Post-stroke early mobilization has been investigated extensively and is recommended in several international clinical practice guidelines because early initiation of exercise (24 to 72 h post-stroke) promotes functional recovery, prevents post-stroke complications, and facilitates return to society . However, exposure to high doses at the early phase and forced exercise within 24 h post-stroke might adversely affect stroke outcomes. The effects of early exposure to EE on functional recovery and the underlying mechanism after stroke have yet to be fully elucidated.
In the current study, early exposure to EE significantly improved functional recovery and preserved neuronal survival. The neuroprotective effects were mediated via downregulation of both extrinsic and intrinsic signaling pathways of apoptosis. A significant downregulation of Fas/FasL-mediated apoptosis in cerebral cortex and hippocampus was detected in mice exposed to early EE.
Previous studies have shown that VE may improve motor and cognitive ability, and promoted anti-apoptotic as well as neuroprotective effects [57, 58]. Compared with forced exercise, VE is not associated with systemic stress and does not decrease the neuroprotective effect [58–60]. VE via exploratory movements such as EE may have greater benefit . An early study demonstrated the beneficial effects of VE on hippocampal function, which were associated with suppression of cleaved caspase-3 expression, reduction of Bax expression and increased Bcl-2 expression in the hippocampus [62, 63]. Following stroke, VE may improve motor rehabilitation, enhance cognitive ability and hippocampal BDNF expression compared with involuntary muscle movement and forced exercise . The effects of voluntary movements post-stroke on neuronal regeneration and repair were related to upregulation of the expression of growth-associated protein 43 and neurotrophin 3 . A recent study reported that early commencement of VE post-stroke improved cerebral blood flow, vascular quality, and brain functions such as connectivity and motor abilities .
In terms of forced exercise post-stroke, mild-, moderate- and high-intensity exercise training initiated at various time points following stroke played a beneficial role [65–67]. Especially, mild- and moderate-intensity exercise enhanced neuroprotection compared with high-intensity exercise training [65, 66, 68, 69]. However, an early start of forced exercise post-stroke aggravated brain damage and apoptotic cell death, and triggered energy deficits along with generation of reactive oxygen species .
Apoptosis may contribute significantly to neuronal death following brain ischemia; however, the time window for effective stroke rehabilitation including VE and the underlying mechanisms are not fully understood. To our knowledge, the current study was the first of its kind to investigate the effect of hyperacute exposure to EE on both extrinsic and intrinsic pathways of apoptosis in an adult mouse model of HI brain injury. Our data reveal that hyperacute (within 3 hours) exposure to EE post-stroke suppresses both extrinsic and intrinsic pathways of apoptosis in cerebral cortex and hippocampus. This may be due to the higher sensitivity of these brain areas to therapies that suppress neuronal apoptosis [70, 71]. These results provide novel insights into the mechanisms underlying the neuroprotective effects of hyperacute EE treatment after ischemic stroke.
Previous studies highlighted the importance of suppressing the extrinsic pathway of apoptosis for favorable outcomes during the acute phase of ischemic stroke. Several molecules involved in the TNF/Fas family death receptor-mediated extrinsic pathway are increased particularly in the damaged penumbra and remained elevated in the stroke model . The upregulated expression of Fas, FasL, and TNF-related apoptosis-inducing ligands was observed within 12 hours after cerebral ischemia and peaked between 24 and 48 hours in the post-ischemic rat brain, which highly correlates with the time course of neuronal apoptosis [72, 73]. In patients with acute ischemic stroke, Fas-induced apoptosis in peripheral blood was activated in the first week after the onset, followed by a decrease towards the end of the acute period . In a rodent model of ischemic stroke, acute treatment with edaravone was neuroprotective in transient focal ischemia, and the mechanism involved suppression of the Fas/FasL signaling pathway . Recently, very early treatment with zonisamide decreased morbidity by suppressing the expression of caspase-3, caspase-8, and calpain-1, and inhibiting the apoptosis of neuronal cells after cerebral ischemia injury . Further, intranasal administration of a Fas-blocking peptide 12 hours post-ischemic stroke attenuated Fas-mediated apoptosis, decreased infarct volumes, and reduced neurologic deficits.24 Importantly, a significant reduction in infarct volume occurred in hybrid mice expressing FasL dysfunction and in TNF knockout mice 24 hours after stroke[19, 73]. However, the mechanism of suppression of extrinsic apoptosis mediated by EE treatment in the hyperacute phase of ischemic stroke has yet to be reported. In the current study, we found that hyperacute exposure to EE significantly suppressed extrinsic apoptosis via downregulation of Fas/FasL-mediated signaling in both cerebral cortex and hippocampus; however, the delayed exposure to EE failed to inhibit the apoptosis execution pathway.
The Fas/FasL system plays an important role in apoptosis during the acute phase in other neurological disorders, and the current study findings were in accordance with previous studies [26, 28–30, 33]. In a mouse model of traumatic brain injury, the peaked expression of Fas was noticed in the cortex and hippocampus 24 hours after the injury . Furthermore, Both the level of Fas and FasL in cortical neurons and astrocytes were sustained for up to 72 hours after injury . Fas-mediated apoptosis of neurons occurred in mouse models of acute and subacute SCI and reduced apoptosis and neurological dysfunction were detected in Fas-deficient mice compared with control mice after SCI .
The extrinsic pathway of apoptosis plays a critical role in the induction of apoptosis in non-neuronal cells, especially in the acute phase. Previous studies demonstrated the effects of extrinsic pathway of apoptosis in acute injury of lung, liver, heart, and kidney, and acute parasite infection. Tiao et al. investigated the protective role of microRNA-29a in acute liver injury in a mouse model of obstructive jaundice, mediated at least partially by modulating the extrinsic rather than intrinsic pathway of apoptosis . Furthermore, CD95 (AP0-1/Fas)-mediated apoptosis was shown to play an important role in the pathogenesis of fulminant hepatic failure in acute Wilson's disease . FADD deletion attenuated cardiomyocyte death and improved cardiac function in acute myocardial ischemia/reperfusion injury . Recent study reveals that the reduced Fas and FasL expression in CD4 + T cells was associated with let-7 microRNA expression, which significantly inhibited the apoptosis of these cells, and improved cell survival rates in patients with acute coronary syndrome . Fas/FasL signaling mediated the pathogenesis of acute ischemic kidney injury via tubular apoptosis and necrosis, suggesting that the modulation of Fas/FasL system can be an effective therapeutic target in ischemic acute kidney injury .
While most of the studies elucidating the possible mechanisms underlying the neuroprotective effect of EE intervention focused on the early phase of stroke, the importance of neuronal survival in the relatively late phase has been neglected. The current study found that delayed exposure to EE reduces infarct volume and DNA damage in brain cells, and thereby improved neurobehavioral function. This result is consistent with a previous study, which demonstrated sustained functional recovery following exposure to EE 5 days after onset, resulting in significant survival of hippocampal newborn cells in a rat stroke model . Moreover, exposure to EE for 10 days strongly rescued the diabetic brain from neurodegenerative progression . However, our study demonstrated that delayed exposure to EE does not significantly suppress apoptosis.
Limitation of this study is that the diverse potential mechanisms of neuronal cell death contributing to long-term neuroprotection and functional recovery after stroke were not investigated, given the complexity. Furthermore, EE positively contributes to improved behavioral recovery after stroke in young animals. A recent study reported that EE had a limited benefit on behavioral recovery of older rats compared with young rats . Since stroke primarily affects mostly the elderly patients, the neuroprotective effects demonstrated in this study may not precisely be applicable to aged subjects. Additionally, ischemic stroke in humans occurs preferentially in patients of both sexes carrying multiple comorbidities requiring various treatments with complex interactions. In contrast, our study involved young, healthy, male inbred mice housed under ideal conditions. Therefore, it is highly desirable to investigate the effects of hyperacute exposure to EE in an aged animal model including both male and females with multiple comorbidities to demonstrate the clinical relevance to stroke rehabilitation. Although the mechanism of EE-induced inhibition of neuronal apoptosis requires further investigation, our study suggests that Fas/FasL-mediated apoptosis may be an important target underlying the neuroprotective effects of very early EE treatment after HI brain injury, contributing to improved emotional, cognitive, and locomotor performance post-stroke.
In summary, we demonstrated that early exposure to EE can induce greater improvement in behavioral outcomes, reduced infarct volume, and decreased neuronal death. It significantly downregulated Fas/FasL-mediated apoptosis and decreased expression of pro-apoptotic proteins in cerebral cortex and hippocampus. Overall, the results of this study demonstrates that very early exposure to EE is a promising neuroprotective candidate for therapeutic translation after stroke by effective inhibition of extrinsic as well as intrinsic apoptotic pathways.