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Astrocyte activation following nitrous oxide exposure is related to oxidative stress and glutamate excitotoxicity
Journal Pre-proofs Research report Astrocyte activation following nitrous oxide exposure is related to oxidative stress and glutamate excitotoxicity Usha Kant Misra, Sandeep Kumar Singh, Jayantee Kalita, Alok Kumar PII: DOI: Reference:
S0006-8993(20)30001-9 https://doi.org/10.1016/j.brainres.2020.146645 BRES 146645
To appear in:
Brain Research
Received Date: Revised Date: Accepted Date:
22 August 2019 10 December 2019 2 January 2020
Please cite this article as: U.K. Misra, S.K. Singh, J. Kalita, A. Kumar, Astrocyte activation following nitrous oxide exposure is related to oxidative stress and glutamate excitotoxicity, Brain Research (2020), doi: https://doi.org/ 10.1016/j.brainres.2020.146645
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© 2020 Published by Elsevier B.V.
Astrocyte activation following nitrous oxide exposure is related to oxidative stress and glutamate excitotoxicity Usha Kant Misra1; Sandeep Kumar Singh1; Jayantee Kalita1; Alok Kumar1,2 1Department
of Neurology, Sanjay Gandhi Post Graduate Institute of Medical Sciences,
Raebareily Road, Lucknow, 226014, Uttar Pradesh, India. 2Department
of Molecular Medicine and Biotechnology, Sanjay Gandhi Post Graduate
Institute of Medical Sciences, Raebareily Road, Lucknow, 226014, Uttar Pradesh, India
Corresponding authors: Usha K. Misra Professor Department of Neurology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareily Road, Lucknow226014 Phone: +91 522 2494177; FAX: 091-0522-2668811 Email: [email protected]; [email protected]
Abstract: Background and Aims: Nitrous oxide is commonly used as an anesthetic agent and its exposure produces prolonged inactivation of vitamin B12. Vitamin B12 is associated with central nervous system changes which are similar to sub-acute combined degeneration (SACD). Astrocytes have important role in neurotoxic toxic injuries, but have not been evaluated in N2O toxicity. In the present study we have evaluated the changes in astrocyte in N2O exposed rats and correlated with neurobehavioral changes, oxidative stress and glutamate level. Material and Methods: Adult wistar male rats were exposed to N2O oxygen mixture in 1:1 ratio at a rate of 2 L/min for 120 min for 60 days. Control rats underwent similar exposure to oxygen. At the end of exposure, spontaneous locomotors activity (total distance travelled, time resting, time moving, number of rearing, stereotypic count) and grip strength were evaluated. Plasma glutathione (GSH), Total antioxidant capacity (TAC), serum malonodialdehyde
(MDA)
and
serum
homocysteine
(Hcy)
were
measured
by
spectrophotometer. Glutamate in the cerebral cortex and cerebellum were measured by colorimetry. Immunohistochemistry for astrocyte (GFAP) phenotypic analysis and its activation in brain and spinal cord were measured by using using image J software in N2O exposed rats and control animals. Results: The N2O exposed rats had significant reduction in total distance travelled, time moving and number of rearing whereas time resting increased compared to the control animals. Hcy, glutamate and MDA levels were significantly increased, however GSH and TAC level decreased in N2O exposed group compared to the controls. Astrocyte phenotype and its activation was significantly altered more so in spinal cord compared to cerebral cortex and was associated with neurobehavioral changes, oxidative stress and glutamate level.
Conclusions: N2O related clinical dysfunction may be related to changes in astrocyte activation which is related to oxidative stress and glutamate neurotoxicity. Keywords: Nitrous oxide; astrocyte activation, oxidative stress; glutamate level; total antioxidant capacity; homocysteine
Introduction: Nitrous oxide initially has been used as a recreational gas and an anesthetic agent for minor surgery such as dental extraction. In 1852, Lassen and colleague reported megaloblastic bone marrow after prolonged N2O exposure [1, 2]. Nitrous oxide inhibits the enzyme methionine synthase (MeS) resulting in hyperhomocysteinemia. Methionine is important for synthesis of DNA, RNA, catecholamine and myelin [3]. Prolonged exposure to N2O is reported to result in subacute combined degeneration (SACD) like illness [4,5]. Controlled N2O exposure to rats, results cobalamin (Cbl) deficiency like illness resulting in demyelination of central and peripheral nervous system [6-8]. In SACD, the most prominent changes occur in spinal cord resulting in spongy vacuolation, intramyelin, interstitial and white matter edema and gliosis [8]. In the patients with SACD, and in the rats exposed to N2O , we have reported increased oxidative stress evidenced by increased malonodialdehyde (MDA), reduced glutathione (GSH) and total antioxidant capacity (TAC) levels [8,9]. In SACD, astrogliosis and increased glial fibrillary acidic protein (GFAP) expression have also been reported [7,10]. Imbalance of tumor necrosis factor α (TNF-α) with epidermal growth factor (EGF) and IL6 have been reported to be associated with the pathological changes in SACD [7]. It is possible similar changes may also occur following N2O toxicity, but its cellular mechanisms have not been evaluated. Following N2O exposure, we have reported hyperhomocystinemia and focal myelin loss, vacuolation in subcortical white matter and spinal cord [8]. Keeping the analogy of SACD, using totally gastrectomized (TGX) mice model reported spongy vacuolation, intramyelin and interstitial edema in white matter of CNS and astrogliosis [7]. Astrogliosis is the dominant and universal response of the nervous system injury. GFAP is the most studied markers of astrocyte activation
and proliferation [22]. The basis of myelin
dysfunction and loss in N2O exposed rats has not been investigated on these lines. The cytokine release, oxidative stress
are closely related to astrocyte activation [23].
Hyperhomocysteinemia causes astrocyte activation, vascular cognitive impairment and dementia [24]. Moreover, in recent In vitro study, Weekman et al (2017) [25] showed that hyperhomocysteinemia induces early proinflammatory changes in astrocytes, which are relevant to their interaction with the vasculature. A study of oxidative stress , glutamate exictotoxicty and glial dysfunction may provide valuable information about the mechanism of myelin dysfunction in N2O exposure model. Oxidative stress and glutamate excitotoxicity may also contribute to astrogliosis, which has not been reported in cobalamine deficiency. In the present study, we report the changes in astrocytes in brain and spinal cord in N2O exposed animals. We also report the psychomotor activity of rats and correlate this with GFAP score in brain and spinal cord.
Results:
Nitrous oxide exposure altered behavioral and neurological function: None of the animal died during the study period. The body weight of the N2O exposed group at the end of the study was 200.60±16.02 gm and that of controls was 245.6±6.3 gm (p<0.01). The N2O exposed group had significant alteration in spontaneous locomotor activity which includes reduction in total distance, time moving, number of rearing, and time resting when compared to the control animals (Fig. 1; p<0.01).
The grip strength was significantly
decreased in the N2O exposed group compared to the control animals (Fig. 1; p<0.01) (Table: 1).
Homocysteine level increases followed by nitrous oxide exposure
There was no significant difference in serum vitamin B12 and folic acid level in the control groups compared to N2O exposed rats. Whereas, Hcy level was significantly higher in the N2O exposed rats compared to the control rats (Fig. 2A; p<0.001[Hcy] (Table: 1).
Glutamate level and lipid peroxidation is increased followed by nitrous oxide exposure and is associate with reduced glutathione, TAC antioxidant level The plasma GSH and TAC level was significantly reduced in the N2O exposed rats compared to the control group (Fig 2D; p<0.01[GSH, TAC]). However, the serum MDA and cerebral cortex glutamate level were higher in the control group versus N2O exposed rat (Fig.2 B,C; p<0.01 [MDA, glutamate level]) (Table: 1).
Highly reactive astrocyte activation phenotypes predominate brain and spinal cord followed by nitrous oxide exposure GFAP score analysis shows increase in GFAP expression (astrocyte activation) in the brain and spinal cord (p<0.01 [brain GFAP score] and p<0.001 [spinal cord GFAP score]) in the N2O exposed rats compared to the controls (Fig. 3C, 4B). Image J software reconstruction of activated astrocyte cells represented that N2O exposure resulted in a increment in GFAP positive cell body area in the cerebral cortex of N2O exposed rat versus control group (Fig. 3 B). Similarly, N2O exposure resulted in a increase in GFAP positive cell body area in spinal cord sub-region of thoracic in control group versus N2O expose rat group (Fig. 4A). However, comparatively GFAP positive score analysis in brain and spinal cord shows GFAP expression significantly increased in spinal cord (1.48 fold) compared to brain cortical subregion (Table:1). Astrocyte activation correlated with neurobehavioral and biochemical parameters in brain and spinal cord following nitrous oxide exposure
Correlation study for astrocyte (GFAP score) in brain and spinal cord with biochemical parameters revealed a positive correlation for glutamate (r=0.72; Fig. 5C), MDA (r= 0.85; Fig. 5D) with brain astrocyte and with Hcy (r=0.87; Fig. 5F) with spinal cord astrocyte where as a negative correlation was observed with TAC (r=-0.86; Fig. 5G) in spinal cord and glutathione in both brain (r=-0.69; Fig. 5E) and spinal cord (r=-0.83; Fig. 5H). However, the blood biomarker correlation analysis between antioxidant glutathione and Hcy, TAC revealed that glutathione was positively correlated with TAC (r=0.57; Fig. 5B) and negatively correlated with Hcy (r=-0.88; Fig. 5A) following N2O exposure.
Further correlation study for astrocyte (GFAP score) in brain and spinal cord with neurobehavioral parameters revealed that activation of astrocyte in cerebral cortex was positively correlated with time resting (r=0.79; Fig. 6A) in cerebral cortex region, total distance covered (r=-0.84; Fig. 6D), numbers of rearing (r=-0.86; Fig. 6G) and grip strength (r=-0.79; Fig. 6H) in spinal cord, whereas there was a a negative correlation with time moving (r=0.79; Fig. 6B), number of rearing (r=-0.76; Fig. 6C) in brain region and time moving (r= -0.883; Fig. 6F) , number of rearing (r=-0.86; Fig. 6G) and grip strength (r=0.79; Fig. 6H)
in spinal cord region.
DISCUSSION: In the present study nitrous oxide exposure resulted in oxidative stress, glutamate excitotoxicity, glial acitvations which was related to altered spontaneous locomotor activity and grip strength. Oxidative stress and DNA damage have been reported following N2O
exposure. The patients undergoing N2O based anesthesia revealed higher postoperative Hcy levels compared to non N2O based anesthesia [13]. In another study, 15 anesthesia or surgery residents were exposed to N2O with isoflurane or sevoflurane as anesthetic agent during their operation theater duty. The reactive oxygen species (ROS) was increased in N2O exposed residents resulting in DNA damage as evaluated by comet assay compared to controls [14]. Similar results have also been reported in 36 nurses exposed to N2O based anesthesia. In the exposed group, the oxidative stress markers namely ROS in leucocytes, α tocopherol and glutathione peroxidase activity were measured. The DNA damage was attributed to increased oxidative stress [15]. In the above mentioned study, the subjects were exposed to anesthetic agents in addition to N2O (isoflurane and sevoflurane), which could also induce DNA damage. In the present study, we have used only N2O and O2 mixture and compared with the controls who received only oxygen; thereby excluding the effects of oxygen. In an earlier study using 1.5L of N2O for 90 min for 1 month revealed reduced levels of GSH, TAC, and increased level of MDA suggesting oxidative stress in the N2O exposed group. In the present study N2O exposed group also developed higher Hcy level. The Histopathological studies following N2O exposure revealed shrinkage and vacuolation of neurons in the cerebral cortex, and focal myelin damage and vacuolation in the subcortical white matter and in the spinal cord [8]. The spinal cord and subcortical white matter myelin damage following N2O toxicity may be due to vitamin B12 inactivation resulting in SACD like changes. In the patients undergoing N2O based anesthesia, and occupational N2O exposure, SACD like illness has been reported [16]. Nitrous oxide inactivates vitamin B12 by oxidizing Cbl+ into Cbl+++. Vitamin B12 is an important cofactor for conversion of Hcy to methionine by the enzyme methionine synthase, and Hcy to cystathione by the enzyme cystathione β synthase [17-19].
We have noted high level of glutamate in N2O exposed rats . Increased level of glutamate requires the scavenging role of astrocytes thereby resulting in astrocytic proliferations. As the capacity of astrocytes to scavenge glutamate is exhausted, the excitotoxicity ensues. We have noted high level of astrocyte in N2O exposed rats. Higher level of astrocyte activation in the spinal cord compared to brain may be consistent with the grater vulnerability of spinal cord. In the patients with SACD compared to brain. The spinal cord MRI characteristically reveals posterior T2 hyper intensity or atrophy in thoracodorsal region in 73.9 % and in subcortical white matter in 25 % [20]. Cobalamin deficiency primarily affects the glial cells. Labelled cobalamin is almost exclusively taken up by glial cells of spinal cord white matter [30]. Human astrocyte culture in cobalamine deficient medium has shown be damaged [31] but gliosis in TGX rats has not correlated with the severity of SACD like illness nor which is different from that in humans [32]. In the patients with SACD, increase in oxidative stress and proinflammatory cytokine has been reported [7,9]. Increased oxidative stress and reduced methionine per se also result in myelin damage. These factors may induce excitotoxic injury in N2O exposed group. In our study there was increase in glutamate, which may be due to increase in Hcy and oxidative stress. Glutamate is the major neurotransmitter of brain, and the excess glutamate from synaptic cleft is cleared by astrocytes which may result in proliferation of astrocytes. Increased GFAP expression in the present study may be due to astrocytic proliferation. In TGX mouse model of vitamin B12 deficiency, increased GFAP expression has been reported [6]. In vitamin B12 deficiency, astrocytes bear the brunt of attack, and the neurons are spared. This may be the reason for good recovery of patients following vitamin B12 treatment [21]. Vitamin B12 priming before N2O based anesthesia reduces its toxicity [4]. In the present study, the GFAP expression in the spinal cord and brain correlated with neurobehavioral
findings. In our earlier study on N2O toxicity, neurobehavioral findings correlated with oxidative stress and histopathological changes [8]. Reactive oxygen species are produced in the astrocytes by the enzyme NADPH oxidase [33] which is also balanced by antioxidant system [34]. The astrocytes also initiate an inflammatory response through
proinflammatory cytokines. In this study, we have
provided evidence on the role of oxidaitves stess , glutamate excitotoxicity and astrocyte activation in the process of behavioral alterations following N2O exposure. The limitation of the present study is that due to snap frozen tissue sample collection procedure, the glutamate, GSH, TAC and MDA levels in brain and spinal cord tissue were not evaluated, which could have provided better insight about the role of oxidative stress in the respective area and its relationship with GFAP. From this study, it can be concluded that N2O toxicity results in alteration of astrocyte phenotype which is related to oxidative stress and glutamate excitotoxicity and is more marked in the spinal cord than in brain (Fig. 7).
Conclusions: N2O related clinical dysfunction may be related to changes in astrocyte activation which is related to oxidative stress and glutamate neurotoxicity.
Materials and methods: Studies were performed using adult male wistar rats (10-12 weeks old, 200-250 gm). Rats were housed in the Animal Care facility at Sanjay Gandhi postgraduate Institute of Medical Sciences, Lucknow a 12h light-dark cycle, with ad libitum access to food and water. All exposure and surgical procedures were carried out in accordance with protocols approved by
the Institutional Animal Ethics Committee of Sanjay Gandhi postgraduate Institute of Medical Sciences, Lucknow. N2O exposure N2O exposure experiments were performed as previously described [8]. The experimental rats were acclimatized for 10-15 days in whole body exposure chamber. After three weeks of acclimatization, rats were exposed to a N2O oxygen mixture in 1:1 ratio at a rate of 2 L/min for 120 min for 60 days. Control rats underwent the same procedure as N2O exposure except N2O was replaced by a mixture of oxygen and room air (1:1) in the whole body exposure chamber. The rats were examined daily for red nose, ataxia or any other abnormal neurological finding. The neurobehavioral study included spontaneous locomotor activity (SLA) parameters like total distance travelled, time resting, time moving, number of rearing, stereotypic count and grip strength. These parameters were also measured after 60 days of exposure before the animals were sacrificed. Study 1 Control (n = 5-10) and N2O exposed (n = 5-10) adult male wister rats were anesthetized (by using ether) at 60 days. Fasting blood was collected in plain and EDTA vial following cardiac puncture. Serum and plasma were separated and stored at -800C. Rats were transcardially perfused with ice cold 0.1 M phosphate-buffered saline (150 ml; pH 7.2) from left ventricle following thoracotomy.
Brain tissue was rapidly dissected and snap-frozen on liquid
nitrogen for glutamate analysis. Data represent three independent experiments. Study 2 Control (n = 5-10) and N2O exposed (n = 5-10) adult male wister rats were used for behavioral studies. At 60 days post N2O exposure, the rats were anesthetized (by using ether)
and were transcardially perfused with ice cold 0.1 M phosphate-buffered saline (150 ml; pH 7.2) followed by 250 ml of 4% paraformaldehyde from left ventricle following thoracotomy. Brain and spinal cord were removed and post-fixed in 4% paraformaldehyde overnight, and cryoprotected in 30% sucrose and were processed for immunohistochemistry analysis. Biochemical studies Analysis for homocysteine, vitamin B12 and folate in control and N2O exposed animals Homocysteine, vitamin B12 and folate levels were measured in fresh serum samples by using chemiluminescence analyzer (IMMULITE 1000, M/s Siemens Ltd. USA and Siemens Healthcare Diagnostics Products Limited, Glyn Rhonwy, Llanberis, Caernarfon, Gwynedd LL55 4EL, UK).
Analysis for lipid peroxidation, glutathione and total antioxidant capacity in control and N2O exposed animals Lipid Peroxidation (LPO) assay: LPO was measured in fresh plasma samples of control and N2O exposed animals by assaying malonodialdehyde (MDA) level, which is the end product of LPO [11]. Plasma was mixed with EDTA, ascorbate (10 mM), and FeSO4 (16.7 mM), and incubated at 37°C for 60 min. The reaction was stopped by adding ice-cold 10% trichloroacetic acid. The mixture was centrifuged at 2000 × g for 10 min, and the supernatant was aspirated. The supernatant was mixed with equal volume of 0.67 % thiobarbituric acid and was kept in a boiling water bath for 15 to 20 min. MDA level was measured with the absorption coefficient of MDA- thiobarbituric acid complex at 532 nm using spectrophotometer. Glutathione (GSH) assay: GSH was measured in fresh plasma samples of control and N2O exposed animals using spectrophotometer employing Tietze method (1969). Plasma sample were added to 10% trichloroacetic acid and were incubated for 2 hr at 40C. The sample were
further centrifuged at 2000xg for 15 min, and the supernatant were collected and added to fresh Tris HCl buffer (0.4 M, pH 8.9 containing EDTA 0.02 M). At the end of the experiment, 5,5 di-thio-bis (2-nitrobenzoic acid 0.01 M) was added to the reaction mixture and end yellow product (5’ thio’ 2 nitrobenzoic acid) was measured by using spectrophotometer at 412 nm. Total antioxidant capacity (TAC) assay: TAC was measured in fresh serum sample of control and N2O exposed animals by using the method of Koracevic et al [12]. The intermediate hydroxyl free radical is produced by the Fenton reaction and reacts with benzoate and releases yellowish-brown thio-barbituric acid reactive substances. On adding the serum sample, the oxidative reaction was initiated by the hydroxyl radical and was suppressed by the antioxidant components of serum, which prevented the color change and provided an effective measure of the TAC. The end product was measured by spectrophotometer at 532 nm and the inhibition of color development defined as the TAC.
Glutamate analysis in brain samples of control and N2O exposed animals The brain tissue sample of control and N2O exposed adult male wister rats were homogenized (10% w/v) in ice-cold 0.1 M phosphate-buffered saline (PBS) at pH 7.4. Samples were centrifuged at 10000 ×g for 10 minutes and were processed for the quantitative analysis of glutamate in brain homogenate. Glutamate concentration was determined by an enzymatic assay, which resulted in a colorimetric (λ = 450 nm) product, proportional to the glutamate present in the sample (Biovision, Inc. CA, US). Tissues were homogenized in 100µL of the glutamate assay buffer, and were centrifuged at 13,000 g for 10 minutes to remove insoluble material. The samples were brought to a final volume of 50µL and 100µl of the reaction mixture was added to each well containing the glutamate standard and test samples which were further incubated for 30 min at 37°C. During complete procedure for glutamate analysis all samples were
protected from the direct exposure of light. The optical density of the reaction end-product was measured at 450nm in a micro plate reader (Bio Tek Elx808). Glutamate concentrations were calculated using standard curves generated from a standard solution of glutamate, and results were expressed in nmol/mg of protein. Immunofluorescence imaging Microtomy: Followed by paraffin-embedded tissue blocks formation, 5µm coronal brain and spinal cord sections of control and N2O exposed rats were cut using microtome (Thermo, Electron Corporation). The sections were transferred onto poly-L-lysine (PLL) coated glass slides and were processed for further GFAP staining. Glial fibrillary acidic (GFAP) immunohistochemistry staining: GFAP immunostaining was performed using standard DAB protocol followed by deparaffinization procedure. Brain section and spinal cord sections (upper half part; thoracic region) were deparaffinised by treating with xylene for 8 min, then 100% ethyl alcohol, 95% ethyl alcohol, 70% ethyl alcohol, and water for 3 min each. Antigen unmasking was performed by microwaving tissue sections for 10 to 30 minutes in sodium citrate buffer. After cooling, tissue sections and cell smears were treated with 3% H2O2 for 30 minutes, washed in phosphate buffer saline (PBS), and then incubated with monoclonal primary antibody raised against rat GFAP (Cell Signaling Technology, Inc. Danvers, MA, US) for 18 hours at 4°C. After washing three times with 1xPBS, the slides were incubated with biotinylated secondary antibody (Cell Signaling Technology, Inc. Danvers, MA, US) for 30 minutes at room temperature then washed again for three times with 1xPBS. Sections were incubated in avidin-biotinhorseradish peroxidase solution (Vectastain elite ABC kit, Vector Laboratories) for 1 hour and then reacted with 3,30-diaminobenzidine (Vector Laboratories) for color development. Sections were washed three times with 1xPBS, counterstained with with hematoxylin
(Sigma–Aldrich; St Louis, MO) and mounted with glass coverslips using DPX. Images were acquired using light microscopy (Nikon Corporation, Tokyo, Japan) at 10x or 40 X magnification. Exposure times were kept constant for all sections in each experiment. Quantification and morphometric analysis for cell size were done using Image-J software (National Institutes of Health, Bethesda, MD, US) and the results were expressed as GFAP positive cell body area per square millimeter and GFAP score is in percent in N2O exposed and control animals. Experiments were repeated three times.
Behavioral studies Behavior analysis for N2O exposed rats and controls were performed by an investigator blinded to groups. Spontaneous Locomotor Activity (SLA): SLA of the control and N2O exposed rats were monitored at the end of exposure on day 60 in a computerized Optovarimex (Columbus Instruments, Ohio, USA) system. The rats were individually placed in the test apparatus, acclimatized for 5 min, and their activity scores were recorded for 3 sessions of 5 min each. Interruptions in the photo beams positioned in parallel, inside the chamber, represented the activity count, which was recorded in the data format. The following parameters; total distance, time resting, time moving, rearing and stereotypic count were measured. a.) Total distance (cm): Distance was measured from the angle of the movement along the walls of the cage or diagonally. b.) Time resting (sec): The duration during which there was no movement and the rat remained stationary and there was no interruption in infrared beams. c.) Time moving (sec): Time moving of the rats was measured while the animal was moving and infrered beam is interrupted.
d.) Rearing: Rearing of the rat was defined by the number of standing position on hind limbs. e.) Stereotypic count: The stereotypic count was measured by computing scratching, grooming of head and swing behavior in which animal was in the same location, but the infrared beam was interrupted by the movement of the body. Grip strength : The grip strength of the control and N2O exposed rats were measured by a computerized grip strength meter (TSE Germany) which consists of a 12x5 cm long wire mesh horizontal platform (1.3 x 1.3 cm2) and connected to the force gauge/ digitizing module by a steel rod.
Statistical analysis All quantitative data for controls and N2O exposed rats were expressed as Mean ± SD of the mean and were compared by using student-t test and Mann-Whitney U test. Astrocyte activation of cerebral cortex and spinal cord was correlated with the oxidative stress markers, Hcy, vitamin B12 and folic acid using Spearman or Karl-Pearson rank correlation test. Statistical analyses were performed using Graph-Pad Prism Program, Version 3.02 for Windows (GraphPad Software, San Diego, CA, USA). A p<0.05 was considered statistically significant.
Acknowledgement: We thank Mr. Shakti Kumar for secretarial help. SKS was supported by Research Associate Fellowship (File No- No.45/61/2015/PHA/BMS) from Indian Council of Medical Research
and AK was supported by the Ramalingaswami Fellowship (BT/RLF/Re-entry/13/2014) from Department of Biotechnology, Ministry of Science and Technology, India
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Legends to the Figure: Fig. 1. Bar graph showing behavioral and grip strength alterations after exposure to nitrous oxide. Spontaneous Locomotor Activity (SLA) of the rats was monitored after end of N2O exposure by computerized Optovarimex (Columbus Instruments, Ohio, USA) system. The rats were individually placed in the test apparatus, acclimatized for 5 min, and their activity scores were recorded for 3 sessions of 5 min each. Interruptions in the photo beams positioned in parallel, inside the chamber, resulted in an activity count and the following parameters of total distance, time resting (sec), time moving (sec) and rearing, stereotypic count were measured. In separate experiment the muscle grip strength was also measured by a computerized grip strength meter. As a result we observed that N2O exposed group had significant reduction in total distance (p<0.01; n=5), time moving (p<0.01; n=5), number of rearing (p<0.001; n=5), time resting (p<0.005; n=5) and decrease grip strength (p<0.01; n=5) compared to the controls. All data are representative of three independent experiments. Fig 2. The serum biochemical analysis after exposure to nitrous oxide. (A) There were no difference in serum vitamin B12 and folate in the N2O exposed and control groups, however, the serum Hcy level was found significantly higher in the N2O exposed compared to the control rats (p<0.001; n=5). (B, C) Glutamate and MDA level were found higher (p<0.01; n=5) in the N2O exposed animals compared to the controls. On the other hand GSH (p<0.01; n=5) and TAC (p<0.01; n=5) level were found significantly reduced in the N2O exposed rats compared to the controls (D). All data are representative of three independent experiments. Fig 3.(A) Representative GFAP-stained coronal sections of the control and N2O exposure rats are shown. Astrocyte found highly activated in phenotype in the cortex sub region of brain,
Images were clicked at 10x and scale bar = 100 m. (B) Image J software
reconstruction of activated astrocyte cells / GFAP staining depicted the cell morphologic
features. N2O exposure resulted in a increment in GFAP positive cell body area in N2O expose rat group versus control group. (C) Similarly, GFAP positive score analysis in brain shows GFAP expression significantly increased (p< 0.001; n=5) in brain cortex in compare to control animals. All data are representative of three independent experiments. Fig 4.(A) Representative GFAP-stained spinal cord region of the control and N2O exposure rats are shown. Astrocyte found highly activated in phenotype in the thoracic region of spinal cord. Images were clicked at 20x and Scale bar = 50 µm, (B) N2O exposure resulted in a significant increase (p< 0.001; n=5) in GFAP positive score in spinal cord compare to control animals. All data are representative of three independent experiments. Fig 5 (A, B). Significant correlations were found with homocystein and TAC level with glutathione in blood. (C-H) Regression curve shows variables having significant correlation of glial fibrillary acidic protein (GFAP) score of brain and spinal cord with glutamate, MDA, glutathione, Hcy and TAC respectively. Fig 6. Regression curve shows variables having significant correlation of glial fibrillary acidic protein (GFAP) score of brain (A-C) and spinal cord (D-H) with spontaneous locomotor activity parameters and grip strength. Fig. 7. Schematic diagram for N2O toxicity, myelin dysfunction, imbalance in oxidant/ antioxidant and alteration in astrocyte activation phenotype.
Astrocyte activation following nitrous oxide exposure is related to oxidative stress and glutamate excitotoxicity
Table 1. Comparison of glial fibrillary acidic protein score in brain and spinal cord, level of oxidative stress markers, glutamate level in the rats exposed nitrous oxide and controls
Variables
GFAP score in brain
Control
N2O exposed
p-value
(Mean±SD; n=5)
(Mean±SD; n=5)
29.72 ± 6.71
41.73 ± 1.14
0.004
39.03 ± 1.06
62.08 + 1.50
<0.001
11.72 ± 2.70
20.56 ± 2.89
0.001
173.80 ± 5.71
164 ± 12.17
0.165
4.65 ± 6.17
6.56 ± 5.63
0.624
3.4 ±0.80
5.10 ± 0.49
0.003
3.41 ± 0.19
2.35 ± 0.55
0.01
1.51 ± 0.31
0.78 ± 0.12
0.004
21.50 ± 0.23
22.51 ± 0.67
0.01
(%) GFAP score in spinal cord (%) Hcy (µmol/l) Vitamin B12 (pg/ml) Folic acid (pg/ml MDA (nmol Trolox eq/l) GSH (mg/dl) TAC (nmol/ml) Glutamate (nmol/mg protein)
GSH=Glutathione, MDA= Malanodialdehyde, TAC= Total antioxidant capacity
Author contribution U.K. and J.K. developed the conceptual idea of the project, designed the study and write the manuscript SK conducted the experiments, collected the samples, analyze the data and help in manuscript writing. AK conducted the experiments, analyze the data and help in manuscript writing. All authors reviewed the manuscript.
1- Glial fibrillary acidic protein (GFAP) was more expressed in N2O exposed group. 2- GFAP expression was higher in spinal cord compared to brain. 3- GFAP expression correlated with neurobehavioral changes, oxidative stress and
glutamate level.
S0006-8993(20)30001-9 https://doi.org/10.1016/j.brainres.2020.146645 BRES 146645
To appear in:
Brain Research
Received Date: Revised Date: Accepted Date:
22 August 2019 10 December 2019 2 January 2020
Please cite this article as: U.K. Misra, S.K. Singh, J. Kalita, A. Kumar, Astrocyte activation following nitrous oxide exposure is related to oxidative stress and glutamate excitotoxicity, Brain Research (2020), doi: https://doi.org/ 10.1016/j.brainres.2020.146645
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© 2020 Published by Elsevier B.V.
Astrocyte activation following nitrous oxide exposure is related to oxidative stress and glutamate excitotoxicity Usha Kant Misra1; Sandeep Kumar Singh1; Jayantee Kalita1; Alok Kumar1,2 1Department
of Neurology, Sanjay Gandhi Post Graduate Institute of Medical Sciences,
Raebareily Road, Lucknow, 226014, Uttar Pradesh, India. 2Department
of Molecular Medicine and Biotechnology, Sanjay Gandhi Post Graduate
Institute of Medical Sciences, Raebareily Road, Lucknow, 226014, Uttar Pradesh, India
Corresponding authors: Usha K. Misra Professor Department of Neurology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareily Road, Lucknow226014 Phone: +91 522 2494177; FAX: 091-0522-2668811 Email: [email protected]; [email protected]
Abstract: Background and Aims: Nitrous oxide is commonly used as an anesthetic agent and its exposure produces prolonged inactivation of vitamin B12. Vitamin B12 is associated with central nervous system changes which are similar to sub-acute combined degeneration (SACD). Astrocytes have important role in neurotoxic toxic injuries, but have not been evaluated in N2O toxicity. In the present study we have evaluated the changes in astrocyte in N2O exposed rats and correlated with neurobehavioral changes, oxidative stress and glutamate level. Material and Methods: Adult wistar male rats were exposed to N2O oxygen mixture in 1:1 ratio at a rate of 2 L/min for 120 min for 60 days. Control rats underwent similar exposure to oxygen. At the end of exposure, spontaneous locomotors activity (total distance travelled, time resting, time moving, number of rearing, stereotypic count) and grip strength were evaluated. Plasma glutathione (GSH), Total antioxidant capacity (TAC), serum malonodialdehyde
(MDA)
and
serum
homocysteine
(Hcy)
were
measured
by
spectrophotometer. Glutamate in the cerebral cortex and cerebellum were measured by colorimetry. Immunohistochemistry for astrocyte (GFAP) phenotypic analysis and its activation in brain and spinal cord were measured by using using image J software in N2O exposed rats and control animals. Results: The N2O exposed rats had significant reduction in total distance travelled, time moving and number of rearing whereas time resting increased compared to the control animals. Hcy, glutamate and MDA levels were significantly increased, however GSH and TAC level decreased in N2O exposed group compared to the controls. Astrocyte phenotype and its activation was significantly altered more so in spinal cord compared to cerebral cortex and was associated with neurobehavioral changes, oxidative stress and glutamate level.
Conclusions: N2O related clinical dysfunction may be related to changes in astrocyte activation which is related to oxidative stress and glutamate neurotoxicity. Keywords: Nitrous oxide; astrocyte activation, oxidative stress; glutamate level; total antioxidant capacity; homocysteine
Introduction: Nitrous oxide initially has been used as a recreational gas and an anesthetic agent for minor surgery such as dental extraction. In 1852, Lassen and colleague reported megaloblastic bone marrow after prolonged N2O exposure [1, 2]. Nitrous oxide inhibits the enzyme methionine synthase (MeS) resulting in hyperhomocysteinemia. Methionine is important for synthesis of DNA, RNA, catecholamine and myelin [3]. Prolonged exposure to N2O is reported to result in subacute combined degeneration (SACD) like illness [4,5]. Controlled N2O exposure to rats, results cobalamin (Cbl) deficiency like illness resulting in demyelination of central and peripheral nervous system [6-8]. In SACD, the most prominent changes occur in spinal cord resulting in spongy vacuolation, intramyelin, interstitial and white matter edema and gliosis [8]. In the patients with SACD, and in the rats exposed to N2O , we have reported increased oxidative stress evidenced by increased malonodialdehyde (MDA), reduced glutathione (GSH) and total antioxidant capacity (TAC) levels [8,9]. In SACD, astrogliosis and increased glial fibrillary acidic protein (GFAP) expression have also been reported [7,10]. Imbalance of tumor necrosis factor α (TNF-α) with epidermal growth factor (EGF) and IL6 have been reported to be associated with the pathological changes in SACD [7]. It is possible similar changes may also occur following N2O toxicity, but its cellular mechanisms have not been evaluated. Following N2O exposure, we have reported hyperhomocystinemia and focal myelin loss, vacuolation in subcortical white matter and spinal cord [8]. Keeping the analogy of SACD, using totally gastrectomized (TGX) mice model reported spongy vacuolation, intramyelin and interstitial edema in white matter of CNS and astrogliosis [7]. Astrogliosis is the dominant and universal response of the nervous system injury. GFAP is the most studied markers of astrocyte activation
and proliferation [22]. The basis of myelin
dysfunction and loss in N2O exposed rats has not been investigated on these lines. The cytokine release, oxidative stress
are closely related to astrocyte activation [23].
Hyperhomocysteinemia causes astrocyte activation, vascular cognitive impairment and dementia [24]. Moreover, in recent In vitro study, Weekman et al (2017) [25] showed that hyperhomocysteinemia induces early proinflammatory changes in astrocytes, which are relevant to their interaction with the vasculature. A study of oxidative stress , glutamate exictotoxicty and glial dysfunction may provide valuable information about the mechanism of myelin dysfunction in N2O exposure model. Oxidative stress and glutamate excitotoxicity may also contribute to astrogliosis, which has not been reported in cobalamine deficiency. In the present study, we report the changes in astrocytes in brain and spinal cord in N2O exposed animals. We also report the psychomotor activity of rats and correlate this with GFAP score in brain and spinal cord.
Results:
Nitrous oxide exposure altered behavioral and neurological function: None of the animal died during the study period. The body weight of the N2O exposed group at the end of the study was 200.60±16.02 gm and that of controls was 245.6±6.3 gm (p<0.01). The N2O exposed group had significant alteration in spontaneous locomotor activity which includes reduction in total distance, time moving, number of rearing, and time resting when compared to the control animals (Fig. 1; p<0.01).
The grip strength was significantly
decreased in the N2O exposed group compared to the control animals (Fig. 1; p<0.01) (Table: 1).
Homocysteine level increases followed by nitrous oxide exposure
There was no significant difference in serum vitamin B12 and folic acid level in the control groups compared to N2O exposed rats. Whereas, Hcy level was significantly higher in the N2O exposed rats compared to the control rats (Fig. 2A; p<0.001[Hcy] (Table: 1).
Glutamate level and lipid peroxidation is increased followed by nitrous oxide exposure and is associate with reduced glutathione, TAC antioxidant level The plasma GSH and TAC level was significantly reduced in the N2O exposed rats compared to the control group (Fig 2D; p<0.01[GSH, TAC]). However, the serum MDA and cerebral cortex glutamate level were higher in the control group versus N2O exposed rat (Fig.2 B,C; p<0.01 [MDA, glutamate level]) (Table: 1).
Highly reactive astrocyte activation phenotypes predominate brain and spinal cord followed by nitrous oxide exposure GFAP score analysis shows increase in GFAP expression (astrocyte activation) in the brain and spinal cord (p<0.01 [brain GFAP score] and p<0.001 [spinal cord GFAP score]) in the N2O exposed rats compared to the controls (Fig. 3C, 4B). Image J software reconstruction of activated astrocyte cells represented that N2O exposure resulted in a increment in GFAP positive cell body area in the cerebral cortex of N2O exposed rat versus control group (Fig. 3 B). Similarly, N2O exposure resulted in a increase in GFAP positive cell body area in spinal cord sub-region of thoracic in control group versus N2O expose rat group (Fig. 4A). However, comparatively GFAP positive score analysis in brain and spinal cord shows GFAP expression significantly increased in spinal cord (1.48 fold) compared to brain cortical subregion (Table:1). Astrocyte activation correlated with neurobehavioral and biochemical parameters in brain and spinal cord following nitrous oxide exposure
Correlation study for astrocyte (GFAP score) in brain and spinal cord with biochemical parameters revealed a positive correlation for glutamate (r=0.72; Fig. 5C), MDA (r= 0.85; Fig. 5D) with brain astrocyte and with Hcy (r=0.87; Fig. 5F) with spinal cord astrocyte where as a negative correlation was observed with TAC (r=-0.86; Fig. 5G) in spinal cord and glutathione in both brain (r=-0.69; Fig. 5E) and spinal cord (r=-0.83; Fig. 5H). However, the blood biomarker correlation analysis between antioxidant glutathione and Hcy, TAC revealed that glutathione was positively correlated with TAC (r=0.57; Fig. 5B) and negatively correlated with Hcy (r=-0.88; Fig. 5A) following N2O exposure.
Further correlation study for astrocyte (GFAP score) in brain and spinal cord with neurobehavioral parameters revealed that activation of astrocyte in cerebral cortex was positively correlated with time resting (r=0.79; Fig. 6A) in cerebral cortex region, total distance covered (r=-0.84; Fig. 6D), numbers of rearing (r=-0.86; Fig. 6G) and grip strength (r=-0.79; Fig. 6H) in spinal cord, whereas there was a a negative correlation with time moving (r=0.79; Fig. 6B), number of rearing (r=-0.76; Fig. 6C) in brain region and time moving (r= -0.883; Fig. 6F) , number of rearing (r=-0.86; Fig. 6G) and grip strength (r=0.79; Fig. 6H)
in spinal cord region.
DISCUSSION: In the present study nitrous oxide exposure resulted in oxidative stress, glutamate excitotoxicity, glial acitvations which was related to altered spontaneous locomotor activity and grip strength. Oxidative stress and DNA damage have been reported following N2O
exposure. The patients undergoing N2O based anesthesia revealed higher postoperative Hcy levels compared to non N2O based anesthesia [13]. In another study, 15 anesthesia or surgery residents were exposed to N2O with isoflurane or sevoflurane as anesthetic agent during their operation theater duty. The reactive oxygen species (ROS) was increased in N2O exposed residents resulting in DNA damage as evaluated by comet assay compared to controls [14]. Similar results have also been reported in 36 nurses exposed to N2O based anesthesia. In the exposed group, the oxidative stress markers namely ROS in leucocytes, α tocopherol and glutathione peroxidase activity were measured. The DNA damage was attributed to increased oxidative stress [15]. In the above mentioned study, the subjects were exposed to anesthetic agents in addition to N2O (isoflurane and sevoflurane), which could also induce DNA damage. In the present study, we have used only N2O and O2 mixture and compared with the controls who received only oxygen; thereby excluding the effects of oxygen. In an earlier study using 1.5L of N2O for 90 min for 1 month revealed reduced levels of GSH, TAC, and increased level of MDA suggesting oxidative stress in the N2O exposed group. In the present study N2O exposed group also developed higher Hcy level. The Histopathological studies following N2O exposure revealed shrinkage and vacuolation of neurons in the cerebral cortex, and focal myelin damage and vacuolation in the subcortical white matter and in the spinal cord [8]. The spinal cord and subcortical white matter myelin damage following N2O toxicity may be due to vitamin B12 inactivation resulting in SACD like changes. In the patients undergoing N2O based anesthesia, and occupational N2O exposure, SACD like illness has been reported [16]. Nitrous oxide inactivates vitamin B12 by oxidizing Cbl+ into Cbl+++. Vitamin B12 is an important cofactor for conversion of Hcy to methionine by the enzyme methionine synthase, and Hcy to cystathione by the enzyme cystathione β synthase [17-19].
We have noted high level of glutamate in N2O exposed rats . Increased level of glutamate requires the scavenging role of astrocytes thereby resulting in astrocytic proliferations. As the capacity of astrocytes to scavenge glutamate is exhausted, the excitotoxicity ensues. We have noted high level of astrocyte in N2O exposed rats. Higher level of astrocyte activation in the spinal cord compared to brain may be consistent with the grater vulnerability of spinal cord. In the patients with SACD compared to brain. The spinal cord MRI characteristically reveals posterior T2 hyper intensity or atrophy in thoracodorsal region in 73.9 % and in subcortical white matter in 25 % [20]. Cobalamin deficiency primarily affects the glial cells. Labelled cobalamin is almost exclusively taken up by glial cells of spinal cord white matter [30]. Human astrocyte culture in cobalamine deficient medium has shown be damaged [31] but gliosis in TGX rats has not correlated with the severity of SACD like illness nor which is different from that in humans [32]. In the patients with SACD, increase in oxidative stress and proinflammatory cytokine has been reported [7,9]. Increased oxidative stress and reduced methionine per se also result in myelin damage. These factors may induce excitotoxic injury in N2O exposed group. In our study there was increase in glutamate, which may be due to increase in Hcy and oxidative stress. Glutamate is the major neurotransmitter of brain, and the excess glutamate from synaptic cleft is cleared by astrocytes which may result in proliferation of astrocytes. Increased GFAP expression in the present study may be due to astrocytic proliferation. In TGX mouse model of vitamin B12 deficiency, increased GFAP expression has been reported [6]. In vitamin B12 deficiency, astrocytes bear the brunt of attack, and the neurons are spared. This may be the reason for good recovery of patients following vitamin B12 treatment [21]. Vitamin B12 priming before N2O based anesthesia reduces its toxicity [4]. In the present study, the GFAP expression in the spinal cord and brain correlated with neurobehavioral
findings. In our earlier study on N2O toxicity, neurobehavioral findings correlated with oxidative stress and histopathological changes [8]. Reactive oxygen species are produced in the astrocytes by the enzyme NADPH oxidase [33] which is also balanced by antioxidant system [34]. The astrocytes also initiate an inflammatory response through
proinflammatory cytokines. In this study, we have
provided evidence on the role of oxidaitves stess , glutamate excitotoxicity and astrocyte activation in the process of behavioral alterations following N2O exposure. The limitation of the present study is that due to snap frozen tissue sample collection procedure, the glutamate, GSH, TAC and MDA levels in brain and spinal cord tissue were not evaluated, which could have provided better insight about the role of oxidative stress in the respective area and its relationship with GFAP. From this study, it can be concluded that N2O toxicity results in alteration of astrocyte phenotype which is related to oxidative stress and glutamate excitotoxicity and is more marked in the spinal cord than in brain (Fig. 7).
Conclusions: N2O related clinical dysfunction may be related to changes in astrocyte activation which is related to oxidative stress and glutamate neurotoxicity.
Materials and methods: Studies were performed using adult male wistar rats (10-12 weeks old, 200-250 gm). Rats were housed in the Animal Care facility at Sanjay Gandhi postgraduate Institute of Medical Sciences, Lucknow a 12h light-dark cycle, with ad libitum access to food and water. All exposure and surgical procedures were carried out in accordance with protocols approved by
the Institutional Animal Ethics Committee of Sanjay Gandhi postgraduate Institute of Medical Sciences, Lucknow. N2O exposure N2O exposure experiments were performed as previously described [8]. The experimental rats were acclimatized for 10-15 days in whole body exposure chamber. After three weeks of acclimatization, rats were exposed to a N2O oxygen mixture in 1:1 ratio at a rate of 2 L/min for 120 min for 60 days. Control rats underwent the same procedure as N2O exposure except N2O was replaced by a mixture of oxygen and room air (1:1) in the whole body exposure chamber. The rats were examined daily for red nose, ataxia or any other abnormal neurological finding. The neurobehavioral study included spontaneous locomotor activity (SLA) parameters like total distance travelled, time resting, time moving, number of rearing, stereotypic count and grip strength. These parameters were also measured after 60 days of exposure before the animals were sacrificed. Study 1 Control (n = 5-10) and N2O exposed (n = 5-10) adult male wister rats were anesthetized (by using ether) at 60 days. Fasting blood was collected in plain and EDTA vial following cardiac puncture. Serum and plasma were separated and stored at -800C. Rats were transcardially perfused with ice cold 0.1 M phosphate-buffered saline (150 ml; pH 7.2) from left ventricle following thoracotomy.
Brain tissue was rapidly dissected and snap-frozen on liquid
nitrogen for glutamate analysis. Data represent three independent experiments. Study 2 Control (n = 5-10) and N2O exposed (n = 5-10) adult male wister rats were used for behavioral studies. At 60 days post N2O exposure, the rats were anesthetized (by using ether)
and were transcardially perfused with ice cold 0.1 M phosphate-buffered saline (150 ml; pH 7.2) followed by 250 ml of 4% paraformaldehyde from left ventricle following thoracotomy. Brain and spinal cord were removed and post-fixed in 4% paraformaldehyde overnight, and cryoprotected in 30% sucrose and were processed for immunohistochemistry analysis. Biochemical studies Analysis for homocysteine, vitamin B12 and folate in control and N2O exposed animals Homocysteine, vitamin B12 and folate levels were measured in fresh serum samples by using chemiluminescence analyzer (IMMULITE 1000, M/s Siemens Ltd. USA and Siemens Healthcare Diagnostics Products Limited, Glyn Rhonwy, Llanberis, Caernarfon, Gwynedd LL55 4EL, UK).
Analysis for lipid peroxidation, glutathione and total antioxidant capacity in control and N2O exposed animals Lipid Peroxidation (LPO) assay: LPO was measured in fresh plasma samples of control and N2O exposed animals by assaying malonodialdehyde (MDA) level, which is the end product of LPO [11]. Plasma was mixed with EDTA, ascorbate (10 mM), and FeSO4 (16.7 mM), and incubated at 37°C for 60 min. The reaction was stopped by adding ice-cold 10% trichloroacetic acid. The mixture was centrifuged at 2000 × g for 10 min, and the supernatant was aspirated. The supernatant was mixed with equal volume of 0.67 % thiobarbituric acid and was kept in a boiling water bath for 15 to 20 min. MDA level was measured with the absorption coefficient of MDA- thiobarbituric acid complex at 532 nm using spectrophotometer. Glutathione (GSH) assay: GSH was measured in fresh plasma samples of control and N2O exposed animals using spectrophotometer employing Tietze method (1969). Plasma sample were added to 10% trichloroacetic acid and were incubated for 2 hr at 40C. The sample were
further centrifuged at 2000xg for 15 min, and the supernatant were collected and added to fresh Tris HCl buffer (0.4 M, pH 8.9 containing EDTA 0.02 M). At the end of the experiment, 5,5 di-thio-bis (2-nitrobenzoic acid 0.01 M) was added to the reaction mixture and end yellow product (5’ thio’ 2 nitrobenzoic acid) was measured by using spectrophotometer at 412 nm. Total antioxidant capacity (TAC) assay: TAC was measured in fresh serum sample of control and N2O exposed animals by using the method of Koracevic et al [12]. The intermediate hydroxyl free radical is produced by the Fenton reaction and reacts with benzoate and releases yellowish-brown thio-barbituric acid reactive substances. On adding the serum sample, the oxidative reaction was initiated by the hydroxyl radical and was suppressed by the antioxidant components of serum, which prevented the color change and provided an effective measure of the TAC. The end product was measured by spectrophotometer at 532 nm and the inhibition of color development defined as the TAC.
Glutamate analysis in brain samples of control and N2O exposed animals The brain tissue sample of control and N2O exposed adult male wister rats were homogenized (10% w/v) in ice-cold 0.1 M phosphate-buffered saline (PBS) at pH 7.4. Samples were centrifuged at 10000 ×g for 10 minutes and were processed for the quantitative analysis of glutamate in brain homogenate. Glutamate concentration was determined by an enzymatic assay, which resulted in a colorimetric (λ = 450 nm) product, proportional to the glutamate present in the sample (Biovision, Inc. CA, US). Tissues were homogenized in 100µL of the glutamate assay buffer, and were centrifuged at 13,000 g for 10 minutes to remove insoluble material. The samples were brought to a final volume of 50µL and 100µl of the reaction mixture was added to each well containing the glutamate standard and test samples which were further incubated for 30 min at 37°C. During complete procedure for glutamate analysis all samples were
protected from the direct exposure of light. The optical density of the reaction end-product was measured at 450nm in a micro plate reader (Bio Tek Elx808). Glutamate concentrations were calculated using standard curves generated from a standard solution of glutamate, and results were expressed in nmol/mg of protein. Immunofluorescence imaging Microtomy: Followed by paraffin-embedded tissue blocks formation, 5µm coronal brain and spinal cord sections of control and N2O exposed rats were cut using microtome (Thermo, Electron Corporation). The sections were transferred onto poly-L-lysine (PLL) coated glass slides and were processed for further GFAP staining. Glial fibrillary acidic (GFAP) immunohistochemistry staining: GFAP immunostaining was performed using standard DAB protocol followed by deparaffinization procedure. Brain section and spinal cord sections (upper half part; thoracic region) were deparaffinised by treating with xylene for 8 min, then 100% ethyl alcohol, 95% ethyl alcohol, 70% ethyl alcohol, and water for 3 min each. Antigen unmasking was performed by microwaving tissue sections for 10 to 30 minutes in sodium citrate buffer. After cooling, tissue sections and cell smears were treated with 3% H2O2 for 30 minutes, washed in phosphate buffer saline (PBS), and then incubated with monoclonal primary antibody raised against rat GFAP (Cell Signaling Technology, Inc. Danvers, MA, US) for 18 hours at 4°C. After washing three times with 1xPBS, the slides were incubated with biotinylated secondary antibody (Cell Signaling Technology, Inc. Danvers, MA, US) for 30 minutes at room temperature then washed again for three times with 1xPBS. Sections were incubated in avidin-biotinhorseradish peroxidase solution (Vectastain elite ABC kit, Vector Laboratories) for 1 hour and then reacted with 3,30-diaminobenzidine (Vector Laboratories) for color development. Sections were washed three times with 1xPBS, counterstained with with hematoxylin
(Sigma–Aldrich; St Louis, MO) and mounted with glass coverslips using DPX. Images were acquired using light microscopy (Nikon Corporation, Tokyo, Japan) at 10x or 40 X magnification. Exposure times were kept constant for all sections in each experiment. Quantification and morphometric analysis for cell size were done using Image-J software (National Institutes of Health, Bethesda, MD, US) and the results were expressed as GFAP positive cell body area per square millimeter and GFAP score is in percent in N2O exposed and control animals. Experiments were repeated three times.
Behavioral studies Behavior analysis for N2O exposed rats and controls were performed by an investigator blinded to groups. Spontaneous Locomotor Activity (SLA): SLA of the control and N2O exposed rats were monitored at the end of exposure on day 60 in a computerized Optovarimex (Columbus Instruments, Ohio, USA) system. The rats were individually placed in the test apparatus, acclimatized for 5 min, and their activity scores were recorded for 3 sessions of 5 min each. Interruptions in the photo beams positioned in parallel, inside the chamber, represented the activity count, which was recorded in the data format. The following parameters; total distance, time resting, time moving, rearing and stereotypic count were measured. a.) Total distance (cm): Distance was measured from the angle of the movement along the walls of the cage or diagonally. b.) Time resting (sec): The duration during which there was no movement and the rat remained stationary and there was no interruption in infrared beams. c.) Time moving (sec): Time moving of the rats was measured while the animal was moving and infrered beam is interrupted.
d.) Rearing: Rearing of the rat was defined by the number of standing position on hind limbs. e.) Stereotypic count: The stereotypic count was measured by computing scratching, grooming of head and swing behavior in which animal was in the same location, but the infrared beam was interrupted by the movement of the body. Grip strength : The grip strength of the control and N2O exposed rats were measured by a computerized grip strength meter (TSE Germany) which consists of a 12x5 cm long wire mesh horizontal platform (1.3 x 1.3 cm2) and connected to the force gauge/ digitizing module by a steel rod.
Statistical analysis All quantitative data for controls and N2O exposed rats were expressed as Mean ± SD of the mean and were compared by using student-t test and Mann-Whitney U test. Astrocyte activation of cerebral cortex and spinal cord was correlated with the oxidative stress markers, Hcy, vitamin B12 and folic acid using Spearman or Karl-Pearson rank correlation test. Statistical analyses were performed using Graph-Pad Prism Program, Version 3.02 for Windows (GraphPad Software, San Diego, CA, USA). A p<0.05 was considered statistically significant.
Acknowledgement: We thank Mr. Shakti Kumar for secretarial help. SKS was supported by Research Associate Fellowship (File No- No.45/61/2015/PHA/BMS) from Indian Council of Medical Research
and AK was supported by the Ramalingaswami Fellowship (BT/RLF/Re-entry/13/2014) from Department of Biotechnology, Ministry of Science and Technology, India
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Legends to the Figure: Fig. 1. Bar graph showing behavioral and grip strength alterations after exposure to nitrous oxide. Spontaneous Locomotor Activity (SLA) of the rats was monitored after end of N2O exposure by computerized Optovarimex (Columbus Instruments, Ohio, USA) system. The rats were individually placed in the test apparatus, acclimatized for 5 min, and their activity scores were recorded for 3 sessions of 5 min each. Interruptions in the photo beams positioned in parallel, inside the chamber, resulted in an activity count and the following parameters of total distance, time resting (sec), time moving (sec) and rearing, stereotypic count were measured. In separate experiment the muscle grip strength was also measured by a computerized grip strength meter. As a result we observed that N2O exposed group had significant reduction in total distance (p<0.01; n=5), time moving (p<0.01; n=5), number of rearing (p<0.001; n=5), time resting (p<0.005; n=5) and decrease grip strength (p<0.01; n=5) compared to the controls. All data are representative of three independent experiments. Fig 2. The serum biochemical analysis after exposure to nitrous oxide. (A) There were no difference in serum vitamin B12 and folate in the N2O exposed and control groups, however, the serum Hcy level was found significantly higher in the N2O exposed compared to the control rats (p<0.001; n=5). (B, C) Glutamate and MDA level were found higher (p<0.01; n=5) in the N2O exposed animals compared to the controls. On the other hand GSH (p<0.01; n=5) and TAC (p<0.01; n=5) level were found significantly reduced in the N2O exposed rats compared to the controls (D). All data are representative of three independent experiments. Fig 3.(A) Representative GFAP-stained coronal sections of the control and N2O exposure rats are shown. Astrocyte found highly activated in phenotype in the cortex sub region of brain,
Images were clicked at 10x and scale bar = 100 m. (B) Image J software
reconstruction of activated astrocyte cells / GFAP staining depicted the cell morphologic
features. N2O exposure resulted in a increment in GFAP positive cell body area in N2O expose rat group versus control group. (C) Similarly, GFAP positive score analysis in brain shows GFAP expression significantly increased (p< 0.001; n=5) in brain cortex in compare to control animals. All data are representative of three independent experiments. Fig 4.(A) Representative GFAP-stained spinal cord region of the control and N2O exposure rats are shown. Astrocyte found highly activated in phenotype in the thoracic region of spinal cord. Images were clicked at 20x and Scale bar = 50 µm, (B) N2O exposure resulted in a significant increase (p< 0.001; n=5) in GFAP positive score in spinal cord compare to control animals. All data are representative of three independent experiments. Fig 5 (A, B). Significant correlations were found with homocystein and TAC level with glutathione in blood. (C-H) Regression curve shows variables having significant correlation of glial fibrillary acidic protein (GFAP) score of brain and spinal cord with glutamate, MDA, glutathione, Hcy and TAC respectively. Fig 6. Regression curve shows variables having significant correlation of glial fibrillary acidic protein (GFAP) score of brain (A-C) and spinal cord (D-H) with spontaneous locomotor activity parameters and grip strength. Fig. 7. Schematic diagram for N2O toxicity, myelin dysfunction, imbalance in oxidant/ antioxidant and alteration in astrocyte activation phenotype.
Astrocyte activation following nitrous oxide exposure is related to oxidative stress and glutamate excitotoxicity
Table 1. Comparison of glial fibrillary acidic protein score in brain and spinal cord, level of oxidative stress markers, glutamate level in the rats exposed nitrous oxide and controls
Variables
GFAP score in brain
Control
N2O exposed
p-value
(Mean±SD; n=5)
(Mean±SD; n=5)
29.72 ± 6.71
41.73 ± 1.14
0.004
39.03 ± 1.06
62.08 + 1.50
<0.001
11.72 ± 2.70
20.56 ± 2.89
0.001
173.80 ± 5.71
164 ± 12.17
0.165
4.65 ± 6.17
6.56 ± 5.63
0.624
3.4 ±0.80
5.10 ± 0.49
0.003
3.41 ± 0.19
2.35 ± 0.55
0.01
1.51 ± 0.31
0.78 ± 0.12
0.004
21.50 ± 0.23
22.51 ± 0.67
0.01
(%) GFAP score in spinal cord (%) Hcy (µmol/l) Vitamin B12 (pg/ml) Folic acid (pg/ml MDA (nmol Trolox eq/l) GSH (mg/dl) TAC (nmol/ml) Glutamate (nmol/mg protein)
GSH=Glutathione, MDA= Malanodialdehyde, TAC= Total antioxidant capacity
Author contribution U.K. and J.K. developed the conceptual idea of the project, designed the study and write the manuscript SK conducted the experiments, collected the samples, analyze the data and help in manuscript writing. AK conducted the experiments, analyze the data and help in manuscript writing. All authors reviewed the manuscript.
1- Glial fibrillary acidic protein (GFAP) was more expressed in N2O exposed group. 2- GFAP expression was higher in spinal cord compared to brain. 3- GFAP expression correlated with neurobehavioral changes, oxidative stress and
glutamate level.