Brief Inhalation Of Sevoflurane Can Reduce Glial Scar Formation After Hypoxic-ischemic Brain Injury in Neonatal Rats Part 3

SPC upregulates HIF-1α expression in the hippocampus

In a previous study, we found that sevoflurane post-conditioning attenuated astrogliosis and glial scar formation in the hippocampus, and improved learning and memory impairment after HI. 

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HIF-1α was confirmed as a key factor in this neuroprotective mechanism (Yang et al., 2019). To further investigate the specific regulation induced by sevoflurane, we examined HIF-1α protein expression in the hippocampus 12, 24, and 48 hours after SPC. Our results revealed dynamic changes in HIF-1α expression: 12 hours after treatment, increased HIF-1α expression was observed in HI and HIS groups compared with the sham group (P < 0.0001), but there were virtually no differences between HI and HIS groups (Figure 7A and B). 

Twenty-four hours after SPC, HIF1α expression was decreased in the HI group compared with the HIS group (P < 0.001) but still increased compared with the sham group (P < 0.001; Figure 7C). 

Forty-eight hours after SPC, HIF-1α expression in the HI group returned to a very low level and was not different from the sham group; however, HIF-1α expression was still sustained at a very high level in the HIS group (P < 0.01; Figure 7D). Next, we examined the expression of HIF-1α mRNA (relative to GAPDH) in the hippocampus 12, 24, and 48 hours after SPC by real-time PCR analysis. 

The results revealed that HIF-1α mRNA expression peaked 24 hours after HI (P < 0.001; Figure 7E), and there were no obvious differences between HI and HIS groups at the three-time points examined. Thus, the dynamic regulation of HIF-1α induced by sevoflurane probably involved post-translational regulation, which is in line with our hypothesis.

To further investigate our hypothesis that SPC reduces astrogliosis after HI injury in neonatal rats by upregulating DJ-1 to inhibit HIF-1α ubiquitylation, we examined the expression of VHL and DJ-1 proteins (Figure 8A). 

VHL protein physically interacts with DJ-1 (Parsanejad et al., 2014), as analyzed in an unbiased mass spectrometry screen and confirmed in a PD model. Our findings indicated that DJ-1 could inhibit VHL-dependent ubiquitylation and stabilize HIF-1α expression. Moreover, our results showed that VHL expression decreased when DJ-1 expression was increased in the HIS group compared with the HI group (P < 0.05; Figure 8B and C).

HIF-1α inhibitor YC-1 attenuates the improvement of learning and memory induced by sevoflurane

Neonatal HIE leads to many negative long-term effects; one of the most severe dysfunctions is the impairment of learning and memory. Abundant evidence has demonstrated that SPC can protect against HI damage and improve learning and memory function (Wang et al., 2019a; Xue et al., 2019; Yang et al., 2019). 

We injected YC-1, an inhibitor of HIF-1α, to verify whether the protective mechanism of sevoflurane is related to HIF-1α. The Morris water maze was used to evaluate learning and recognition on days 29–34 after surgery (Figure 9A). There were manifest differences in escape latency between HI and HIS groups (P < 0.0001). YC-1 treatment notably increased escape latency after sevoflurane exposure (P < 0.01; Figure 9B). 

In the spatial probe test, the HIS group performed better than the HI (P < 0.01) and HIS + YC-1 (P < 0.05; Figure 9C) groups. However, the YC-1 injection group did not show marked improvement compared with the HI group. Apart from this, no motor impairment was observed according to swimming speed or suspension test results (P > 0.05; Figure 9D and E). 

Open field test results revealed no differences in time spent in the center or total distance traveled between groups (P > 0.05; Figure 9F–H), indicating there was no locomotor activity or anxiety behavior divergence among the four groups.

Discussion

The classical Rice–Vannucci model was used in this study to evaluate the effects of SPC on HIE in P7 rats (Jevtovic-Todorovic et al., 2003; Grandvuillemin et al., 2017). Our results showed that: (1) SPC improved long-term learning and memory in neonatal HIE by attenuating excessive hippocampal astrogliosis and glial scarring; (2) sevoflurane may induce the observed neuroprotection by stabilizing HIF-1α expression through inhibition of its ubiquitylation; and (3) inhibition of HIF-1α ubiquitylation by sevoflurane may be induced via upregulation of DJ-1, which has been demonstrated to be neuroprotective in PD (Parsanejad et al., 2014) and is regulated by SPC. 

Glial scars affect neuronal alignment and synapse generation in the developing brain. Herein, we found that sevoflurane upregulates the brain-protective protein DJ-1, reduces the level of HIF-1α ubiquitination, improves glial scarring, and affects long-term learning and memory functions to exert brain protection. Most live-born babies surviving HIE suffer severe neurological dysfunction, such as cerebral palsy, epilepsy, and especially long-term learning and memory impairment (Edwards et al., 2010; Descloux et al., 2015). 

However, the current treatment of HIE mainly focuses on symptomatic treatment (Yuan, 2009). Only a few treatments, such as whole-body moderate hypothermia, can provide neuroprotection and improve long-term outcomes. 

Application of the inhaled anesthetic sevoflurane after neonatal HIE provides neuroprotection that can reverse the damage caused by HI, as verified in animal studies (Lai et al., 2016; Kim et al., 2017). The influence of anesthetics on the developmental nervous system has also been a hot topic of research for many years (Archer et al., 2017). 

The effects of inhaled anesthetics on the brain are concentration-dependent. Low-concentration and short-term treatments have protective effects on the injured brain, while high-concentration and long-term treatments can damage brain tissue. However, the mechanism underlying protection from brain damage has not yet been clarified. 

Therefore, we explored the potential mechanisms of sevoflurane on the improvement of learning and memory function. Astrocytes are key central nervous system components of the injury response, including neonatal asphyxia (Zhao and Rempe, 2010). In the acute phase following HI damage, these glial cells become activated and begin to proliferate. 

The proliferation of glial cells is crucial for sealing the site of injury, remodeling the hippocampal tissue structure, and temporally and spatially controlling the local immune response; however, this response becomes harmful in the chronic phase (Pekny and Nilsson, 2005; Pekny et al., 2014). Indeed, this excessive astrocyte hypertrophy impairs hippocampal anatomical structures. 

More specifically, our immunofluorescence results showed that astrocytes around the CA1 and CA3 pyramid layer, as well as in the DG region (especially the border between the granule cell layer and hilum), severely destroyed the normal tissue structure. The processes of glial scaffolds grew into the regular neuron array. 

Morphological results illustrated that these excessive GFAP-expressing radial glia reoriented their processes into a dense mesh, which formed a wall-like structure that obstructed synapse development and directly impaired spatial-dependent learning and memory (Wanner et al., 2008). The Morris water maze test, a classic behavior test used to detect hippocampus-related learning and memory (Wu et al., 2018), was combined in this study with immunofluorescence staining results to provide new evidence that sevoflurane could improve learning and memory by reducing excessive astrogliosis and glial scarring. 

Not only did the immunofluorescence intensity of neurocan and GFAP in hypertrophic astrocytes decrease after SPC, but protein expression levels also decreased. Meanwhile, Nissl staining revealed a denser, more regular hippocampal architecture, hyperchromatic cytoplasm, very little neuronal loss, and marked cellular atrophy after SPC. 

Golgi staining also provided evidence that SPC could elevate the number of dendritic spines. Another novel finding of this study is that SPC upregulated the expression of synaptic markers PSD95 and GAP43 in the hippocampus compared with the HI group. 

This phenomenon indicates that apart from attenuating glial scars, sevoflurane may facilitate hippocampal synapse formation, which directly improves learning and memory functions (Zhang et al., 2015; Lu et al., 2017).

SPC attenuated glial scarring and excessive astrocyte proliferation in neonatal HIE during development. Dendrite spine numbers can be elevated after SPC, thus providing evidence that sevoflurane can induce synapse formation after HI (Roberts et al., 2010). The application of YC-1, an HIF1α inhibitor, reversed the therapeutic effect induced by SPC, indicating that the protection of synapse formation is highly related to HIF-1α. 

As a crucial transcription factor, HIF-1α has various target genes that perform distinct functions, such as processes involved in cell survival, glucose transport and metabolism, and angiogenesis, after ischemia (Ostrowski and Zhang, 2020). Additionally, HIF-1α expression is tightly regulated by various mechanisms. Previous studies demonstrated that HIF-1α cannot consistently exist in brain tissue at a high, effective level because of the oxygen-dependent ubiquitylation degradation pathway (Hirayama and Koizumi, 2017). 

Therefore, finding a way to maintain HIF expression levels can extend its protective effects after HI. Recently, a new treatment modality has been proposed, in which mild hypoxic postconditioning is applied after a period of HI injury to alleviate brain damage (Zhan et al., 2012). Hypoxic postconditioning has been verified to reduce glial activation, including both astrocytes and microglia. 

Moreover, decreases in inflammatory markers have been observed after hypoxic postconditioning (Teo et al., 2015). The potential mechanism underlying this protection was demonstrated to be closely related to the maintenance of HIF-1α expression (Zhu et al., 2014). At present, post-conditioning of hypoxia is nearly impossible to apply in clinical practice as a conventional detrimental treatment and has many potential hazards. Additionally, individual differences in hypoxia tolerance make it difficult to standardize the dose control of hypoxia post-conditioning treatment (Zhan et al., 2012). 

For neonatal HIE, inaccurate post-conditioning of hypoxia treatment is extremely likely to cause secondary injury. In contrast, sevoflurane is safer, milder, and more controllable in clinical practice, and as demonstrated in our research, can also upregulate HIF-1α expression. 

Similar to its neuroprotective role in PD, DJ-1 seems to play an essential role in SPC in our research. The best-defined substrate of VHL, a widely known E3 ubiquitin ligase complex, is HIF-1α (Zhang et al., 2018). DJ-1 has been verified to interact with VHL and can effectively decrease VHL expression, which induces HIF-1α stabilization (Parsanejad et al., 2014). 

We hypothesized that sevoflurane protects the brain against hypoxic damage by upregulating DJ-1 and inhibiting the ubiquitylation activity of VHL. Our novel findings obtained from real-time PCR and western blot analysis support this hypothesis. First, the difference in HIF-1α between SPC and HI groups did not result from transcriptional alterations. 

Second, VHL expression decreased while DJ-1 expression increased in the SPC group. Third, stabilization of HIF-1α protected synapse formation and learning and memory function and reversed astrogliosis and glial scarring. Thus, sevoflurane most likely regulates HIF-1α through inhibition of ubiquitylation by VHL via a DJ-1 interaction. A previous study showed that HIF-1α plays essential roles in the regulation of neural stem progenitor cells and in promoting responses to hypoxia (Cunningham et al., 2012). 

HIF-1α is an intrinsic regulator of neural stem progenitor cell multipotency and developmental outcomes upon differentiation. Further investigation showed that HIF-1α promoted the differentiation of neural stem cells into neurons and inhibited their differentiation into glial cells through the regulation of Notch and Wnt signaling pathways in the subventricular zone (Lie et al., 2005; Morris et al., 2007; Kuwabara et al., 2009; Mazumdar et al., 2010). 

In neonatal rat hippocampus, HIF-1α may also provide neuroprotection by modulating neural progenitor cell differentiation outcomes to attenuate excessive astrogliosis. However, the specific mechanism has yet to be clarified and requires further research. The current study had several limitations. First, dynamic changes in astrocyte activation were not observed or recorded. 

Second, the definitive mechanism by which HIF1α reduces astrogliosis was not investigated. Third, although astrocyte and microglia activation have been observed to occur simultaneously, our study focused on astrocytes instead of microglia activation or the subsequent inflammatory reaction, which would be extremely valuable to investigate. 

Second, the behavior test performance of female rats was slightly better than that of male rats, and the increase of glial cells of female rats was also slightly less than that of male rats. 

However, the behavior test results of the two sexes showed no statistical differences after sevoflurane post-conditioning. The specific mechanism underlying sex-specific effects on development after HI will be investigated in our future research. In addition, genetic methods such as knockout of the gene encoding DJ-1 would be beneficial in demonstrating whether the neuroprotective effects of SPC treatment result from inhibition of HIF-1α ubiquitylation. 

Regardless, our findings indicate that sevoflurane alleviates astrogliosis and glial scar formation in the rat hippocampus, and reduces learning and memory impairment after HI injury by stabilizing HIF-1α expression. This mechanism may be related to sevoflurane-induced upregulation of the neuroprotective protein DJ-1, thus reducing VHL, the ubiquitin ligase of HIF-1α. These findings provide new targets for the treatment of neonatal ischemic hypoxic encephalopathy.

Author contributions: Study conception and design: PZ and QSG; experimental implementation: QSG, YHZ, and ZYW; data analysis: QSG; providing reagents/materials/analysis tools: HX, CL; paper writing: QSG; statistical expertise, obtaining funding, administrative, technical or material support, and supervision: PZ. All authors approved the final version of the paper.

Conflicts of interest: The authors declare that they have no conflict of interest. Financial support: This study was supported by the National Nature Science Foundation of China, Nos. 81671311, 81870838; the Key Research and Development Program of Liaoning Province of China, No. 2018225004; and the Outstanding Scientific Fund of Shengjing Hospital of China, No. 201708 (all to PZ). The funding sources had no role in study conception and design, data analysis or interpretation, paper writing, or deciding to submit this paper for publication.

Copyright license agreement: The Copyright License Agreement has been signed by all authors before publication. Data sharing statement: Datasets analyzed during the current study are available from the corresponding author on reasonable request. Plagiarism check: Checked twice by iThenticate. Peer review: Externally peer-reviewed. 

Open access statement: This is an open access journal, and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

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