one-sample t-test; Controls (n=9), STD-I (n=5), HFD-S (n=6), and HFD-I (n=6). A one-way ANOVA (F(3,22) =1.180, P=0.3399) with Tukey’s multiple comparisons test revealed no difference between groups. Data presented as mean ± SD.