A steroid receptor coactivator small molecule "stimulator" attenuates post-stroke ischemic brain injury

Abstract

Introduction: Pathologic remodeling of the brain following ischemic stroke results in neuronal loss, increased inflammation, oxidative stress, astrogliosis, and a progressive decrease in brain function. We recently demonstrated that stimulation of steroid receptor coactivator 3 with the small-molecule stimulator MCB-613 improves cardiac function in a mouse model of myocardial ischemia. Since steroid receptor coactivators are ubiquitously expressed in the brain, we reasoned that an MCB-613 derivative (MCB-10-1), could protect the brain following ischemic injury. To test this, we administered MCB-10-1 to rats following middle cerebral artery occlusion and reperfusion. Methods: Neurologic impairment and tissue damage responses were evaluated on day 1 and day 4 following injury in rats treated with control or 10-1. Results: We show that 10-1 attenuates injury post-stroke. 10-1 decreases infarct size and mitigates neurologic impairment. When given within 30 min post middle cerebral artery occlusion and reperfusion, 10-1 induces lasting protection from tissue damage in the ischemic penumbra concomitant with: (1) promotion of reparative microglia; (2) an increase in astrocyte NRF2 and GLT-1 expression; (3) early microglia activation; and (4) attenuation of astrogliosis. Discussion: Steroid receptor coactivator stimulation with MCB-10-1 is a potential therapeutic strategy for reducing inflammation and oxidative damage that cause neurologic impairment following an acute ischemic stroke.

Keywords: astrocytes; cerebral ischemia; inflammation; neuroprotection; oxidative stress; steroid receptor coactivator stimulation; transcriptional regulation.

Figure 1

The SRC activator 10-1 attenuates cerebral ischemic injury and performance activity post-MCAO R. (A) Schematic representation of experimental procedures. Rats were injected with 10-1 (20 mg/kg) or control 30 min after a 90-min occlusion of the middle cerebral artery and every 24 h up to harvest at day 1 (n = 20) and day 4 (n = 20) after surgery. (B) Brains were harvested and stained with TTC to delineate and calculate infarct size. (C) Mean area of infarct was calculated at day 1 and day 4 post-MCAO R. (D) Neurological testing was done 24 h and 4 days after stroke onset using a modified Bederson score. n = 10 control and n = 10, 10-1.

Figure 2

10-1 treatment attenuates progression of tissue damage post-MCAO R. (A) H&E tissue sections from the contralateral side (control, 1 day post-MCAO) and infarct areas at day 1 and day 4. Arrows indicate areas containing darkly stained pyknotic nuclei, cell body shrinkage, perineuronal vacuolization, and granular necrotic debris. (B) Representative H and E sections day 1 and day 4 post-MCAO. (C) Cortical and sub-cortical infarct size were measured in H&E stained brain tissue sections 1 day and 4 days post-MCAO R. (D) Representative images of TUNEL and NeuN immunostaining showing neuronal apoptosis one and 4 days post-MCAO R. Quantification of TUNEL positive NeuN positive cells. Day 1 and day 4 n = 10 control, 10 drug-treated, three images per section.

Figure 3

SRC activation promotes a pro-reparative immune response. (A) Representative images of IBA1 and ARG1 immunostaining showing per cent of pro-reparative M2 microglia 1 day and 4 days post-MCAO R. (B) Quantification of mean IBA1 intensity 1 and 4 days post-MCAO R. Day 1 and day 4 n = 10 control, 10 drug-treated, three images per section. (C) Quantification of IBA1 positive and ARG1 positive cells at day 4. (D) Quantification of average numbers of Tregs in the core infarct. Day 1 and day 4 n = 10 control, 10 drug-treated, three images per section.

Figure 4

Decreased astrogliosis is associated with an increased proportion of NRF2 and GLT-1 positive astrocytes. (A) Representative images of GFAP and NRF2 in the contralateral side (normal side) and penumbra 1 day and 4 days post-MCAO R. (B) Number of astrocytes in the penumbra. (C) The proportion of GFAP positive, NRF2 positive astrocytes in the penumbra. (D) Representative H&E section with area of sampling in core (box inside dashed area) and penumbra (box outside dashed area). (E) Quantification of the intensity of GLT-1 staining in the core and penumbra as shown in Supplementary Figure 2. Day 1 and day 4 n = 10 control, 10 drug-treated, three HPF per section.

Figure 5

Schematic of regulation of ischemic stroke injury following 10-1 treatment. 10-1 treatment attenuated neuronal injury following robust transient activation of microglia, increased Treg numbers, and increased representation of NRF2 positive astrocytes 24 h after ischemic injury. Four days after ischemia injury, the 10-1 therapy also enhanced NRF2 positive astrocytes, polarized M2 reparative microglia, and reduced astrogliosis. Created with BioRender.com.