CA/ROSC model can be established by fibrillation, asphyxia and potassium chloride induction. Previous studies have shown that various modeling methods differ in pathophysiology and the degree of damage to individual organs. Based on the purpose of this study to investigate PCABI, an 8-minute asphyxiation cardiac arrest model was chosen. The rats were subjected to systemic hypoxia after asphyxia, and the heartbeat gradually stopped after 3 minutes, and the blood pressure dropped below 20 mmHg, followed by complete arrest for about 5 minutes and cessation of systemic blood perfusion. After cardiopulmonary resuscitation, blood circulation is restarted, resulting in reperfusion injury. The neurological deficit score showed that the CA group had a significantly lower score than the Sham group. He staining results showed significant cell death in the CA1 area of hippocampus, indicating that the rats had severe neurological impairment after CA/ROSC, which was in line with previous studies and suggested that the model was stable and reliable.
Apoptosis, a form of programmed cell death, plays a critical role in central nervous system diseases. Studies have reported that that Caspase-3 and TUNEL-positive cells were increased in rat cerebral ischemia-reperfusion models[44, 45]. Li et al[46] discovered that apoptosis was increased in rats undergoing CA/ROSC compared with the Sham group, and previous research also showed that apoptosis in brain tissue after CA/ROSC was significantly increased[47]. In this study, the expression of Caspase-3 and Bax in rat hippocampal tissues was increased 12–72 hours after ROSC, and the expression of Bcl-2 decreased, and the of neurological deficit score decreased, illustrating that apoptosis occurred in the hippocampal after CA/ROSC, causing neurological injury.
Autophagy, also known as type II programmed cell death, is often thought to be an endogenous protective mechanism that promotes cell survival. The autophagic process is a continuous cytological behavior that is artificially divided into three phases: initiation stage, autophagosome formation stage, autophagy lysosome formation and cleavage. Bcelin-1 and LC3-II are generally used as molecular markers for autophagy for their key role in the formation and extension of autophagic lysosomes[48]. On the one hand, autophagy activation will clear the damaged organelles, provide raw materials and energy for cell reconstruction[49], on the other hand, if autophagy is overactivated, it will cause apoptosis or even necrosis, resulting in irreversible damage. Current research points that autophagy may have different functions in different diseases and in different processes of disease[50] and Studies have shown that autophagy is elevated during cerebral ischemia-reperfusion[37, 51]. However, changes of autophagy after CA/ROSC remain unclear. This study showed that Caspaes-3, Bax, and Beclin-1 and LC3-II all increased, and Bcl-2 decreased, indicating that apoptosis and autophagy in hippocampus increased after CA/ROSC. These findings suggest that neurological impairment after experiencing cardiac arrest may be related to the activation of autophagy.
In 2012, Dixo et al. [25] found ferroptosis, and recent studies have reported an important connection between autophagy and ferroptosis. Hou et al. [52] reported that knocked down ATG5 and ATG7 could decrease intracellular free iron levels and LPO end products (such as MDA) levels significantly, while increase the expression of the stable ferritin marker FTH1, which revealed the genetic relationship between autophagy and ferroptosis. Li et al.[53] further confirmed that I/R injury induced excessive degradation of ferritin via the autophagy pathway and subsequent increased of free iron in neurons, thereby promoting the accumulation of LPO in cells, and then ferroptosis occurred. This special autophagic cell death is called ferritinophagy.
Apoptosis, ferroptosis and autophagy are programmed regulatory mechanisms in cells, and there is a close relationship among them which is controversial in cardiac arrest models. The results showed that the autophagy inhibitor 3-MA reduced apoptosis and ferroptosis in the hippocampus of CA rats, and attenuated brain injury, while the autophagy agonist rapamycin did not. Those results demonstrate that overactivation of autophagy may lead to apoptosis and ferroptosis, whereas inhibition of autophagy may attenuate apoptosis and improve neurological function. Previous studies [54] and the results of this study have demonstrated that overactivation of autophagy could provoke apoptosis and ferroptosis, and inhibition of autophagy could alleviate PCABI.
Liraglutide, a synthetic long-acting analogue of GLP-1 with a half-life of up to 13 hours, has glucose-dependent hypoglycemic effect and repair of pancreatic beta cells, which can effectively control blood glucose while reducing body weight, and has been increasingly used in the treatment of type 2 diabetes[55]. It has been found that liraglutide can pass through the blood-brain barrier and exerts a protective effect in neurological diseases. Deng et al[56] in a study exploring the effects of liraglutide pretreatment on focal cerebral ischemic injury in diabetic rats, discovered that the liraglutide group had a decrease in neurological deficit score and cerebral infarction volume 48 hours after cerebral ischemia compared with the model group. Kong et al.[57] in vitro experiments, found that liraglutide pretreatment can prevent cognitive impairment caused by diabetes, alleviate neuronal damage and synaptic ultrastructural damage in the CA1 region of the hippocampus. Timper et al.[58] found that astrocytes in different brain regions express the GLP-1 receptor, and demonstrated a role for astrocyte GLP-1R in maintaining mitochondrial integrity, whereas astrocytes lack GLP-1R Impairs systemic glucose homeostasis and memory formation. Although liraglutide has been shown to reduce neurological injury in multiple models, there is no evidence that liraglutide can reduce brain injury after cardiac arrest. This study revealed that liraglutide could significantly improve the 7-day survival rate and neurological deficit score, inhibit cell apoptosis and ferroptosis, and reduce TNF-αand IL-1β expression in serum and hippocampus of CA rats. The study found that liraglutide could reduce autophagy and ferroptosis in the hippocampus of CA rats indicating that liraglutide may exert anti-apoptotic and anti-inflammatory effects by inhibiting autophagy and ferroptosis. To further verify the relationship between the neuroprotective effect of liraglutide and autophagy, rapamycin was used to activate autophagy in this study, The results showed that the protections of liraglutide were counteracted after activation of autophagy, suggesting that the neuroprotective effect of liraglutide on CA rats was related to the inhibition of autophagy and ferroptosis.
This study also has limitations: Neurological impairment after cardiac arrest can last for several years, or even permanent, the observation time in this study was short, and further research is needed in the future. This study is a basic experiment, and whether liraglutide has a similar effect in patients with clinical cardiac arrest remains to be further studied. As a GLP-1 receptor agonist, the neuroprotective effect of liraglutide may be mediated by the GLP-1 receptor pathway. Current studies have shown that liraglutide can also exert its effects through other pathways, which were not studied in this study.