Exercise Improves Cerebral Blood Flow and Functional Outcomes in an Experimental Mouse Model of Vascular Cognitive Impairment and Dementia (VCID).

Vascular cognitive impairment and dementia (VCID) are a growing threat to public health without any known treatment. The bilateral common carotid artery stenosis (BCAS) mouse model is valid for VCID. Previously, we have reported that remote ischemic postconditioning (RIPostC) during chronic cerebral hypoperfusion (CCH) induced by BCAS increases cerebral blood flow (CBF), improves cognitive function, and reduces white matter damage. We hypothesized that physical exercise (EXR) would augment CBF during CCH and prevent cognitive impairment in the BCAS model. BCAS was performed in C57/B6 mice of both sexes to establish CCH. One week after the BCAS surgery, mice were randomized to treadmill exercise once daily or no EXR for four weeks. CBF was monitored with an LSCI pre-, post, and 4 weeks post-BCAS. Cognitive testing was performed for post-BCAS after exercise training, and brain tissue was harvested for histopathology and biochemical test. BCAS led to chronic hypoperfusion resulting in impaired cognitive function and other functional outcomes. Histological examination revealed that BCAS caused changes in neuronal morphology and cell death in the cortex and hippocampus. Immunoblotting showed that BCAS was associated with a significant downregulate of AMPK and pAMPK and NOS3 and pNOS3. BCAS also decreased red blood cell (RBC) deformability. EXR therapy increased and sustained improved CBF and cognitive function, muscular strength, reduced cell death, and loss of white matter. EXR is effective in the BCAS model, improving CBF and cognitive function, reducing white matter damage, improving RBC deformability, and increasing RBC NOS3 and AMPK. The mechanisms by which EXR improves CBF and attenuates tissue damage need further investigation.

Time Dimension Influences Severity of Stroke and Heightened Immune Response in Mice.

Ischemic stroke is caused by obstructed cerebral blood flow, which results in neurological injury and poor outcomes. Pro-inflammatory signaling from both residential and infiltrating immune cells potentiates cerebral injury and worsens patient outcomes after stroke. While the occurrence of a stroke exhibits a time-of-day-dependent pattern, it remains unclear whether disrupted circadian rhythms modulate post-stroke immunity. In this study, we hypothesized that stroke timing differentially affects immune activation in mice. Following middle cerebral artery occlusion (MCAO), circadian genes BMAL1, CLOCK, Cry1, and Cry2 elevated at ZT06, with a significant difference between ZT06 and ZT18. Conversely, expression of the negative limb circadian clock gene, Per1, decreased at ZT06 and ZT18 in stroke mice compared to sham. Paralleling these circadian gene expression changes, we observed a significant increase in TNF-α and a decrease in IL-10 expression at 48 h post-MCAO, when the procedure was performed at ZT06 (MCAO-ZT6), which corresponds to a deep sleep period, as compared to when the stroke was induced at ZT12 (MCAO-ZT12), ZT18 (MCAO-ZT18), or ZT0 (MCAO-ZT12). Similarly, increased pro-inflammatory, decreased anti-inflammatory monocytes, and increased NLRP3 were observed in blood, while changes in the expression of CD11b and Iba1 were noted within brain tissue at 48 h of MCAO-ZT06, as compared to MCAO-ZT18. Consistent with the increased immune response, infarct volume and sensorimotor deficits were greater in MCAO-ZT06 mice compared to MCAO-ZT18 mice at 48 h. Finally, we found reduced weight and length of the spleen while splenocytes showed significant time-dependent changes in Tregs, Bregs, and monocytes in MCAO-ZT06 mice. Taken together, this study demonstrates that circulating and splenic immune responses following ischemic stroke exhibit a circadian expression pattern which may contribute to time-of-day-dependent stroke outcomes.

The time dimension to stroke: Circadian effects on stroke outcomes and mechanisms.

The circadian system is widely involved in the various pathological outcomes affected by time dimension changes. In the brain, the master circadian clock, also known as the "pacemaker," is present in the hypothalamus's suprachiasmatic nucleus (SCN). The SCN consists of molecular circadian clocks that operate in each neuron and other brain cells. These circadian mechanisms are controlled by the transcription and translation of specific genes such as the clock circadian regulator (Clock) and brain and muscle ARNT-Like 1 (Bmal1). Period (Per1-3) and cryptochrome (Cry1 and 2) negatively feedback and regulate the clock genes. Variations in the circadian cycle and these clock genes can affect stroke outcomes. Studies suggest that the peak stroke occurs in the morning after patients awaken from sleep, while stroke severity and poor outcomes worsen at midnight. The main risk factor associated with stroke is high blood pressure (hypertension). Blood pressure usually dips by 15-20% during sleep, but many hypertensives do not display this normal dipping pattern and are non-dippers. A sleep blood pressure is the primary determinant of stroke risk. This article discusses the possible mechanism associated with circadian rhythm and stroke outcomes.

Preclinical evaluation of circadian rhythm in ischemic stroke outcomes.

Stroke is a leading cause of disability and death worldwide. There is evidence that there is a circadian rhythm in stroke with peak occurrence in the morning (6 to 10 am). However, it is not clear if the size of infarcts and the outcome of stroke also varies during the 24-hour period. We hypothesized that the size of cerebral infarct and outcome from stroke would show circadian variation in a mouse suture occlusion model. Seven to eight-month-old C57BL/6J (n =10-12 mice/group) mice were randomly assigned to undergo middle cerebral artery occlusion (MCAO) for 60 minutes at different time points during the 24h day following zeitgeber time at ZT0, ZT6, ZT12, and Z18. Cerebral blood flow was monitored by Laser Speckle Contrast Imaging at baseline after occlusion, and again at 24h post-occlusion. Neurological deficit was observed by using Bederson score at 24h and 48h. The corner test was used to detect unilateral abnormalities in sensory and motor functions in the stroke mice at 48h. To estimate brain infarction, 2,3,5-tryphenyltetrazolium chloride staining was performed 48h after stroke and the infarct area was quantified using NIH-Image J software. We did not find a significant difference in cerebral blood flow at any time point. There was a significant decrease in neurological deficit as assessed using the Bederson Score from 24h (1.82 ± 1.11) to 48h (1.10 ± 0.12) in the ZT18 (midnight) period (p = 0.0025), however there were no differences between groups at 48h. In the corner test, we found right turn preference significantly higher (p = 0.0348) at noon/ZT06 (9.5 ± 1.06) compared to the fully awake (5.5 ± 4.06) (midnight, ZT18) period and ZT0 (6 am, 4.8 ± 0.97, p = 0.0087). Similarly, the infarction volume was significantly higher (p = 0.0220) during the sleep (ZT06, noon) period (35.22 ± 20.77) than when the ischemic mice were fully awake during the midnight/ZT18 period (15.68 ± 7.54). This is the first report demonstrating that mice have larger infarcts and worse short-term outcomes during their sleep period (noon/ZT06) than during their awake period (midnight/ZT18).

Remote ischemic perconditioning is effective after embolic stroke in ovariectomized female mice.

Remote ischemic conditioning is neuroprotective in young male rodents after experimental stroke. However, it has never been tested in females whom remain at higher risk of stroke injury after menopause. We tested remote ischemic perconditioning therapy (RIPerC) at 2 h after embolic stroke in ovariectomized (OVX) female mice with and without intravenous tissue plasminogen activator (IV-tPA) treatment. We assessed cerebral blood flow (CBF), neurobehavioral outcomes, infarction, hemorrhage, edema, and survival. RIPerC therapy with and without IV-tPA improved the CBF and neurobehavioral outcomes and reduced the infarction, hemorrhage, and edema significantly. Late IV-tPA alone at 4 h post-stroke neither improved the neurobehavior nor reduced the infarction but aggravated hemorrhage and mortality in OVX mice. RIPerC therapy prevented the increased mortality during late IV-tPA. Our study demonstrates for the first time that RIPerC therapy is effective in OVX females.

Remote ischemic perconditioning is effective alone and in combination with intravenous tissue-type plasminogen activator in murine model of embolic stroke.

BACKGROUND AND PURPOSE: Remote ischemic conditioning is cardioprotective in myocardial infarction and neuroprotective in mechanical occlusion models of stroke. However, there is no report on its therapeutic potential in a physiologically relevant embolic stroke model (embolic middle cerebral artery occlusion) in combination with intravenous tissue-type plasminogen activator (tPA). METHODS: We tested remote ischemic perconditioning therapy (RIPerC) at 2 hours after embolic middle cerebral artery occlusion in the mouse with and without intravenous tPA at 4 hours. We assessed cerebral blood flow up to 6 hours, neurological deficits, injury size, and phosphorylation of Akt (Serine(473)) as a prosurvival signal in the ischemic hemisphere at 48 hours poststroke. RESULTS: RIPerC therapy alone improved the cerebral blood flow and neurological outcomes. tPA alone at 4 hours did not significantly improve the neurological outcome even after successful thrombolysis. Individual treatments with RIPerC and intravenous tPA reduced the infarct size (25.7% and 23.8%, respectively). Combination therapy of RIPerC and tPA resulted in additive effects in further improving the neurological outcome and reducing the infarct size (50%). All the therapeutic treatments upregulated phosphorylation of Akt in the ischemic hemisphere. CONCLUSIONS: RIPerC is effective alone after embolic middle cerebral artery occlusion and has additive effects in combination with intravenous tPA. RIPerC may be a simple, safe, and inexpensive combination therapy with intravenous tPA.