In this study, we found that inhalation of 2% ISO for 1 hour before and after hypoxic-ischemic brain damage in neonatal rats significantly promoted neuronal regeneration, inhibited neuronal apoptosis, and improved motor and cognitive function, and ISO pretreatment inhibited neuronal cell death in the cerebral cortex, CA1, CA2, CA3, and DG of neonatal rats; that is, it improved acute and long-term neurological deficits induced by hypoxic-ischemic brain damage in neonatal rats.
Previous studies have successfully established neonatal HI models using Rice-Vannucci method and confirmed the degree of neurological and motor impairment of HI rats. In this study, we applied a lot of behavioral experiments to determine the acute as well as long-term neurological defects after HI injury. Among them, WMW and Y-maze were usually carried out to investigate the learning and memory abilities(Potschka et al., 2000; Meirsman et al., 2016), and Rotarod test were used to observe the exercise and coordination function of rats(Balduini et al., 2000; Chu et al., 2004). Through these tests, we found that the Morris water maze and Y-maze test were strongly corresponded regarding the activities of neurological improvement. Behavioral evaluation can be used to assess long-term behavioral changes in HIE rats. Behavioral assessment of rats 1 month after HI injury further determined the efficacy of ISO for long-term neuroprotection. The experimental results showed that ISO reduced the severity of long-term neurological injury, improved long-term spatial learning and memory abilities, and increased motor coordination in HI rats. the HI group showed severe learning and memory impairment, and the littermates in the Pretreatment group were not significantly different from those in the sham group, show that ISO pretreatment can protect neurocognitive function. Our results are consistent with the study by Shao et al.(Shao et al., 2006). Animals in the HI group had more pronounced pathological damage to brain morphology, and these findings confirm previous studies showing a correlation between learning impairment and ipsilateral hemispheric and hippocampal tissue loss(Ten et al., 2004; Cengiz et al., 2011).
An increasing number of studies have shown that HI causes irreversible brain damage to the developing brain of newborns, resulting in long-term neurological deficits. Microvascular responses and blood-brain barrier damage in neonatal HI models lead to brain injury(Wang et al., 2016). HE and Nissl staining can usually calculate the degree of injury and the number of necrotic neurons. The hippocampus and cortex are very sensitive to ischemia-hypoxia injury(Li et al., 2017a), so in this study, the above regions were selected to observe the morphological changes of neurons. At 24 h after HI, the neurons of hippocampal CA1、CA3 and cortex in the HI group showed significant morphological damage, with loss of neurons and Nissl bodies, pyknotic nuclei, and vacuolization. The results of Nissl staining clearly showed that HIE induced extensive apoptosis in the ipsilateral hemisphere, which was effectively prevented by conditioning after ISO preconditioning, and the results of Galle et al were consistent(Galle and Jones, 2013), further confirming that ISO preconditioning has a neuroprotective effect. Pathologically, ISO pretreatment alleviated neuronal edema and necrosis, irregularly arranged Nissl bodies, incomplete cellularity, and inflammatory cell infiltration occurring in the cerebral cortex. The number of Nissl positive cells in the cortex and hippocampus increased after ISO pretreatment, indicating that ISO inhibits neuronal cell death in HI rats. Bauer, T.M.et al.(Galle and Jones, 2013) it was confirmed that ISO postconditioning had a protective effect on hypoxic-ischemic brain damage in neonatal rats, and this protective effect may be related to the inhibition of the opening of mitochondrial permeability transition pore (mPTP).Opening of mPTP can lead to impaired energy synthesis and cellular oxidative response, triggering a cascade, resulting in decreased ATP levels, while the increase in intracellular Ca2+ concentration is closely related to ATP levels(Galle and Jones, 2013; Bauer and Murphy, 2020).It is generally accepted that the elimination ability is reduced by massive release of glutamate during ischemia and hypoxia, the extracellular glutamate concentration is greatly increased to toxic levels, glutamate receptors are hyperactivated, and massive influx of Ca2+ leads to cell swelling and apoptosis, and this process is mainly mediated by AMPA receptors(Henley et al., 2011). It has been documented that administration of AMPA receptor antagonists to neonatal rats with hypoxic-ischemic brain damage alone can reduce the inflammatory response and peroxide levels in brain cells and reduce the degree of brain injury(Bauer and Murphy, 2020). The excitatory neurotransmitter AMPA, on the other hand, activates AMPA receptors and causes changes in their configuration resulting in damaging effects, and it has also been shown to cause brain tissue damage. The results of this experiment showed that ISO pretreatment in neonatal rats with hypoxic-ischemic brain damage significantly improved the degree of brain injury, and the number of normal neurons in the hippocampus of the right brain was significantly increased at 1 d of ischemia and hypoxia.
Through correlation analysis between behavior and morphology, we found that there was a strong correspondence between WMW and Y-maze had a strong corresponding relationship in the improvement of nerve activity, especially a significant negative correlation between WMW and Rotated various indicators. Thus, the behavioral difference between WMW and Y-maze can be used as a behavioral assessment reference for brain injury. These results all reveal that ISO preconditioning can improve spatial learning and memory abilities, as well as long-term motor function with positive effects.
ISO is an inhalation anesthetic with certain analgesic and muscle relaxant effects. There is no contraindication for its use. It is applicable to various types of intraoperative anesthesia. It has a close relationship with the biological behavior of malignant tumor histiocytes such as colorectal cancer, prostate cancer, and glioma(Hu et al., 2018; Liu et al., 2019).A large number of studies have reported that low-dose ISO has antioxidant, anti-apoptotic and immunomodulatory effects, and has a good protective effect on various acute central nervous system injuries such as cerebral ischemia-hypoxia injury and reperfusion injury(Zimin et al., 2018).In addition, some studies have confirmed that ISO can regulate the activation of NF-KB signaling pathway(Li et al., 2008; Yao et al., 2020), and ISO or multiple treatments during development can affect spatial cognitive behavior in rats as adults(Katiyar et al., 2018). Previous studies(Li et al., 2017b; Agnic et al., 2018) have shown that ISO may contribute to postoperative cognitive dysfunction in elderly patients by eliciting neuroinflammation, disrupting choline function, and synaptic plasticity. It can induce postoperative cognitive dysfunction and promote neuronal apoptosis. Recent studies(Yang et al., 2020)have shown that ISO can alleviate the inflammatory factor analysis and the degree of lipid peroxidation in rats with cerebral ischemia/reperfusion injury through TGF-beta1/Smad2/3 Signaling Pathway, reduce the hydrolytic activity of matrix metalloproteinases in brain tissue, reduce tight junction protein loss, and improve cerebral ischemia-reperfusion injury in rats. The in vitro studies(Yan et al., 2016), demonstrated that ISO preconditioning reduced the release of OGD-induced lactate dehydrogenase (LDH) and enhanced the OGD-inhibited cell viability. It has also been observed that the hypoxia inducible factor-1α (HIF-1α) was increased under ISO preconditioning. In fact, these results thus suggest that the ISO preconditioning may provide potential neuroprotection against Cerebral ischemic/reperfusion injury via up-regulating the HIF-1α expression through the Akt/mTOR/s6K activation. McAuliffe et al. (McAuliffe et al., 2007)studied the long-term effects of delayed preconditioning with Iso, hypoxia, or room air on motor and cognitive function in mice subjected to hypoxia-ischemia for 65 minutes at postnatal day 10. Delayed preconditioning of Iso and hypoxia in neonatal mice improved learning and memory with functional neuroprotection. Xiong et al. (Xiong et al., 2003) study demonstrated that repeated Iso anesthesia induces ischemic tolerance in rats in a dose-response manner. GLB, an adenosine triphosphate-regulated potassium channel blocker, abolished the tolerance induced by Iso. Brief isoflurane anesthesia induces ischemic tolerance in the brain. The effect was found to be dose dependent in a rat focal cerebral ischemia model. Ischemic tolerance induced by isoflurane preconditioning is dependent on activation of adenosine triphosphate-regulated potassium channels. The effects of ISO on the nervous system are wide-ranging and complex, so active studies of the mechanism of action of ISO on the nervous system are helpful to better understand the effects.
HIE is a common neonatal disease in clinical practice at present, and the pathogenesis includes mitochondrial damage, oxidative stress, neurotoxicity of excitatory amino acids, and inflammatory immune response due to the results of a combination of cellular and molecular mechanisms. Its occurrence is mainly related to perinatal asphyxia and plays an indispensable role in perinatal neurological diseases, which can cause brain tissue hypoxia, interruption or reduction of cerebral blood flow, brain injury in newborns, neurological injury, and severe death in children(Schump, 2018; Narayanamurthy et al., 2021).There is increasing evidence that HI causes irreversible brain damage to the developing brain, leading to long-term neurological deficits. The incidence of neurological sequelae is relatively high, resulting in an increased incidence of disability, and the earlier this type of disease, the better the therapeutic effect. At present, mild hypothermia therapy is often used to treat moderate and severe full-term HIE children in clinical practice, which has a regulatory effect on the body's cerebral blood flow and can reduce neuronal apoptosis and relieve neurological symptoms such as disturbance of consciousness and floppy limbs in children(Brekke et al., 2014).It is undeniable that hypothermia treatment is recognized as an effective treatment for HIE(Saw et al., 2019; Finder et al., 2020).However, its clinical application is limited due to the short treatment window and other reasons. In addition, the pathogenesis and clinical characteristics of HIE are complex. Current studies have shown that drugs combined with hypothermia are more effective in the treatment of HIE(Lin et al., 2014; Landucci et al., 2018; Barata et al., 2019). In order to make the neuroprotective effect of HIE more clinically applicable, combined hypothermia treatment needs to be further considered and explored to achieve more targets and enhance neuroprotection. There is a lack of drugs for the treatment of HIE in clinical practice, and it is still urgent to find safe and effective drugs from natural products.
The main finding of this study was that ISO pretreatment not only protected neonatal rats with HIE from brain injury, but also promoted their neurological recovery but also improved their learning and memory, possibly by inhibiting cell death in the cortex and hippocampus after HI. After ISO preconditioning, motor function will be restored to reduce brain injury and improve neurological function provides a new idea. It is worth noting that the specific mechanism of the protective effect of ISO pretreatment has not been fully clarified, and in the next step we will further study it in depth.