GPR39 Knockout Worsens Microcirculatory Response to Experimental Stroke in a Sex-Dependent Manner

Abstract

No current treatments target microvascular reperfusion after stroke, which can contribute to poor outcomes even after successful clot retrieval. The G protein-coupled receptor GPR39 is expressed in brain peri-capillary pericytes, and has been implicated in microvascular regulation, but its role in stroke is unknown. We tested the hypothesis that GPR39 plays a protective role after stroke, in part due to preservation of microvascular perfusion. We generated GPR39 knockout (KO) mice and tested whether GPR39 gene deletion worsens capillary blood flow and exacerbates brain injury and functional deficit after focal cerebral ischemia. Stroke was induced in male and female GPR39 KO and WT littermates by 60-min middle cerebral artery occlusion (MCAO). Microvascular perfusion was assessed via capillary red blood cell (RBC) flux in deep cortical layers in vivo using optical microangiography (OMAG). Brain injury was assessed by measuring infarct size by 2,3,5-triphenyltetrazolium chloride staining at 24 h or brain atrophy at 3 weeks after ischemia. Pole and cylinder behavior tests were conducted to assess neurological function deficit at 1 and 3 weeks post-stroke. Male but not female GPR39 KO mice exhibited larger infarcts and lower capillary RBC flux than WT controls after stroke. Male GPR39 KO mice also exhibited worse neurologic deficit at 1 week post-stroke, though functional deficit disappeared in both groups by 3 weeks. GPR39 deletion worsens brain injury, microvascular perfusion, and neurological function after experimental stroke. Results indicate that GPR39 plays a sex-dependent role in re-establishing microvascular flow and limiting ischemic brain damage after stroke.

Keywords: Capillary flux; G protein–coupled receptor 39; No-reflow; OMAG; Pericytes; Sex difference.

Figure 1.. Creation and confirmation of mouse GPR39 knockout model.

A. Schematic of mouse GPR39 gene locus targeting strategy. Guide RNAs were designed to cleave at sites flanking exon 1 of Gpr39 resulting in a 1322 base pair deletion. Exon 2 was left intact. B. RT-PCR and C. Western blot of GPR39 (top row) and beta-Actin (bottom row) confirming presence of GPR39 mRNA (B) and protein (C) in brain, heart and kidneys from wild type mice (left) but absent in GPR39 knockout mice (right).

Figure 2.. GPR39 deletion increases infarct size in male mice after MCAO.

A. Relative laser Doppler perfusion of MCA territory. There were no differences among groups. B. Infarct size was significantly larger in male GPR39 KO mice (n=14) compared to WT littermates (n=9) in cerebral cortex, caudate putamen (CP) and whole hemisphere. C. There was no difference in infarct size between WT (n=9) and GPR39 KO (n=12) in females. GPR39 KO vs. WT. n.s., not significant. *p<0.05, ** p<0.001. Cortex and CP are components of the hemisphere.

Figure 3.. Optical microangiography (OMAG) demonstrates worsened microvascular brain tissue perfusion in GPR39 KO vs WT males after stroke.

A. Schematic of OMAG setup for capillary blood flow measurement in 4 cortical layers within ischemic penumbra. B. Post-stroke cortical capillary flux in in layers 1-4 (% baseline) in GPR39 KO (n=10) and WT littermates (n=11). (Genotype F_1,76 = 5.36; *p<0.0234). C. Post-stroke capillary flux in GPR39 KO vs. WT littermates in deepest cortical layer only (#4). (n=10 KO, n=11 WT, *p<0.05). D. Experimental timeline illustrating OMAG timing relative to MCAO. Mice were allowed 1 hour of recovery after cranial window creation under anesthesia prior to baseline OMAG scan. Mice were then subjected to MCAO, and recovered for 24 hours when they underwent a second post-stroke OMAG scan. Brains were collected, sectioned and stained with TTC to measure infarct size.

Figure 4.. GPR39 KO decreases survival and motor function 1 week after stroke, and increases brain atrophy 3 weeks after stroke.

A. Timeline of behavior testing, MCAO, infarct size and brain atrophy measurement, superimposed on survival curve. GPR39 KO decreased survival at 1 week compared to WT (*p<0.05). B. Ipsilateral atrophy in cortex, caudate putamen (CP) and hemisphere in KO and WT mice at 3 weeks after stroke. C. KO mice show greater forepaw disability in the Cylinder test 1 week after stroke. Higher asymmetry indicates greater disability. D. KO mice show decreased coordination in the Pole test 1 week after stroke. Longer time indicates greater disability. Total number of KO and WT mice at beginning of protocol was 24 and 21, respectively. Number of mice at full survival to 21 days was 17 in each group (*P <0.05).

Figure 5.. Hemodynamic measures in GPR39 KO and WT mice during and after stroke.

A. Relative laser-Doppler perfusion over the MCA territory during and 15 min after MCAO, n.s. not significant. B. Mean arterial pressure (MAP), as % baseline, in cohort used for arterial blood gas measurement. (Genotype F_1,32 = 10.05; **p<0.0034; post final ABG adjusted p value for multiple comparisons *p<0.05; n=5 per group).