Brief Inhalation Of Sevoflurane Can Reduce Glial Scar Formation After Hypoxic-ischemic Brain Injury in Neonatal Rats Part 1

Abstract

Previous studies have demonstrated that sevoflurane postconditioning can provide neuroprotection after hypoxic-ischemic injury and improve learning and memory function in developing rodent brains. 

Hypoxic-ischemic injury refers to a pathological state caused by cerebral ischemia, hypoxia, metabolic disorder, and nerve cell damage due to disorders of the cardiovascular and cerebrovascular systems. With social progress and lifestyle changes, hypoxic-ischemic injury is becoming more and more common. This condition can cause many harm to human health, including affecting one's intelligence and memory.

Although hypoxia and ischemia can cause a decline in brain function, it does not mean that memory will be negatively affected. On the contrary, we can improve memory and increase the brain's efficiency through the right methods. Here are some ways to help improve your memory:

1. Healthy diet: The impact of diet on memory is very important. Foods rich in protein, vitamins, and minerals can help maintain a healthy nervous system and thinking function.

2. Exercise more: Physical exercise can promote blood circulation, increase oxygen flow, enhance body immunity, and reduce the risk of disease. Through exercise, you can improve your memory ability.

3. Strengthen self-confidence: Confidence is an important factor in a person's memory. If you believe that you can complete a certain task, you will easily achieve the results you want, and you can also effectively improve your memory.

It can be seen that hypoxic-ischemic injury is not a condition that will inevitably hurt human memory. As long as we master the correct methods and take good care of our brains, we can embrace a healthy future with a positive attitude. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory because Cistanche deserticola is a traditional Chinese medicinal material that has many unique effects, one of which is to improve memory. The efficacy of Cistanche deserticola comes from the multiple active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health through a variety of pathways.

Click know 10 ways to improve memory

The classical Rice-Vannucci model was used to induce hypoxic-ischemic injury and newborn (postnatal day 7) rats were treated with 2.4% sevoflurane for 30 minutes after hypoxic-ischemic injury. 

Our results showed that sevoflurane postconditioning significantly improved the learning and memory function of rats, decreased astrogliosis and glial scar formation, increased numbers of dendritic spines, and protected the histomorphology of the hippocampus. Mechanistically, sevoflurane postconditioning decreased the expression of von Hippel-Lindau of hypoxia-inducible factor-1α and increased the expression of DJ-1. 

Injection of 1.52 μg of the hypoxia-inducible factor-1α inhibitor YC-1 (Lificiguat) into the left lateral ventricle 30 minutes before hypoxic-ischemic injury reversed the neuroprotection induced by sevoflurane. This finding suggests that sevoflurane can effectively alleviate astrogliosis in the hippocampus and reduce learning and memory impairments caused by glial scar formation after hypoxic-ischemic injury. 

The underlying mechanism may be related to upregulated DJ-1 expression, reduced ubiquitination of hypoxia-inducible factor-1α, and stabilized hypoxia-inducible factor-1α expression. This study was approved by the Laboratory Animal Care Committee of China Medical University, China (approval No. 2016PS337K) on November 9, 2016.

Key Words: brain injury; brain; central nervous system; in vivo; injury; model; plasticity; rat; recovery; regeneration; repair Chinese Library Classification No. R453; R741; R614.2+1.

Introduction

Neonatal hypoxic-ischemic encephalopathy (HIE) is a common complication in the neonatal period caused by many factors, such as neonatal asphyxia, intrauterine distress, and hyaline membrane disease (Douglas-Escobar and Weiss, 2015; Barkhuizen et al., 2017). 

HIE is the leading cause of infant fatalities and the primary source of neurological sequelae (Edwards et al., 2010; Descloux et al., 2015). Ninety percent of HIE survivors have long-term neurological dysfunction, such as learning, cognitive, and motor dysfunctions, and epilepsy (Doi et al., 2012; Davies et al., 2019). Current treatment of HIE mainly focuses on symptomatic treatment, including mechanical ventilation, correction of hypotension, supplementation of glucose, and limiting fluid intake, as appropriate. 

However, an effective treatment capable of reversing or reducing long-term brain damage is required (Stankowski and Gupta, 2011; Davidson et al., 2015). As a pivotal part of learning and recognition, the hippocampus, including the dentate gyrus (DG), CA1, and CA3 regions, participates in transforming and integrating peripheral information into nerve centers (Morris et al., 2012; Jung et al., 2020). 

The hippocampus is hypersensitive to hypoxic-ischemic (HI) damage, which may provoke apoptosis of hippocampal neurons, stimulate excessive reactive gliosis, and lead to glial scar formation (Hopkins and Haaland, 2004; Wang et al., 2012). The proliferation of astrocytes is crucial for sealing the site of infarction, remodeling the hippocampus structure, and temporally controlling local immune responses during the acute phase (Rolls et al., 2009). 

However, hypertrophy and glial scar formation contribute to aberrant neurogenesis, obstruct dendritic spine growth, and hamper synapse shape, further impairing learning and recognition functions (Yiu and He, 2006; Wanner et al., 2008; Burda and Sofroniew, 2014; Pekny et al., 2014; Shi et al., 2017). 

Glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans, such as neurocan, are pathologic markers of astrogliosis and glial scarring whose expression can be upregulated by highly activated astrocytes (Pekny and Nilsson, 2005; Choudhury and Ding, 2016). Thus, inhibition of astrogliosis and glial scarring may be a therapeutic target for HIE and its long-term neurological sequelae. 

The application of sevoflurane has been demonstrated to mitigate HI injury in rodents. Studies investigating the significance of sevoflurane postconditioning (SPC) to alleviate HI damage by upregulating hypoxia-inducible factor-1α (HIF-1α) in adult rodents and neonatal rats have identified several potential mechanisms (Wang et al., 2019b; Yang et al., 2019; Du et al., 2020; Liu and Gong, 2020). 

As a key physiological sensor of hypoxia, HIF-1α is a transcription factor with hundreds of downstream molecules involved in ischemic tolerance mechanisms, such as vascular endothelial growth factor and erythropoietin (Na et al., 2015). Hypoxic postconditioning has been verified to reduce astrocyte and microglial activation after HI in neonatal rat brains (Teo et al., 2015). 

Therefore, we hypothesized that HIF-1α may contribute to attenuating the formation of glial scars and astrogliosis. HIF-1α can constitutively exist in neurons, whereby it quickly increases in expression and accumulates under hypoxic conditions. However, when oxygen returns to normal, HIF1α expression cannot be sustained at a high enough level to continuously exert a neuroprotective effect because of oxygen-dependent degradation by prolyl-hydroxylase proteins and subsequent ubiquitylation by von Hippel-Lindau (VHL) proteins (Berra et al., 2003; Zhang et al., 2018). 

DJ-1 (encoded by Park7) is reportedly a neuroprotective protein, especially under oxidative conditions (Aleyasin et al., 2007, 2010). VHL protein physically interacts with DJ-1, as determined by an unbiased mass spectrometry screen and confirmed in a Parkinson's disease (PD) model. In addition, Parsanejad et al. (2014) showed that DJ-1 inhibits VHL-dependent ubiquitylation and stabilizes HIF-1α expression. 

Thus, we hypothesized that sevoflurane stabilizes HIF-1α protein in the hippocampus by upregulating DJ-1 to inhibit HIF-1α ubiquitylation, thereby improving long-term learning and memory function. 

To investigate the role of HIF-1α in the relationship between sevoflurane and astrogliosis, we used YC-1 (Lificiguat), a selective antagonist of HIF-1α, to completely inhibit HIF-1α expression at the post-transcriptional level. In this study, we investigated the role of HIF-1a in neuroprotection of SPCtreated HIE neonatal rats.

Materials and Methods

Animals
Animal research was performed at the synaptic stage of rat development, which commences on postnatal day 7 (P7), corresponding to the growth period of human infants from late pregnancy to 3 years after birth (Jevtovic-Todorovic et al., 2003). Seven-day-old Sprague-Dawley rats weighing 12–16 g were selected from 20 pregnant rats (purchased from the Laboratory of Shengjing Hospital, China Medical University, China; male/female ratio, 1:1). 

All rats were able to access water and food freely, and maintained at standard humidity and temperature in the lab, with 12-hour light/dark (8 a.m./8 p.m.) cycles. All animal experiments were performed according to the National Institutes of Health (Bethesda, MD, USA) Guidelines for the Care and Use of Laboratory Animals. 

The Laboratory Animal Care Committee of China Medical University formally approved the described experiments (Shenyang, China; approval No. 2016PS337K) on November 9, 2016. 

From 25 pregnant rats, 249 P7 rats were chosen and randomly divided into groups based on a random number table (Wang et al., 2019a). Our final analysis included a total of 201 newborn rats; the mortality rate after HI treatment was 19%. 

These 201 newborn rats were randomly divided into four groups: sham (n = 54), HI (n = 54), HI + sevoflurane (HIS) (n = 54), and HIS + YC-1 (n = 39) groups. In each group, 24 rats were used for western blot assay, 5 were used for immunohistochemistry and Golgi staining, and 10 were used for behavioral testing. The remaining 45 rats in sham, HI, and HIS groups were used for reverse transcription polymerase chain reaction (RT-PCR).

Experimental design and exposure to anesthetic

The employed HIE model was previously described (Zhao et al., 2007; Grandvuillemin et al., 2017). Based on the distance between the anus and reproductive organs, sex identification of postnatal rats was completed. To establish the HIE model, the head of each P7 rat was put into a transparent plastic pipe, which was sealed with cotton before sevoflurane was released (Wang et al., 2019a; Xue et al., 2019). 

The left common carotid artery of each rat was permanently ligated, and between the two ligations, the artery was cut; each surgery was finished within 5 minutes. Subsequently, the rats awoke unaffected by anesthesia and were put back into their mothers' cages for 2 hours. For administration of SPC, rats were put into a transparent chamber (Lingzhi Company, Hangzhou, China) connected to a sevoflurane vaporizer; one duct assisted in ventilation, while the other conveyed the gas sample out of the chamber to the monitor. 

The chamber was ventilated with 30% O2 and 70% N2 for 2 hours for the sham group, while 8% O2 and 92% N2 were administered for 2 hours to the HI group. SPC was set promptly after HI: 2.4% sevoflurane (1 minimum alveolar concentration) was inhaled by rats in the chamber with an atmosphere of 30% O2 and 70% N2 and an airflow rate of 2 L/min for 30 minutes. The heating pool stabilized the temperature inside the chamber at 37°C.

Drug administration

Exactly 30 minutes before HI, 1.52 μg of HIF-1α inhibitor (YC1; Sigma-Aldrich, St. Louis, MO, USA) was injected into the left lateral ventricle (Paxinos and Franklin, 2013) using a 5-μL Hamilton syringe (Yingweida Technology, Beijing, China). As previously described, YC-1 was dissolved in artificial cerebral spinal fluid at a concentration of 0.304 μg/μL (Shen et al., 2012; Na et al., 2015).

Western blot analysis

P7 rats were anesthetized by 2% sevoflurane, and hippocampal tissues were extracted from pups at 12, 24, and 48 hours, and 28 days after surgery. After extraction, tissues were immediately placed on ice. 

Total supernatant proteins were isolated by adding a radio-immunoprecipitation assay agent (Beyotime, Haimen, China). Protein concentrations were determined with a One BCA Kit (Beyotime). 

Next, specimens were separated on 12.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes. After blocking with 5% bovine serum albumin (Sigma-Aldrich) at 4°C, the membrane was incubated with the appropriate primary antibodies, including anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2000; Cat# 5174S; Cell Signaling Technology, Danvers, MA, USA), anti-DJ-1 (1:1000; Cat# ab18257; Abcam, Cambridge, UK), anti-VHL (1:5000; polyclonal; Cat# ab77262; Abcam), anti-HIF-1α (1:900; polyclonal; Cat# ab2185; Abcam), anti-neurocan (1:200; monoclonal; Cat# sc-33663; Santa Cruz Biotechnology, Dallas, TX, USA), anti-postsynaptic density protein 95 (PSD95; 1:1000; monoclonal; Cat. No 3409S; Cell Signaling Technology), antigrowth associated protein 43 (GAP43; 1:2000; polyclonal; Cat# 16971-1-AP; Proteintech Biotechnology, Chicago, IL, USA), and anti-GFAP (1:5000, polyclonal, Cat# ab53554; Abcam). 

Afterward, blots were incubated with anti-rabbit IgG (1:5000; Cat# ZB-2301; Zhongshanjinqiao, Beijing, China) or anti-mouse IgG (1:5000; Cat# ZB-2301; Zhongshanjinqiao) for 2 hours at room temperature. 

Protein blots were visualized with enhanced chemiluminescence detection reagents (Super Signal West Pico; Pierce, Rockford, IL, USA). Protein bands were quantified with ImageJ software (National Institutes of Health). For analysis of western blots, data were normalized to GAPDH.

Immunohistochemistry

Twenty-eight days after surgery, rats were administered anesthetic using the method described above, perfused with 4% formalin, and their brains were removed for immunofluorescence staining. 

Brains were immersed into 4% paraformaldehyde at 4°C for 48 hours, subsequently dehydrated in a graded ethanol series, and finally embedded in paraffin. Next, paraffin-embedded tissues were cut into 2.5-μm-thick sections with a vibratome, and the resulting sections were stored at room temperature. 

Each brain was sectioned continuously, and three discontinuous brain sections were selected for NeuN (neuron marker), GFAP (astrocyte marker), and neurocan (glial scar marker) staining. For double labeling, tissue sections were incubated with two mixed primary antibodies, including goat anti-GFAP (1:250; polyclonal; Cat# ab53554; Abcam), rabbit antigens (1:100; Cat# 12943; Cell Signaling Technology), and/ or mouse anti-neurocan (1:200; monoclonal; Cat# sc-33663; Santa Cruz Biotechnology) at 4°C overnight in a humidified chamber. 

Subsequently, sections were washed with 0.1 M phosphate-buffered saline and incubated for 2 hours at room temperature with the appropriate secondary antibodies, including anti-rabbit IgG conjugated to Alexa Fluor-594 (1:200; Life Technologies, Grand Island, NY, USA), anti-mouse IgG conjugated to Alexa Fluor-594 (1:200; Life Technologies), and anti-goat IgG conjugated to fluorescein isothiocyanate (1:200; Life Technologies). 

Counterstaining of cell nuclei was implemented with 4′,6-diamidino-2-phenylindole (Beyotime) for 5 minutes. 

An Olympus BX51 microscope (Olympus Corporation, Tokyo, Japan) was used for the observation of stained sections. Three fields of view of the left hippocampus were randomly selected from each slice for photography. ImageJ software was utilized to estimate quantitative colocalization and, therefore, calculate Manders' overlap coefficient.

For more information:1950477648nn@gmail.com