Oxidative Stress, Dysfunctional Energy Metabolism, And Destabilizing Neurotransmitters Altered The Cerebral Metabolic Profile in A Rat Model Of Simulated Heliox Saturation Diving To 4.0 MPa

Abstract

The main objective of the present study was to determine metabolic profile changes in the brains of rats after simulated heliox-saturated diving (HSD) to 400 meters of seawater compared to the blank controls. Alterations in the polar metabolome in the rat brain due to HSD were investigated in the cortex, hippocampus, and striatum tissue samples by applying an NMR-based metabolomic approach coupled with biochemical detection in the cortex. The reduction in glutathione and taurine levels may hypothetically boost antioxidant defenses during saturation diving, which was also proven by the increased malondialdehyde level, the decreased superoxide dismutase, and the decreased glutathione peroxidase in the cortex. The concomitant decrease in aerobic and anaerobic metabolic pathways comprised downregulated energy metabolism, which was also proven by the biochemical quantification of the metabolic enzymes Na-K ATPase and LDH in cerebral cortex tissue. The significant metabolic abnormalities of amino acid neurotransmitters, such as GABA, glycine, and aspartate, decreased aromatic amino acids, including tyrosine and phenylalanine, both of which are involved in the metabolism of dopamine and noradrenaline, which are downregulated in the cortex. Notably, a decline in N-acetyl aspartate is associated with neuronal damage. In summary, hyperbaric decompression of a 400 msw HSD affected the brain metabolome in a rat model, potentially including a broad range of disturbing amino acid homeostasis, metabolites related to oxidative stress and energy metabolism, and destabilizing neurotransmitter components. These disturbances may contribute to the neurochemical and neurological phenotypes of HSD.

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Introduction

High pressure above 1.3 MPa (at approximately 120-meter seawater) is induced in humans and mammals at risk of central nervous system (CNS) changes [1, 2]. The CNS might be one of the most sensitive targets in excessive atmospheric pressure, gas bubbles in the body, and decompression sickness (DCS) caused by deep-sea diving. In such conditions, a series of psychomotor and cognitive manifestations are highly complex, with distal and proximal tremors, electroencephalographic abnormalities, fasciculations, myoclonus, sleep disorders, nausea, headache, dizziness, and reduced performance on cognitive tests [2–4]. Moen et al. measured regional neurological abnormalities by diffusion- and perfusion-weighted magnetic resonance imaging (MRI). Perfusion deficits in cerebral microvascular function with arterial microemboli [5] were found in North Sea divers, proven by reduced mean transition time due to reduced complexity of the microvascular or capillary system. Alvhild Alette Bjørkum et al. found disturbing protein homeostasis, e.g., in synaptic vesicles, and destabilizing cytoskeletal components after heliox saturation diving in a rat model. However, Arvid Hope et al. reported that no visible CNS injuries of morphological changes under MRI scan were observed in rats with massive neurological symptoms of decompression sickness following heliox saturation decompression [6]. Thus, we hypothesize that potential molecular profile alterations behind such significant but ambiguous physiological abnormalities might be observed in the CNS tissue after a heliox saturation dive.

The identification of biomarkers for monitoring the status of cellular physiological mechanisms is important for understanding biochemical events. It is always a challenge considering the complexity and diversity of molecular pathways involved in the response of biological systems to diverse factors in a specific moment or condition. Previous attempts linking individual biomarkers with functional CNS perturbation have provided some insight into oxidative damage and energy metabolism postsaturation dive [3, 4, 7]. Similarly, several studies have compared the amino acid neurotransmitter profiles of rats with high-pressure neurological syndrome to those of healthy individuals [2]. Illustrating the neurological metabolic fingerprint of CNS leisure might favor etiological hypotheses [8, 9], prevention [10], and therapeutic approaches [11]. However, the integrated neurological metabolic perturbation induced by a great depth of heliox saturation diving has not been investigated. Such information output from complex biological systems can be rapidly recorded because of advances in technological means [12]. The application of high-resolution nuclear magnetic resonance (NMR) spectroscopy can provide extreme amounts of high complexity but interpretable and robust metabolic profiling data. The spectral data of biofluids and tissue metabolite extracts can be obtained by means of chemometric and bioinformatic methods to reveal physiological or pathological status information. NMR spectroscopy gives immediate qualitative and quantitative information on approximately 102 different small molecules present in a biological sample. NMR detection enables a broad unbiased approach without a priori selection of specific biochemical pathways. Additionally, NMR allows high-throughput analysis and high reproducibility, and it is an intrinsically quantitative technique over a wide dynamic range due to the linear response of NMR signals within a concentration. This technology has been successfully applied to neurological diseases such as cerebellar ataxia [13], Huntington’s disease [14, 15], and Alzheimer’s disease [16, 17] in both preclinical and clinical studies

Despite the promising applicability of NMR-based metabolomics, its application to evaluating the central nervous metabolic perturbation effects of heliox saturation diving has not been reported. The aim of the present study was to investigate metabolomic changes in different anatomic compartments of the brain (cortex, hippocampus, and striatum) and biochemical index level changes in the cortex in a rat model after simulated 400 meters of seawater (MSW) heliox saturation diving, as shown by the schematic experimental design of the present research in Fig 1. The illustration of the metabolic fingerprint of the target organ might provide a set of biomarkers available that could contribute to improvements in diving procedures.

Fig 1. Schematic experimental design of the present research

Experimental procedures 

We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. 

Reagents and materials Analytical-grade sodium chloride, DMSO, NaN3, NaH2PO4•2H2O, and Na2HPO4•12H2O were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). HPLC-grade CHCl3 and CH3OH were obtained from Merck (Darmstadt, Germany). D2O (99.9% in D) containing sodium 3-(trimethyl-silyl) propionate-2, 2, 3, 3, d4 (TSP) as an internal standard for chemical shift reference was provided by Sigma–Aldrich (MO, USA). A buffer system containing 0.2 M Na2HPO4/NaH2PO4 in D2O at pH 7.4 was prepared to prevent the pH effect on the chemical shifts of metabolites at different concentrations. The assay kits for the determination of sodium-potassium ATPase (Na-K-ATPase), cholinesterase (AChE) and lactate dehydrogenase (LDH) were purchased from Abcam (USA). The assay kits for dopamine (DA) were purchased from RD, USA, the assay kits for epinephrine (E) and norepinephrine (NE) were from Abnova, Taiwan, 5-hydroxytryptamine (5HT) assay kits were from BioSource, and gamma-aminobutyric acid (GABA) assay kits were from Santa Cruz, USA. The assay kits of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GPx) were purchased from Cayman, USA.

Ethics approval 

All experimental protocols were approved by the Animal Ethics Committee of Naval Medical Center of PLA, Naval Medical University (Approval no: SYXK(Shanghai)2017-0019, Approval year: 2020). All animals received good care according to the Guide of the Care and Use of Laboratory Animals of Naval Medical University. We confirm that all methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments. The participants researched in this study were rats, not humans; thus, there was no consent to participate.

Animals and grouping design 

Adult male Sprague–Dawley rats with an average age of 8 weeks and weighing 200–220 g were included in this research. Animals were purchased from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). Maximum efforts were carried out to minimize animal suffering and the number of animals necessary to capture reliable data. Standard rat chow and drinking water were available for all animals ad libitum. Animals with 3 rats per cage were housed in a specific pathogen-free (SPF) animal room (temperature, 22–24˚C, humidity, 45– 55%) and controlled conditions of light (12/12 hour light-dark cycle) for one week before the experiment. Prior to the commencement of the diving experiments, rats were acclimatized for five days in the lab environment, including a hyperbaric chamber. Sixteen rats were weighed, labeled, restricted randomly assigned to a control (exposure to normobaric air but enduring similar light and noise, named the CON group, n = 8) group and an experimental (subjected to a simulated heliox saturation diving to 4.0 MPa (400 msw), named the HSD group, n = 8).

Stimulated saturation diving of 400 msw 

Rats in the HSD group were pressurized in helium-oxygen gas in a hyperbaric chamber. In brief, four phases of compression, storage, decompression, and bend watch were included in 

the saturation period. The rat’s compression rate was 1 msw/min up to 10 msw with 20% HeO gas mixtures and 3.54 msw/min from 10 msw to 400 msw with pure He. The time compression from the surface to 400 msw took approximately 120 minutes. The bottom phase at 400 msw was 120 minutes. The oxygen concentration was supplemented with pure oxygen to maintain the oxygen partial pressure at 35~50 kPa during the compression process and the storage depth. The period of decompression bend watch was 40 min at 300 msw, 45 min at 200 msw, 50 min at 105 msw, 60 min at 45 msw, 75 min at 10 msw, 75 min at 3 msw, and then at the surface. The ascent rate used in this decompression model is approximately 10 meters (33 ft) per min. the oxygen partial pressure was maintained at 38~67 kPa during the decompression stage. After decompression to 10 m, the oxygen concentration was maintained at 20~24%. The saturation diving timeline overview is shown in Fig 2. Rats in the air control group (Group CON, named the control group, with eight rats) were bred within the atmospheric environment in the same experimental lab with the same chamber as the HSD groups. The control group did not receive any compressing or decompressing procedures but endured similar noise and light as the HSD group.

Sample collection 

After the decompression period, the rats were anesthetized with pentobarbital sodium (0.3%, 1.0 ml/kg rat weight) intraperitoneally, followed by removal of the brain. The left cerebral hemispheres were rapidly dissected. In two animal groups, the cortex, hippocampus, and striatum tissues were dissected and abbreviated respectively as HSDC, HSDH, HSDS, CONC, CONH, and CONS. As the largest tissue among the three compartments, each cortex sample was then cut into two pieces with approximately equal weights (one part of the sample for metabolomics analysis, the other pieces of samples for biochemical assessments). All tissues were snap-frozen in liquid nitrogen and stored at −80˚C until further analysis.

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