Upregulation of serum and glucocorticoid-regulated kinase 1 exacerbates brain injury and neurological deficits after cardiac arrest

Abstract

Cardiopulmonary arrest (CA) is the leading cause of death and disability in the United States. CA-induced brain injury is influenced by multifactorial processes, including reduced cerebral blood flow (hypoperfusion) and neuroinflammation, which can lead to neuronal cell death and functional deficits. We have identified serum and glucocorticoid-regulated kinase-1 (SGK1) as a new target in brain ischemia previously described in the heart, liver, and kidneys (i.e., diabetes and hypertension). Our data suggest brain SGK1 mRNA and protein expression (i.e., hippocampus), presented with hypoperfusion (low cerebral blood flow) and neuroinflammation, leading to further studies of the potential role of SGK1 in CA-induced brain injury. We used a 6-min asphyxia cardiac arrest (ACA) rat model to induce global cerebral ischemia. Modulation of SGK1 was implemented via GSK650394, a SGK1-specific inhibitor (1.2 μg/kg icv). Accordingly, treatment with GSK650394 attenuated cortical hypoperfusion and neuroinflammation (via Iba1 expression) after ACA, whereas neuronal survival was enhanced in the CA1 region of the hippocampus. Learning/memory deficits were observed 3 days after ACA but ameliorated with GSK650394. In conclusion, SGK1 is a major contributor to ACA-induced brain injury and neurological deficits, while inhibition of SGK1 with GSK650394 provided neuroprotection against CA-induced hypoperfusion, neuroinflammation, neuronal cell death, and learning/memory deficits. Our studies could lead to a novel, therapeutic target for alleviating brain injury following cerebral ischemia.NEW & NOTEWORTHY Upregulation of SGK1 exacerbates brain injury during cerebral ischemia. Inhibition of SGK1 affords neuroprotection against cardiac arrest-induced hypoperfusion, neuroinflammation, neuronal cell death, and neurological deficits.

Keywords: cerebral blood flow; cerebral ischemia; neuroinflammation; neuronal cell death; serum and glucocorticoid-regulated kinase.

Fig. 1.

Hippocampal serum and glucocorticoid-regulated kinase-1 (SGK1) mRNA and proteins were enhanced 1 and 3 days after asphyxia cardiac arrest (ACA). A: representative images of gel electrophoresis from RT-PCR experiments. Total RNA was extracted from 3 rats denoted as R1, R2, and R3. SGK1 and SGK3 were detected in the hippocampus. SGK2 expression was not detected in the hippocampus. K indicates total RNA extracted from the rat kidney serving as a positive control, whereas β-actin served as an internal control. M indicates 100-base pair DNA ladder. B: total RNA was extracted 1, 3, and 7 days (d) after ACA. Relative hippocampal mRNA levels of SGK1 and SGK3 were measured by RT-qPCR. Results were normalized to the internal control (β-actin). C: representative images of the capillary-based immunoassay. Hippocampal total protein was extracted 1 and 3 days after ACA. SGK1 presented with bands at 49 and 55 kDa due to posttranslational modifications. Results from capillary-based immunoassay were summarized in D. SGK1 protein levels were normalized to total protein (C). *P < 0.05, as compared with control rats; and #P < 0.05, as compared with 7 days after ACA; n, number of experiments.

Fig. 2.

Schematic diagram of the experimental design. A: rats received an intracerebroventricular (icv) injection of GSK650394 (1.2 μg/kg) 5 min before asphyxia cardiac arrest (ACA). H&E, hematoxylin and eosin; FJC, Fluoro-Jade C. Cortical cerebral blood flow (CBF) was measured via laser speckle contrast imaging 30 min before and 24 h after ACA to examine the hypoperfusion event. Y maze was implemented 3 days after ACA/sham surgery. Upon completion of behavioral trials, rats were euthanized 7 days after ACA/sham surgery for brain histology. In a separate set of experiments, rats were euthanized 1, 3, and 7 days after ACA/sham for protein and mRNA analyses. SGK1, serum and glucocorticoid-regulated kinase-1. B: representative images of brain sections (2 mm thick) after intracerebroventricular injection of Evans blue. GSK650394 was injected the into third ventricle based on the fact that diffusion of Evans blue (tracer dye) through the ventricular system can be detected in the hippocampus (red arrows). *Evans blue was injected into the third ventricle. The rat was euthanized 5 h after injection.

Fig. 3.

GSK650394 (GSK) alleviated asphyxia cardiac arrest (ACA)-induced hypoperfusion and neuroinflammation. Cerebral blood flow (CBF) was measured via laser speckle contrast imaging. A: representative flux images of cortical vasculature 30 min before and 24 h after ACA and GSK + ACA treatment. Dashed ovals represent the cranial window where CBF was measured. Changes in CBF were summarized in B and presented as percent change from baseline (CBF, 30 min before ACA). In a separate set of experiments, total hippocampal protein was extracted 1, 3, and 7 days (d) after ACA for Iba1 measurements. C: representative images of synthetic bands from capillary-based immunoassay. Iba1 bands were detected at 17 kDa, with β-actin present at 45 kDa. Iba1 protein levels before/after ACA were normalized to β-actin and summarized in D. Each lane corresponds to an individual capillary electrophoresis protein sample in which the conditions of adjacent lanes are fully independent. *P < 0.05, as compared with control; and #P < 0.05, as compared with respective days after ACA; n, number of experiments.

Fig. 4.

Inhibition of serum and glucocorticoid-regulated kinase-1 alleviated asphyxia cardiac arrest (ACA)-induced neuronal deficits. Brain histopathology was performed 7 days after ACA. Representative images of coronal brain sections of the CA1 region of the hippocampus from control, ACA- and GSK650394 (GSK) +ACA-treated rats. Brain sections were stained with hematoxylin and eosin (H&E, purple and pink; A), FJC (green; B, left), and DAPI nuclear counterstain (blue; B, middle and right). A: healthy neurons were identified based on a lightly stained nucleus, a dark-stained nucleolus, and a red-stained cytoplasm. Dead/injured neurons contain a shrunken cytoplasm and a pyknotic nuclei. Red arrows indicate neuronal cell death, whereas green fluorescent-positive neurons in B represent degenerative neurons. Scale bars in H&E and FJC staining indicate 20 and 50 μm, respectively. C: rats were subjected to the Y-maze spontaneous alternation test 3 days after ACA to assess working/short-term memory. Control rats exhibited high alternation rates, which are indicative of intact learning/memory function. On the contrary, ACA-treated rats with learning/memory deficits displayed lower spontaneous alternation ratio (red bar). These ACA-induced learning/memory deficits can be ameliorated with GSK650394 (GSK; 1.2 μg/kg) treatment (green bar). *P < 0.05, as compared with control; #P < 0.05, as compared with respective days after ACA; n, number of experiments.