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Delayed Administration of Angiotensin II Type 2 Receptor (AT2R) Agonist Compound 21 Prevents the Development of Post-stroke Cognitive Impairment in Diabetes Through the Modulation of Microglia Polarization

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Abstract

A disabling consequence of stroke is cognitive impairment, occurring in 12%–48% of patients, for which there is no therapy. A critical barrier is the lack of understanding of how post-stroke cognitive impairment (PSCI) develops. While 70% of stroke victims present with comorbid diseases such as diabetes and hypertension, the limited use of comorbid disease models in preclinical research further contributes to this lack of progress. To this end, we used a translational model of diabetes to study the development of PSCI. In addition, we evaluated the application of compound 21 (C21), an angiotensin II Type 2 receptor agonist, for the treatment of PSCI by blinding the treatment assignment, setting strict inclusion criteria, and implementing a delayed administration time point. Diabetes was induced by a high-fat diet (HFD) and low-dose streptozotocin (STZ) combination. Control and diabetic rats were subjected to 1 h middle cerebral artery occlusion (MCAO) or sham surgery. Adhesive removal task (ART) and two-trial Y-maze were utilized to test sensorimotor and cognitive function. Three days post-stroke, rats that met the inclusion criteria were administered C21 or vehicle in drinking water at a dose of 0.12 mg/kg/day for 8 weeks. Samples from freshly harvested brains were analyzed by flow cytometry and immunohistochemistry (IHC). Diabetes exacerbated the development of PSCI and increased inflammation and demyelination. Delayed administration of C21 3 days post-stroke reduced mortality and improved sensorimotor and cognitive deficits. It also reduced inflammation and demyelination through modulation of the M1:M2 ratio in the diabetic animals.

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Acknowledgments

This study is from the doctoral thesis of Ladonya Jackson, presented to the University of Georgia. The authors would like to thank Dr. Darrell Brann for providing the BV2 cell line and Vicore Pharma for providing C21. We would also like to thank the Electron Microscope and Histology Core at Augusta University for the histological staining of our samples, with a special thanks to Ms. Penny Roon.

Funding

This study was supported by National Institute of Health (NIH) R01 NS104573 to Adviye Ergul and Susan C. Fagan; R01 NS083559, Veterans Affairs (VA) Merit Review (BX000347), VA Senior Research Career Scientist Award (IK BX004471) to Adviye Ergul; and TL1 TR002382 and UL1TR002378 to Ladonya Jackson.

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Authors and Affiliations

  1. Program in Clinical and Experimental Therapeutics, University of Georgia College of Pharmacy, Augusta, GA, USA

    Ladonya Jackson, Waleed Althomali, Mohammed A. Sayed, Wael Eldahshan & Susan C. Fagan

  2. Charlie Norwood Veterans Affairs Medical Center, Augusta, USA

    Ladonya Jackson, Waleed Althomali, Mohammed A. Sayed, Wael Eldahshan & Susan C. Fagan

  3. Physiology, Medical College of Georgia, Augusta University, Augusta, GA, USA

    Ladonya Jackson, Guangkuo Dong, Wael Eldahshan & Jessica Filosa

  4. Oral Biology, Dental College of Georgia, Augusta, GA, USA

    Babak Baban

  5. Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Augusta University, Augusta, GA, USA

    Maribeth H. Johnson

  6. Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Ave. MSC, Charleston, SC, 908, USA

    Adviye Ergul

  7. Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC, USA

    Adviye Ergul

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  1. Ladonya Jackson
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  2. Guangkuo Dong
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  3. Waleed Althomali
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  4. Mohammed A. Sayed
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  9. Susan C. Fagan
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  10. Adviye Ergul
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Corresponding author

Correspondence to Adviye Ergul.

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Statement on the Welfare of Animals

All rats were housed in the animal care facility at Augusta University, which is approved by the American Association for Accreditation of Laboratory Animal Care. All experiments were conducted in accordance with the National Institute of Health (NIH) guidelines for the care and use of animals in research. Furthermore, all protocols were approved by the institutional animal care and use committee.

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Electronic Supplementary Materials

ESM 1

Fig. S1 The degree of weight loss correlated with lesion size. In order to set inclusion criteria, we first evaluated the correlation between weight loss at day 3 and the infarct size at day 3. We discovered that weight loss correlated with the size of the infarct (R2 = 0.8), Pearson’s Correlation. Since a 10% drop in weight loss only correlated with a 20% infarct size, we required the drop in weight loss of the included animals to be greater than 10%. Fig. S2 In an effort to ensure inclusion of animals with a significant degree of ischemic injury, a strict inclusion criteria was employed. 3 days after the animals underwent MCAO surgery, those that met the weight loss and sensorimotor deficit criteria were included in the study. Sham diabetic animals trended to show increased inflammation. A) 30 um sections from the B slice were co-stained with IBA-1 and GFAP and 10x images were taken from the structures within the prefrontal cortex, limbic system and OVLT. Images derived from control sham (A) and a diabetic sham groups (B) were Z stacked and quantified throughout the layer. Diabetic sham animals trended to exhibit a chronic upregulation of C) IBA-1+ cells (p < 0.07). Student’s t test, (Control sham C vs Diabetic sham D) + compared to control sham (p < 0.05). No difference was found in D) GFAP+ cells compared to their control counterparts. (n = 5/group). Fig. S3 Delayed C21 administration did not impact blood glucose. Blood glucose ranged from 300–350 mg/dL throughout the majority of the study. Fig. S4 Delayed C21 administration did not impact weight loss post-stroke. There was no significant difference in weight with the treatment of C21. Since weight loss is typically maximized by day 3 post-stroke, the delayed administration of C21 was not administered early enough to prevent significant weight loss, and it also did not later contribute to a larger weight gain. Fig. S5 Flow cytometry gating strategy. Strategy showing how cells were gated for flow cytometric analysis to identify infiltrating macrophages. Path A was utilized to differentiate infiltrating cells from resident cells, while path B was utilized to analyze the M1:M2 ratio. (PPTX 711 kb)

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Jackson, L., Dong, G., Althomali, W. et al. Delayed Administration of Angiotensin II Type 2 Receptor (AT2R) Agonist Compound 21 Prevents the Development of Post-stroke Cognitive Impairment in Diabetes Through the Modulation of Microglia Polarization. Transl. Stroke Res. 11, 762–775 (2020). https://doi.org/10.1007/s12975-019-00752-5

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