Mitophagy in Cerebral Ischemia And Ischemia/Reperfusion InjuryⅡ

PATHOPHYSIOLOGY OF ISCHEMIC-REPERFUSION INJURY (FIGURE 2) 

Clinical Classification of Ischemic Stroke 

Ischemic stroke, also known as cerebral ischemia, is a significant type of all stroke case. This disease occurs when blood clots or plaques block or narrow the brain arteries. Depending on the pathological condition, ischemic stroke can be divided into several subtypes: Intracranial arterial stenosis, acute arterial occlusion, and chronic arterial occlusion. Intracranial arterial stenosis refers to the narrowing of arteries caused by the formation of fatty deposits called atherosclerotic plaques and the concurrent thickening of vessel walls. 

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In Intracranial arteries, including middle cerebral arteries, basilar artery, carotid arteries, and intracranial vertebral arteries, narrowed blood vessels can significantly reduce blood flow, leading to an ischemic event (Chimowitz et al., 2005; Banerjee and Chimowitz, 2017). A systematic analysis focusing on the role of intracranial atherosclerosis in ischemic stroke indicates that atherosclerosis-inducing stenosis graded higher than 30% can be a cause of fatal brain infarction (Mazighi et al., 2008). The atherosclerotic plaque is thrombogenic. Once its cap is ruptured, an unstable clot can be formed to narrow or completely occlude the arteries. The blood clot that blocks the affected site can form locally or originate elsewhere, such as in the heart, and embolize through the circulatory system. 

Rupture of plaques and clot embolisms is usually linked with acute arterial occlusion, manifesting stroke symptoms within hours (Malhotra et al., 2017). The occlusion can also be chronic (lasting more than 4 weeks) if the brain alters the cerebral hemodynamics and compensates for the blood flow by building collateral circulation in response to the reduced arterial blood supply (Sundaram et al., 2017). In that case, with sufficient collateral compensation, the disease can be asymptomatic and benign (Powers et al., 2000); Chronic occlusion without enough compensation from collateral circulation might still result in chronic cerebral hypoperfusion, leading to ischemic infarction. In some cases, patients with chronic occlusion may spontaneously recanalize over a long time (more than 3 months) (Delgado et al., 2015).

Management of Ischemic Stroke 

Thrombolytic agents and recanalization procedures are developed as reperfusion strategies to recover the blood flow in affected arteries. Usually, different therapeutic approaches are given to these three subtypes of stroke in the clinical setting. Due to technical limits, severe stenosis and acute occlusion of arteries are difficult to distinguish accurately (Clevert et al., 2006). Still, the correct diagnosis can be beneficial for optimal treatment and a better prognosis. Intravenous thrombolysis is the only approved therapy for AIS patients and can be given within 3 h of symptom onset. However, clinical outcomes of thrombolytic medical treatment alone for patients with severe stenosis and occlusion have shown worse than expected prognosis and less efficacy (Mokin et al., 2012). 

Clinical trials focusing on the lysis of clots suggest that intravenous thrombolytic therapy alone has a low recanalization rate of only 30–40% among patients (Chen et al., 2012). Another analysis of clinical outcomes of intravenous thrombolysis for internal carotid artery occlusion suggests the rate of favorable outcomes is 25% (Mokin et al., 2012). Revascularization treatments, including stenting or endarterectomy, have thus been advised for patients with moderate or severe stenosis. Compared with intravenous thrombolysis, thrombectomy recipients have a significantly reduced incidence of ipsilateral stroke, meaning a better prognosis. Arterial therapies also achieve a better outcome in patients with acute occlusion (Mokin et al., 2012). However, in the clinical setting, endarterectomy is not considered an option by many in treating complete ICA occlusion since this operation is still technically challenging to perform in preventing postoperative thrombus generation and maintaining a good prognosis (Kao et al., 2007; Chen et al., 2012; Faggioli et al., 2013). 

Until now, the search for effective treatments for chronic occlusion continues. Medical treatments like anti-platelet aggression drugs or intravenous tissue plasminogen activators can be given to patients to decrease the risk of stroke. Surgical approaches like endarterectomy and stenting can also be used in treating chronic occlusion, though they still show some seeming drawbacks. Like in acute occlusion, endarterectomy might fail in cases with complex clot organization, and the success rate of recanalization only achieves 40% in patients with chronic occlusion (Thompson et al., 1986; Xu et al., 2018). Hypoperfusion still occurs in patients who have failed to restore blood flow in recanalization therapies, which is presumed to result in the recurrence of ischemic events (Grubb et al., 1998). Also, in the process of stenting, the clot may detach when the stent is released, blocking the intracranial artery and may therefore cause post-operative complications (Xu et al., 2018). 

Ischemia-Reperfusion Injury 

In patients receiving recanalization therapies, sudden restoration of blood flow can sometimes be harmful, leading to the so-called ‘reperfusion injury.’ I/R injury refers to the tissue reoxygenation injury caused by the sudden return of blood supply to formerly ischemic or anoxic tissues. During the ischemia phase, the blood supply below standard functional requirements will cause deficiencies in oxygen and nutrients, leading to metabolic disturbances (Irie et al., 2014) and inflammatory response (Jin et al., 2013) in affected areas. Restoration of blood flow thus has been considered a fundamental treatment to preserve tissue function. Loads of research and clinical trials on reperfusion treatments have shown that reperfusion therapies, including intravenous thrombolytic agents and endovascular interventions like mechanical thrombectomy, are relatively safe and can help with the recovery of acute ischemic stroke (AIS) patients when given inside a narrow time window (Kwiatkowski et al., 1999; Lees et al., 2010; Berkhemer et al., 2014; Jovin et al., 2015). 

However, reperfusion might also cause secondary injury in the previously ischemic tissues since the resupply of nutrients and oxygen can trigger considerable ROS production and accumulation and meanwhile alters calcium homeostasis, resulting in excessive oxidative stress and local inflammation. Such cellular changes cause cell damage and may activate the cell death pathway in the former ischemic tissues.

Process and Mechanisms of I/R Injury (Figure 2) 

Excessive Oxidative Stress Plays a Critical Part in I/R Injury

Oxidative stress is a disturbance in the balance between free radicals and antioxidant ability, and it often occurs when the production of ROS surpasses antioxidant defense. In the ischemic stage, obstructed blood flow with less oxygen and nutrient supply induces a shift in mitochondrial metabolism from aerobic to anaerobic, thus producing a lower concentration of ATP and antioxidative agents in cells. Later return of blood flow to the ischemic tissue can cause the reactivation of mitochondrial aerobic respiration and thus increase the production of ROS. Because of the decreased level of antioxidative agents, oxidation exceeds antioxidation during the reperfusion period, thus causing increased oxidative stress. 

Enzyme systems, including the xanthine oxidase system, the NADPH oxidase system, the nitric oxide (NO) synthase system, and the mitochondria electron transport chain, are involved mainly in the occurrence of oxidative stress. In normal cells, purine metabolism initiates from converting ATP to inosine with the participation of deaminases and nucleotidases, followed by its further transformation into hypoxanthine. Oxidation of hypoxanthine to xanthine and xanthine to uric acid later occurs, and xanthine dehydrogenase (XDH) and xanthine oxidase (XOD) separately function in these two oxidation processes. XDH utilizes NAD+ as an electron acceptor to produce NADH, and the ischemia state can induce its shift to XOD which uses O2 as an acceptor (Kinuta et al., 1989). Restoration of blood flow and oxygen can stimulate the oxidation process in purine metabolism. Since the level of XOD is previously promoted, the formation of uric acid in the reperfusion phase is accompanied by the production of highly reactive superoxide anion (O2−). Superoxide can be later shifted to hydrogen peroxide (H2O2) and the hydroxyl radical (OH•), which further stimulates oxidative stress and causes damage. NADPH oxidases are the primary source of ROS. They oxidize NADPH to NADP+ and deliver electrons to O2, thus generating superoxide or H2O2. 

The Nox/Duox family of NADPH oxidases has been reported to involve in ROS production during I/R injury by their facilitated activity (Wang et al., 2006; Simone et al., 2014). Nox2 has been a focus in I/R injury that occurs in stroke. Nox subunit-deficient mice and mice with apocynin (a Nox2 inhibitor) pretreatment show remarkably decreased infarct volume and improved clinical outcome of stroke (Chen et al., 2009; Jackman et al., 2009), suggesting that Nox-induced ROS plays a considerable role in I/R injury. Besides immediately producing ROS, NADPH oxidases are also regulating ROS production by stimulating the NO synthase system. NO, also known as an endothelium-derived relaxing factor, is made from L-arginine by nitric oxide synthase (NOS) of three sources: Neuron NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). The role of NO is variable: It generally works as an anti-oxidant agent, but its interaction with the superoxide anion can lead to the formation of the peroxynitrite (ONOO−) (Marla et al., 1997). ROS created by NADPH oxidases can oxidize tetrahydrobiopterin (BH4), an essential cofactor that mediates eNOS activity. BH4 oxidation later induces the uncoupling of eNOS, resulting in decreased NO production and increased ONOO− production from eNOS (Landmesser et al., 2003). 

Mitochondria are the major site of oxidative stress generation, action, and injury. ROS can be generated from ETC. In ischemia, cellular stress can induce post-translational modifications of oxidative phosphorylation proteins in ETC, making them more sensitive to reoxygenation (Prabu et al., 2006). Disrupted ETC complexes can result in higher mitochondrial membrane potentials, positively associated with more ROS generation (Prabu et al., 2006). Enhanced oxidative stress can target mitochondria and further damage ETC, causing more ROS generation (Indo et al., 2007) subsequently. ROS from exogenous origins and mitochondrial ROS generation can lead to mitochondria DNA damage (Indo et al., 2007). In addition, too much oxidative stress can cellular damage or death (Figure 2).

Calcium Overload: Another Disturbance in Ischemia-Reperfusion Injury 

In addition to oxidative stress caused by different sources, calcium overload, and abnormally increased intracellular Ca2+ level is other major pathological that plays an important role in reperfusion injury. Anaerobic respiration in ischemia decreases intracellular pH; thus, the Na+/H+ exchanger (NHE) allows for the influx of Na+ to maintain the pH. NHE is generally inactivated during ischemia, but its activity can be increased during reperfusion, leading to a large Na+ influx (Allen and Xiao, 2003). A lower level of ATP in ischemia also weakens the activity of the energy-dependent Na+ pumps, resulting in a higher level of intracellular Na+. 

A study in 1987 suggested that the precedent sodium imbalance be a cause of calcium overload using an energy-repleted Na+ loading model (Grinwald and Brosnahan, 1987). Failing to return to normal Na+ balance upon oxygen restoration can promote the function of Na+/Ca2+ exchanger (NCX) that is sensitive to intracellular Na+ level, thus leading to higher Ca2+ influx. Calcium overload is also induced by elevated Ca2+ release and limited Ca2+ uptake from an internal source, including the endoplasmic reticulum (ER) or Golgi apparatus (Chami et al., 2008). Promoted uptake of Ca2+ by mitochondria later occurs following cytosolic calcium overload (Brookes et al., 2004). Cytosolic and mitochondrial calcium overload can cause cellular damage in various ways, including disrupting mitochondrial function (Wang M. et al., 2015), promoting ROS production (Zhu et al., 2018), and inducing cell death (Boehning et al., 2004; Zhu et al., 2018) (Figure 2).

Mitochondria-Dependent Cell Death in I/R Injury 

Cellular alternations, including increased oxidative stress and calcium overload, can lead to apoptosis with the involvement of mitochondria. This process is initiated by changes in mitochondrial membrane permeability controlled by the mitochondrial permeability transition pore (mPTP). The activity of mPTP is likely to be mediated by mitochondrial matrix Ca2+ level, and the mitochondrial calcium overload resulting from cytosolic calcium overload can facilitate the opening of mPTP (Qian et al., 1999). ROS production during I/R injury, especially hydroxyl radicals and hydrogen peroxide, have also been found indispensable in mPTP opening (Assaly et al., 2012). The permeabilized membrane allows for the activation and insertion of pro-apoptotic Bcl-2 family members BAX and BAK into the mitochondria membrane (Wei et al., 2000; Kirkland et al., 2002).

 This helps with transferring mitochondrial proteins including cytochrome c from mitochondria to the cytosol, followed by the interaction between cytochrome c and two cofactors, apoptotic protease activating factor 1 (APAF-1) and pro-caspase-9, to form the apoptosome, which eventually activates caspase–9-caspase-3 signaling cell death pathway with proteolytic events and DNA fragmentation (Broughton et al., 2009). This pathway is referred to as the caspase-dependent apoptotic pathway. Another cell death pathway, caspase-independent apoptosis, can be activated when cellular energy is running out (Daugas et al., 2000). Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that locates upstream of the pathway (Yu et al., 2002). 

ROS-induced DNA damage can trigger PARP-1 overactivation, in which NAD+ is used, thus depleting energy storage. Yu et al. (2002) also found that PARP-1 activation can lead to the release of its downstream target apoptosis-inducing factor (AIF, a mitochondrial flavoprotein) from the mitochondrial intermembrane to the nucleus, causing chromatin condensation and large-scale DNA fragmentation. Studies have indicated that AIF does not have a direct DNA fragmentation effect (Susin et al., 1999; Wang et al., 2002). Thus, it probably needs a downstream effector during this process. Studies have suggested that endonuclease G might interact with AIF and cause DNA fragmentation (Wang et al., 2002; Lee et al., 2005), though their interaction is still unclear. PARP-1-induced cell death is a unique cell death pathway. It generally exhibits characteristics of apoptosis, and it is also considered necrotic by some researchers since classic apoptosis is energy-dependent (Ha and Snyder, 1999).

The Brain Is Susceptible to I/R Injury 

I/R injury can occur in many organs and tissues, including the brain, heart, skeletal muscles, and kidneys. Some common features are shared by I/R injury in these areas, including the elevated production of ROS, calcium overload, inflammation, and the opening of mPTP. Yet, organ-specific characteristics can affect the severity of I/R injury in different organs. The brain, the organ where irreversible damage occurs within 20 min after ischemia and a narrow time window (generally 3–4.5 h) can be given for reperfusion therapy, is considered very susceptible to I/R injury (Ordy et al., 1993). 

ROS in the brain is mostly generated from mitochondria rather than other enzymatic ROS sources as a metabolically active area. The brain accounts for more than 20% of total body oxygen consumption but with a relatively low antioxidative agent level compared with other organs, making it vulnerable to oxidative stress (Markesbery and Lovell, 2007; Damle et al., 2009; Kalogeris et al., 2012). Moreover, accumulated labile iron in the brain can react with H2O2 to produce highly reactive •OH. This reaction stimulates the oxidation and peroxidation of massively accumulated polyunsaturated fatty acid in the brain, causing even more oxidative stress (Ferretti et al., 2008). Because of the brain’s susceptibility to I/R injury, finding targets to prevent reperfusion injury to the brain is significant in treating stroke.

Extending the Therapeutic Time Window in Ischemic Stroke: Delayed Recanalization 

Successful recanalization of the occluded vessel as early as possible has been widely accepted as the vital principle of AIS treatment. Unfortunately, for many years, most AIS patients were prevented from receiving effective recanalization therapy because of a narrow therapeutic window. In recent years, a series of clinical trials have indicated that delayed recanalization may still have benefits in ischemic brains during an expanded therapeutic window, up to more than 24 h, several days, and even more than 1 month after symptom onset [Reviewed by Kang et al. (2020)]. Clinically, advances in imaging techniques have allowed better characterization of brain tissue and vessel status in AIS. Markers of brain ischemia are instructed by perfusion-weighted imaging/diffusion-weighted imaging (PWI/DWI) mismatch and DWI/fluid-attenuated inversion recovery (DWI/FLAIR) mismatch on magnetic resonance imaging (MRI). 

MRI scanning with PWI or computed tomography (CT) perfusion (CTP) scanning shows different hypoperfusion levels. Given these developments, together with advances in intravascular interventional devices, expanding the recanalization time window in certain patients is possible. Increasing randomized studies have demonstrated that delayed recanalization has beneficial effects on 90-day outcomes. Two high-quality, randomized controlled clinical trials (DAWN and DEFUSE 3) of endovascular mechanical thrombectomy reported that selective delayed recanalization based on imaging mismatch improved patients’ 90-day outcomes, even when performed at 16–24 h after symptom onset (Ragoschke-Schumm and Walter, 2018). In summary, despite the risk of I/R injury, which might increase with the delayed time point for recanalization, delayed recanalization is still beneficial for a certain subtype of patients.

Cistanche neuroprotection effect

Cistanche is a plant extract known for its neuroprotective properties, and its mechanism of action is believed to involve antioxidant, anti-inflammatory, and antiapoptotic effects. There are several relevant tests and application cases related to the neuroprotective effects of Cistanche, which include:

1. In vitro studies: In vitro studies have shown that Cistanche extract protects neurons from stress-induced damage by reducing oxidative stress and inflammation.

2. Animal studies: Animal studies have demonstrated that Cistanche can protect against neuronal damage caused by cerebral ischemia, traumatic brain injury, and neurotoxin exposure.

3. Human studies: There is limited clinical evidence on the neuroprotective effects of Cistanche in humans, but some studies have suggested that it may improve cognitive function and reduce age-related decline in memory.

Luoan Shen1†, Qinyi Gan1†, Youcheng Yang1, Cesar Reis2, Zheng Zhang1, Shanshan Xu3, Tongyu Zhang4 * and Chengmei Sun1,3 * 

1 Zhejiang University-University of Edinburgh Institute, School of Medicine, Zhejiang University, Haining, China, 

2 VA Loma Linda Healthcare System, Loma Linda University, Loma Linda, CA, United States, 

3 Institute for Advanced Study, Shenzhen University, Shenzhen, China, 4 Department of Neurosurgery, Xuanwu Hospital, Capital Medical University, Beijing, China