Therapeutic hypothermia reduces oxidative stress in newborns with hypoxic-ischemic encephalopathy

Therapeutic hypothermia reduces oxidative stress in newborns with hypoxic-ischemic encephalopathy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Therapeutic hypothermia reduces oxidative stress in newborns with hypoxic-ischemic encephalopathy ELİF ÖZALKAYA, Apdulhamid Tüten, Sevilay Topcuoğlu, Hande Özgün Karatepe, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5692677/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: To evaluate the effect of therapeutic hypothermia (TH) on oxidative stress in neonates with hypoxic-ischemic encephalopathy(HİE). Method: This study was designed as a prospective study. Sixteen term neonates diagnosed with HİE who were admitted for TH in a neonatal intensive care unit within one year were included in the study. The oxidative stress markers (Total Antioxidant Capacity ( TAC) and Total Oxidative Stress (TOS) in venous blood were measured before and after TH. Oxidative stress index(OSI) was calculated as the percent ratio of TOS to TAS. Results: After TH, the TAC increased compared to before (p=0.001), while TOS (p=0.005) and OSI (p=0.001) decreased. Conclusion: This study demonstrates that therapeutic hypothermia applied to newborns with hypoxic-ischemic encephalopathy reduces oxidative stress. Adjuvant treatments that reduce oxidative stress may enhance the effect of hypothermia. 1. Introduction The neonatal brain is highly susceptible to hypoxia due to its high concentration of sensitive immature cells, free radicals, unsaturated fatty acids, low levels of antioxidant enzymes, and high oxygen demand. Following hypoxia and ischemia in the neonatal brain, the production of prooxidant agents increases, while antioxidant agents becomes insufficient. An increased prooxidant state (oxidative stress) occurs due to the disruption of the balance between pro-oxidant and antioxidant agents. Oxidative stress directly degenerate or modify cellular macromolecules such as membranes, proteins, lipids, and DNA, triggering the inflammatory response cascade and protease secretion. These processes lead to inflammation, apoptosis, autophagy, and necrosis, culminating in brain injury and the clinical presentation of neonatal hypoxic-ischemic encephalopathy (HIE) (1). Therapeutic hypothermia (TH) reduces brain damage and alleviates severe neurological effects in neonates with moderate to severe HIE (2). By decreasing brain metabolism, TH mitigates damage caused by secondary brain energy failure (3). In vitro and animal studies have shown that TH reduces oxidative stress by decreasing pro-oxidant agents (4). However, human studies evaluating the effects of TH on oxidative stress are limited (5). Although hypothermia is the standard treatment, its high cost and limited success rate have driven numerous studies on adjuvant therapies (6–9). Research focusing on adjuvant treatments that play a key role in TH's mechanisms of action holds promise for advancing HIE treatment. This study aimed to investigate whether TH affects oxidative stress in neonates with HİE. It is anticipated that the findings will guide the development of adjuvant therapies. 2. Materials and Methods 2.1.Settings and Study Population This study was conducted as a single-center, prospective study between January 2021 and December 2021 in the Level IV neonatal intensive care unit of Zeynep Kamil Women's and Children's Health Training and Research Hospital, where approximately 13,000 births occur annually. The study was approved by the Ethics Committee of Zeynep Kamil Women's and Children's Health Training and Research Hospital.Ethics committee approval dated 03.03.2021, number 59, was obtained. 2.2 Data Collection TH was applied within the first 6 hours of postnatal life to neonates born at or after 36 weeks of gestation who met the following criteria(10): 1. Venous blood gas analysis performed on the umbilical cord or within the first hour after birth revealed pH ≤ 7.00 or BE ≤-16 mmol/L. 2. Apgar score < 5 at the 10th minute or the need for ongoing resuscitation. 3. Clinical signs of moderate HIE (lethargic consciousness, hypotonic tone, flexion posture, hyperactive DTRs, presence of myoclonus, poor Moro reflex, miotic pupils, presence of seizures, and continuous low voltage or seizure activity on aEEG) and/or severe HIE (coma state, flaccid tone, decerebrate posture, undetectable DTRs, absence of myoclonus, undetectable Moro reflex, anisocoric pupils, and burst suppression or flat line on aEEG)(11). Before the initiation of TH, venous blood was collected from the neonates for oxidative stress markers. TH was applied using a servo-controlled device (Techotherm Neo) as total body cooling, maintaining a rectal temperature of 34.5°C for 72 hours. After TH, the body temperature of the 16 neonates was gradually increased at a rate of 0.5°C per hour using the same device. On the 4th postnatal day, venous blood samples were collected again for oxidative stress markers. Demographic data of the patients who underwent therapeutic hypothermia, 1st and 5th-minute Apgar scores, the frequency of moderate and severe hypoxic-ischemic encephalopathy, 24-hour amplitude-integrated EEG (Natus aEEG device) monitoring findings, echocardiography (ECHO) findings, cord blood gas or blood gas obtained within the first hour (pH, CO2 (mmHg), BE (mmol/L), lactate (mmol/L)), and the laboratory findings on the first day including creatine kinase (CK, U/L), creatine kinase-MB (CK-MB, U/L), lactate dehydrogenase (LDH, U/L), and uric acid (mg/dL) were recorded 2.3. Study Definitions Seizure was defined as sudden elevation in aEEG activity band upon narrowing during active body cooling. A discontinuous ground on aEEG with a lower amplitude of 0–1 (2) µV and bursts > 25 µV was defined as burst suppression, an activity with an amplitude about 5 µV or lower was defined as continuous very low voltage, and an inactive ground under 5 µV was defined as isoelectric line. Pathological findings on ECHO was defined as decreased biventricular output, increased biventricular myocardial performance index and global systolic and diastolic dysfunction and pulmonary hypertension (12). As oxidative stress markers, TAC (Total Antioxidant Capacity) and TOS (Total Oxidative Stress) were used. TAC indicates antioxidant status, while TOS reflects prooxidant status. Blood concentrations of TAC (mmoltroloxequiv/L), TOS (mmol H2O2 equiv/L). Blood samples were centrifuged for 3 minutes at 5000 rpm, and plasma samples were stored at 80C. Serum TAC and TOS levels were measured, as described by Erel (13). Erel’s method for serum TAC level measurement is based on the bleaching of the characteristic color of a more stable 2,2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radical cation by antioxidants.The results were expressed in mmol trolox equivalents/L. Erel’s TOS measurement is based on the oxidation of ferrous ion to ferricion in the presence of various oxidative species and the measurement of the ferric ion by xylenol orange. The results were expressed in mmol H2O2 equivalents/L. Oxidative stress index [arbitrary unit = TOS(mmol H2O2 equiv/L) / TAC (mmol trolox equiv/L)] was calculated as the percent ratio of TOS to TAC (14). 2.4 . Statistical analysis During the assessment of the data obtained in the study, SPSS version 15 (SPSS Inc., Chicago, IL, USA) program was used for statistical analysis. Median values of nonparametric tests were reported with minimum and maximum values. Nonnormally distributed numerical and ordinal variables were compared using the Mann Whitney U test. Student t test was used for the comparison of parametric variables. Chi-square test was used to compare the categorical variables. Analysis of variance test was used to compare mean values among more than two groups. 3. Results A total of 13,076 term infants were admitted to the neonatal intensive care unit of Zeynep Kamil Maternity and Children’s Diseases Training and Research Hospital within one year. The incidence of infants who received therapeutic hypothermia was 16 (0.1%).The mean gestational age of the infants who underwent therapeutic hypothermia was 38 weeks (range: 36–42 weeks), and their mean birth weight was 3107 grams (range: 2080–4200 grams). The mean 1st-minute Apgar score was 2.5 (range: 0–5), and the mean 5th-minute Apgar score was 5 (range: 3–8). The cesarean delivery rate was 50%, and the male-to-female ratio was 11:5. The proportion of infants with moderate/severe HIE was 7:9 (Table 1 ). Among these infants, 50% showed epileptic foci on aEEG. The average pH of cord blood or postnatal first-hour blood gases was 6.9 (range: 6.6–7.1), the base excess (BE) was − 18 mmol/L (range: -34 to -11), and the lactate level was 12.2 mmol/L (range: 5–18). The mean creatine kinase (CK) level was 1156 U/L (range: 504–14,302), lactate dehydrogenase (LDH) was 1348 U/L (range: 623–18,621), and uric acid was 8.3 mg/dL (range: 5.9–12.8) (Table 2 ). When TAC,TOS blood levels were evaluated in before and after TH; TOS blood level (p = 0.005) and OSI level (p = 0.001) were lower, and TAC blood levels (p = 0.001) were higher studied after TH, compared to before TH. (Table 3 ). Table 1 Demographic Data and APGAR Scores of Newborns Undergoing Therapeutic hypothermia Therapeutic hypothermia n = 16 Gestational week, mean (min-max) 38 gw (36–42) Birth weight, mean (min-max) 3107 gr (2080–4200) Maternal age, mean (min-max) 28 years (18–38) Cesarean section, n (%) 8 (50%) Apgar 1., mean (min-max) 2.5 (0–5) Apgar 5., mean (min-max) 5 (3–8) Male/Female (n/n) 11/5 Moderate/Severe HİE(n/n) 7/9 Table 2 aEEG, Venous Blood Gas, and Laboratory Findings of Newborn Undergoing Therapeutic hypothermia Therapeutic hipothermia n = 16 aEEG-epileptic focus (%) 8 (50) ECHO-TR,AR(%) 7 (43.8) PH 6.9 (6.6–7.1) PCO2 (mmhg) mean (min-max) 80 (28–107) HCO3 mmol/L mean (min-max) 10.5 (4–10) BE mmol/L mean (min-max) -18 (-33- -11) Lactat mmol/L mean (min-max) 12.2 (5–18) CK U/L mean (min-max) 1156 (504-14302) CK-MB U/L mean (min-max) 27.2 (8-366) LDH U/L mean (min-max) 1348 (623-18621) TROPONİN U/L mean (min-max) 0.03 (0.01–0.19) Üric acid mg/dl mean (min-max) 8.3 (5.9–12.8) Ck: Creatine kinase; LDH: Lactate dehydrogenase Table 3 TAS, TOS, OSI, and PON-1 Values Before and After Therapeutic Hypothermia Therapeutic Hipothermia Before Therapeutic Hipothermia After p TAS (mmoltroloxequiv/L) mean (min-max) 2.03 (0.85–2.84) 2.5 (2-3.4) 0.001 TOS (mmoltroloxequiv/L) mean (min-max) 35.1 (13.1–59.8) 21.9 (12.8–46) 0.005 OSI mean (min-max) 30.2 (1.8–141) 4.1 (1.2–53.3) 0.001 TOS: Total oxidative stress; TAS: total antioxidant status; OSI:Oxidative stress index Discussion It has been shown that therapeutic hypothermia (TH) applied to newborns with hypoxic-ischemic encephalopathy (HIE) reduces oxidative stress. A strong aspect of the study is that it is one of the few neonatal studies evaluating the effects of TH on oxidative stress. However, the small sample size limits the study. Oxidative stress markers (TAC and TOS) used in this study are different from those used in other studies. The study suggests that adjunctive therapies that reduce oxidative stress should be prioritized in the treatment of HIE. In a limited number of animal and in vivo studies, therapeutic hypothermia applied after hypoxia has been shown to reduce oxidative stress. Limited reductions in the release of superoxide and nitric oxide have been shown in in vivo hippocampal cells (15), in rodents during ischemia and reperfusion (16), in swines during and after hypoxia (17), and in young adult canines during cardiac arrest (18) in the studies by Mc Manus, Lei, Thoresen and Lei,respectively. A recent in vitro study on rat cardiomyocytes demonstrated that hypothermia reduces oxidative stress during the ischemic period (4). In our study, consistent with other studies, therapeutic hypothermia (TH) in newborns with hypoxic-ischemic encephalopathy (HIE) decreased pro-oxidant status and oxidative stress. In a human study evaluating the effects of TH on oxidative stress, it was found that TH preserved antioxidant defense mechanisms in infants and children after severe traumatic brain injury (5). In our study, TH increased antioxidant capacity and reduced oxidative stress in newborns with HIE. Numerous studies have been conducted on adjunctive treatments for HIE. Many studies are focused on adjunctive therapies targeting various pathways in HIE pathogenesis, such as mitochondrial therapies (Metformin, Coenzyme Q10, Mitoquinone) and stem cell treatments. Our study suggests that priority should be given to studies investigating adjunctive treatments that reduce oxidative stress and enhance the effects of therapeutic hypothermia. There is a need for studies evaluating adjunctive therapies with allopurinol and magnesium, which reduce pro-oxidant production, as well as those using melatonin, acetylcysteine, edaravone, molecular hydrogen, superoxide dismutase, and docosahexaenoic acid (19), which increase antioxidant capacity or modify antioxidant enzyme activity. Declarations Author Contributions: Conceptualization, E.O.; Data curation, E.O.; Formal analysis, S.D.; Methodology, A.T.; Supervision, S.T. and G.K.; Writing original draft, E.O.and H.OK.; Writing—review & editing, A.T. and G.K. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and has received approval from the University of Health Sciences, Zeynep Kamil Maternity and Children’s Hospital Ethics Committee on 03 March 2021 with decision number of 59. Clinical trial number:not applicable Informed Consent Statement: The consent form has been obtained Data Availability Statement: The datasets generated and analyzed for the study are available from the corresponding author upon a reasonable request. Conflicts of Interest: The authors declare no conflict of interest. References Zhao M, Zhu P, Fujino M, Zhuang J, Guo H, Sheikh IA et.al. Oxidative Stress in Hypoxic-Ischemic Encephalopathy: Molecular Mechanisms and Therapeutic Strategies. Int. J. Mol. Sci. 2016 ;17:2078. Azzopardi D,Strohm B, Marlow N, Brocklehurst P,Dierly A! Eddama O! Goodwin J! Halliday H.L, Juszczak E, Kapeleu O. et al. Effects of hypothermia for perinatal asphyxia on childhood outcomes. N. Engl. J. Med. 2014;371: 140–149. Thoresen M, Penrice J, Lorek A, Cady E.B, Wylezinska M, Kirkbride V, Cooper, C.E, Brown, G.C, Edwards A.D, Wyatt J.S.et al. Mild hypothermia after severe transient hypoxia-ischemia ameliorates delayed cerebral energy failure in the newborn piglet. Pediatr. Res. 1995, 37, 667–670 Tissier R, Pons S, Zini R, Darbera L, Lidouren F, Ghaleh B, Berdeaux A, Morin D. Mild hypothermia reduces per-ischemic reactive oxygen species production and preserves mitochondrial respiratory complexes. Resuscitation 2013, 84, 249–255. Bayir H, Adelson P.D, Wisniewski S.R, Shore P, Lai, Y, Brown D, Janesko-Feldman K.L, Kagan V.E, Kochanek P.M. Therapeutic hypothermia preserves antioxidant defenses after severe traumatic brain injury in infants and children. Crit. Care Med. 2009, 37, 689–695. Wei L, Han BH, Li Y, Keogh CL, Holtzman DM, Yu SP. Cell death mechanism and protective effect of erythropoietin after focal ischemia in the whisker-barrel cortex of neonatal rats. J Pharmacol Exp Ther 2006;317:109–116. Zhu C, Kang W, Xu F, Cheng X, Zhang Z, Jia L, et.al. Erythropoietin improved neurologic outcomes in newborns with hypoxic-ischemicencephalopathy. Pediatrics 2009;124:e218–e226. Marro PJ, Mishra OP, Delivoria-Papadopoulos M. Effect of allopurinol on brain adenosine levels during hypoxia in newborn piglets. Brain Res 2006;1073-1074:444–450. Kelen D, Robertson NJ. Experimental treatments for hypoxic ischaemic encephalopathy. Early Hum Dev 2010;86:369–377. Committee on Fetus and Newborn, Papile LA, Baley JE, Benitz W, Cummings J, Carlo WA, Eichenwald E, Kumar P, Polin RA, Tan RC, Wang KS Hypothermia and neonatal encephalopathy. Pediatrics.2014;133(6):1146. Sarnat HB, Sarnat MS. Neonatal encephalopathy following fetal distress. A clinical and electroencephalographic study. Arch Neurol. 1976 ;33:696-705. Karaarslan S, Alp H, Baysal T, Cimen D, Ors R, Oran B. Is myocardial performance index useful in differential diagnosis of moderate and severe hypoxicischaemic encephalopathy? A serial Doppler echocardiographic evaluation.Cardiol Young. 2012;22(3):335-340. O. Erel A new automated colorimetric method for measuring total oxidant status. Clin Biochem 2005;38: 1103-1111. O. Erel. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation Clin Biochem 2004; 37: 277-285 McManus T, Sadgrove M, Pringle AK, Chad JE, Sunstrom LE. Intraischaemic hypothermia reducesfree radical production and protects against ischaemic insults in cultured hippocampal slices. J Neurochem 2004;91:327–36. Lei B, Adachi N, Arai T. The effect of hypothermia on H2O2 production during ischemia and reperfusion: a microdialysis study in the gerbil hippocampus.Neurosci Lett 1997;222:91–4 Thoresen M, Satas S, Puka-Sundvall M, Whitelaw A, Hallstrorn A, Loberg EM et al. Post-hypoxic hypothermia reduces cerebrocortical release of NO and excitotoxins. Neuroreport 1997;8:3359–62. Lei B, Tan X, Cai H, Xu Q, Guo Q.Effect of moderate hypothermia on lipid peroxidation incanine brain tissue after cardiac arrest and resuscitation. Stroke 1994;25:147–52. Rallis D, Dermitzaki N, Baltogianni M, Kapetaniou K, Giapros V. Balance of Antioxidants vs. Oxidants in Perinatal Asphyxia. Appl. Sci. 2024, 14, 9651. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5692677","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":414719687,"identity":"c50fd88e-b943-42db-8443-ce88423f4a50","order_by":0,"name":"ELİF ÖZALKAYA","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIie3QMYvCMBTA8TwepA6P65pQr58hUOioX+Wgazenw0FDh36Ffg2XzAcZXARXwcWe4OSgw8Ed3HDJINzUOArmvySE94MkjMVij1gCy4tbxoIxOLgNvQQJgu78pCOo/IaHCYMb4cIfBEmKqJeX3wnJdlO+f9WTMWfYf+4GiGxA644qyqgu96+mchfjRVEPEGWhPZJAypkj0qAjxLMhMrWgG1ILytNzOZNmESYKPXmzlIm6hKuxYSKsf8vHmmR3mmVg1sQx8Ja0bXv3Y/NcbKvV9cfMp2nS9Mch8q+RQvIr3jfuSw7wff90LBaLPVF/Aww/wspQFuIAAAAASUVORK5CYII=","orcid":"","institution":"Zeynep Kamil Hospital","correspondingAuthor":true,"prefix":"","firstName":"ELİF","middleName":"","lastName":"ÖZALKAYA","suffix":""},{"id":414719688,"identity":"00141122-5fbf-40f2-b98f-e4f5ce6fbb7a","order_by":1,"name":"Apdulhamid Tüten","email":"","orcid":"","institution":"Zeynep Kamil Hospital","correspondingAuthor":false,"prefix":"","firstName":"Apdulhamid","middleName":"","lastName":"Tüten","suffix":""},{"id":414719689,"identity":"e4fe0a7f-21ee-45e6-8666-15449a03125d","order_by":2,"name":"Sevilay Topcuoğlu","email":"","orcid":"","institution":"Zeynep Kamil Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sevilay","middleName":"","lastName":"Topcuoğlu","suffix":""},{"id":414719690,"identity":"248d6415-c9d2-4db0-924c-57008135e250","order_by":3,"name":"Hande Özgün Karatepe","email":"","orcid":"","institution":"Zeynep Kamil Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hande","middleName":"Özgün","lastName":"Karatepe","suffix":""},{"id":414719692,"identity":"727c2236-f696-446b-9489-34b0a873ab55","order_by":4,"name":"Güner Karatekin","email":"","orcid":"","institution":"Zeynep Kamil Hospital","correspondingAuthor":false,"prefix":"","firstName":"Güner","middleName":"","lastName":"Karatekin","suffix":""}],"badges":[],"createdAt":"2024-12-22 08:08:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5692677/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5692677/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76174054,"identity":"7d521194-2bcd-4041-9693-f6dcf734e36a","added_by":"auto","created_at":"2025-02-13 06:03:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":619714,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5692677/v1/1faad6c9-3b3e-456c-a1a3-792d27868611.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Therapeutic hypothermia reduces oxidative stress in newborns with hypoxic-ischemic encephalopathy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe neonatal brain is highly susceptible to hypoxia due to its high concentration of sensitive immature cells, free radicals, unsaturated fatty acids, low levels of antioxidant enzymes, and high oxygen demand. Following hypoxia and ischemia in the neonatal brain, the production of prooxidant agents increases, while antioxidant agents becomes insufficient. An increased prooxidant state (oxidative stress) occurs due to the disruption of the balance between pro-oxidant and antioxidant agents. Oxidative stress directly degenerate or modify cellular macromolecules such as membranes, proteins, lipids, and DNA, triggering the inflammatory response cascade and protease secretion. These processes lead to inflammation, apoptosis, autophagy, and necrosis, culminating in brain injury and the clinical presentation of neonatal hypoxic-ischemic encephalopathy (HIE) (1). Therapeutic hypothermia (TH) reduces brain damage and alleviates severe neurological effects in neonates with moderate to severe HIE (2). By decreasing brain metabolism, TH mitigates damage caused by secondary brain energy failure (3). In vitro and animal studies have shown that TH reduces oxidative stress by decreasing pro-oxidant agents (4). However, human studies evaluating the effects of TH on oxidative stress are limited (5). Although hypothermia is the standard treatment, its high cost and limited success rate have driven numerous studies on adjuvant therapies (6\u0026ndash;9). Research focusing on adjuvant treatments that play a key role in TH's mechanisms of action holds promise for advancing HIE treatment. This study aimed to investigate whether TH affects oxidative stress in neonates with HİE. It is anticipated that the findings will guide the development of adjuvant therapies.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1.Settings and Study Population\u003c/h2\u003e\n \u003cp\u003eThis study was conducted as a single-center, prospective study between January 2021 and December 2021 in the Level IV neonatal intensive care unit of Zeynep Kamil Women\u0026apos;s and Children\u0026apos;s Health Training and Research Hospital, where approximately 13,000 births occur annually. The study was approved by the Ethics Committee of Zeynep Kamil Women\u0026apos;s and Children\u0026apos;s Health Training and Research Hospital.Ethics committee approval dated 03.03.2021, number 59, was obtained.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e2.2 Data Collection\u003c/h3\u003e\n\u003cp\u003eTH was applied within the first 6 hours of postnatal life to neonates born at or after 36 weeks of gestation who met the following criteria(10): 1. Venous blood gas analysis performed on the umbilical cord or within the first hour after birth revealed pH\u0026thinsp;\u0026le;\u0026thinsp;7.00 or BE \u0026le;-16 mmol/L. 2. Apgar score\u0026thinsp;\u0026lt;\u0026thinsp;5 at the 10th minute or the need for ongoing resuscitation. 3. Clinical signs of moderate HIE (lethargic consciousness, hypotonic tone, flexion posture, hyperactive DTRs, presence of myoclonus, poor Moro reflex, miotic pupils, presence of seizures, and continuous low voltage or seizure activity on aEEG) and/or severe HIE (coma state, flaccid tone, decerebrate posture, undetectable DTRs, absence of myoclonus, undetectable Moro reflex, anisocoric pupils, and burst suppression or flat line on aEEG)(11). Before the initiation of TH, venous blood was collected from the neonates for oxidative stress markers. TH was applied using a servo-controlled device (Techotherm Neo) as total body cooling, maintaining a rectal temperature of 34.5\u0026deg;C for 72 hours. After TH, the body temperature of the 16 neonates was gradually increased at a rate of 0.5\u0026deg;C per hour using the same device. On the 4th postnatal day, venous blood samples were collected again for oxidative stress markers. Demographic data of the patients who underwent therapeutic hypothermia, 1st and 5th-minute Apgar scores, the frequency of moderate and severe hypoxic-ischemic encephalopathy, 24-hour amplitude-integrated EEG (Natus aEEG device) monitoring findings, echocardiography (ECHO) findings, cord blood gas or blood gas obtained within the first hour (pH, CO2 (mmHg), BE (mmol/L), lactate (mmol/L)), and the laboratory findings on the first day including creatine kinase (CK, U/L), creatine kinase-MB (CK-MB, U/L), lactate dehydrogenase (LDH, U/L), and uric acid (mg/dL) were recorded\u003c/p\u003e\n\u003ch3\u003e2.3. Study Definitions\u003c/h3\u003e\n\u003cp\u003eSeizure was defined as sudden elevation in aEEG activity band upon narrowing during active body cooling. A discontinuous ground on aEEG with a lower amplitude of 0\u0026ndash;1 (2) \u0026micro;V and bursts\u0026thinsp;\u0026gt;\u0026thinsp;25 \u0026micro;V was defined as burst suppression, an activity with an amplitude about 5 \u0026micro;V or lower was defined as continuous very low voltage, and an inactive ground under 5 \u0026micro;V was defined as isoelectric line. Pathological findings on ECHO was defined as decreased biventricular output, increased biventricular myocardial performance index and global systolic and diastolic dysfunction and pulmonary hypertension (12). As oxidative stress markers, TAC (Total Antioxidant Capacity) and TOS (Total Oxidative Stress) were used. TAC indicates antioxidant status, while TOS reflects prooxidant status. Blood concentrations of TAC (mmoltroloxequiv/L), TOS (mmol H2O2 equiv/L). Blood samples were centrifuged for 3 minutes at 5000 rpm, and plasma samples were stored at 80C. Serum TAC and TOS levels were measured, as described by Erel (13). Erel\u0026rsquo;s method for serum TAC level measurement is based on the bleaching of the characteristic color of a more stable 2,2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radical cation by antioxidants.The results were expressed in mmol trolox equivalents/L. Erel\u0026rsquo;s TOS measurement is based on the oxidation of ferrous ion to ferricion in the presence of various oxidative species and the measurement of the ferric ion by xylenol orange. The results were expressed in mmol H2O2 equivalents/L. Oxidative stress index [arbitrary unit\u0026thinsp;=\u0026thinsp;TOS(mmol H2O2 equiv/L) / TAC (mmol trolox equiv/L)] was calculated as the percent ratio of TOS to TAC (14).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4\u003c/strong\u003e.\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the assessment of the data obtained in the study, SPSS version 15 (SPSS Inc., Chicago, IL, USA) program was used for statistical analysis. Median values of nonparametric tests were reported with minimum and maximum values. Nonnormally\u003c/p\u003e\n\u003cp\u003edistributed numerical and ordinal variables were compared using the Mann Whitney U test. Student t test was used for the comparison of parametric variables. Chi-square test was used to compare the categorical variables. Analysis of variance test was used to compare mean values among more than two groups.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003eA total of 13,076 term infants were admitted to the neonatal intensive care unit of Zeynep Kamil Maternity and Children\u0026rsquo;s Diseases Training and Research Hospital within one year. The incidence of infants who received therapeutic hypothermia was 16 (0.1%).The mean gestational age of the infants who underwent therapeutic hypothermia was 38 weeks (range: 36\u0026ndash;42 weeks), and their mean birth weight was 3107 grams (range: 2080\u0026ndash;4200 grams). The mean 1st-minute Apgar score was 2.5 (range: 0\u0026ndash;5), and the mean 5th-minute Apgar score was 5 (range: 3\u0026ndash;8). The cesarean delivery rate was 50%, and the male-to-female ratio was 11:5. The proportion of infants with moderate/severe HIE was 7:9 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among these infants, 50% showed epileptic foci on aEEG. The average pH of cord blood or postnatal first-hour blood gases was 6.9 (range: 6.6\u0026ndash;7.1), the base excess (BE) was \u0026minus;\u0026thinsp;18 mmol/L (range: -34 to -11), and the lactate level was 12.2 mmol/L (range: 5\u0026ndash;18). The mean creatine kinase (CK) level was 1156 U/L (range: 504\u0026ndash;14,302), lactate dehydrogenase (LDH) was 1348 U/L (range: 623\u0026ndash;18,621), and uric acid was 8.3 mg/dL (range: 5.9\u0026ndash;12.8) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). When TAC,TOS blood levels were evaluated in before and after TH; TOS blood level (p\u0026thinsp;=\u0026thinsp;0.005) and OSI level (p\u0026thinsp;=\u0026thinsp;0.001) were lower, and TAC blood levels (p\u0026thinsp;=\u0026thinsp;0.001) were higher studied after TH, compared to before TH. (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic Data and APGAR Scores of Newborns Undergoing Therapeutic hypothermia\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTherapeutic hypothermia\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;16\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGestational week, mean (min-max)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38 gw\u003c/p\u003e \u003cp\u003e(36\u0026ndash;42)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBirth weight, mean (min-max)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3107 gr\u003c/p\u003e \u003cp\u003e(2080\u0026ndash;4200)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMaternal age, mean (min-max)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28 years\u003c/p\u003e \u003cp\u003e(18\u0026ndash;38)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCesarean section, n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 (50%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eApgar 1., mean (min-max)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5 (0\u0026ndash;5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eApgar 5., mean (min-max)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (3\u0026ndash;8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMale/Female (n/n)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11/5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eModerate/Severe HİE(n/n)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7/9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eaEEG, Venous Blood Gas, and Laboratory Findings of Newborn Undergoing Therapeutic hypothermia\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTherapeutic hipothermia\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;16\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eaEEG-epileptic focus (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 (50)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eECHO-TR,AR(%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (43.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.9 (6.6\u0026ndash;7.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePCO2 (mmhg) mean (min-max)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80 (28\u0026ndash;107)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHCO3 mmol/L mean (min-max)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.5 (4\u0026ndash;10)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBE mmol/L mean (min-max)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-18 (-33- -11)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLactat mmol/L mean (min-max)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.2 (5\u0026ndash;18)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCK U/L mean (min-max)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1156 (504-14302)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCK-MB U/L mean (min-max)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.2 (8-366)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLDH\u003c/b\u003e U/L \u003cb\u003emean (min-max)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1348 (623-18621)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTROPONİN U/L mean (min-max)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.03 (0.01\u0026ndash;0.19)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e\u0026Uuml;ric acid mg/dl mean (min-max)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.3 (5.9\u0026ndash;12.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eCk: Creatine kinase; LDH: Lactate dehydrogenase\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTAS, TOS, OSI, and PON-1 Values Before and After Therapeutic Hypothermia\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTherapeutic\u003c/p\u003e \u003cp\u003eHipothermia\u003c/p\u003e \u003cp\u003eBefore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTherapeutic\u003c/p\u003e \u003cp\u003eHipothermia\u003c/p\u003e \u003cp\u003eAfter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTAS\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(mmoltroloxequiv/L)\u003c/b\u003e\u003c/p\u003e \u003cp\u003emean (min-max)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.03\u003c/p\u003e \u003cp\u003e(0.85\u0026ndash;2.84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003cp\u003e(2-3.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTOS\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(mmoltroloxequiv/L)\u003c/b\u003e\u003c/p\u003e \u003cp\u003emean (min-max)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.1\u003c/p\u003e \u003cp\u003e(13.1\u0026ndash;59.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.9\u003c/p\u003e \u003cp\u003e(12.8\u0026ndash;46)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.005\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOSI\u003c/b\u003e\u003c/p\u003e \u003cp\u003emean (min-max)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.2\u003c/p\u003e \u003cp\u003e(1.8\u0026ndash;141)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.1\u003c/p\u003e \u003cp\u003e(1.2\u0026ndash;53.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eTOS: Total oxidative stress; TAS: total antioxidant status; OSI:Oxidative stress index\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt has been shown that therapeutic hypothermia (TH) applied to newborns with hypoxic-ischemic encephalopathy (HIE) reduces oxidative stress. A strong aspect of the study is that it is one of the few neonatal studies evaluating the effects of TH on oxidative stress. However, the small sample size limits the study. Oxidative stress markers (TAC and TOS) used in this study are different from those used in other studies. The study suggests that adjunctive therapies that reduce oxidative stress should be prioritized in the treatment of HIE. In a limited number of animal and in vivo studies, therapeutic hypothermia applied after hypoxia has been shown to reduce oxidative stress. Limited reductions in the release of superoxide and nitric oxide have been shown in \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003ehippocampal cells (15), in rodents during ischemia and reperfusion (16), in swines during and after hypoxia (17), and in young adult canines during cardiac arrest (18) in the studies by Mc Manus, Lei, Thoresen and Lei,respectively. A recent in vitro study on rat cardiomyocytes demonstrated that hypothermia reduces oxidative stress during the ischemic period (4). In our study, consistent with other studies, therapeutic hypothermia (TH) in newborns with hypoxic-ischemic encephalopathy (HIE) decreased pro-oxidant status and oxidative stress. In a human study evaluating the effects of TH on oxidative stress, it was found that TH preserved antioxidant defense mechanisms in infants and children after severe traumatic brain injury (5). In our study, TH increased antioxidant capacity and reduced oxidative stress in newborns with HIE. Numerous studies have been conducted on adjunctive treatments for HIE. Many studies are focused on adjunctive therapies targeting various pathways in HIE pathogenesis, such as mitochondrial therapies (Metformin, Coenzyme Q10, Mitoquinone) and stem cell treatments. Our study suggests that priority should be given to studies investigating adjunctive treatments that reduce oxidative stress and enhance the effects of therapeutic hypothermia. There is a need for studies evaluating adjunctive therapies with allopurinol and magnesium, which reduce pro-oxidant production, as well as those using melatonin, acetylcysteine, edaravone, molecular hydrogen, superoxide dismutase, and docosahexaenoic acid (19), which increase antioxidant capacity or modify antioxidant enzyme activity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor Contributions: Conceptualization, E.O.; Data curation, E.O.; Formal analysis, S.D.; Methodology, A.T.; Supervision, S.T. and G.K.; Writing original draft, E.O.and H.OK.; Writing—review \u0026amp; editing, A.T. and G.K. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eFunding: This research received no external funding.\u003c/p\u003e\n\u003cp\u003eInstitutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and has received approval from the University of Health Sciences, Zeynep Kamil Maternity and Children’s Hospital Ethics Committee on 03 March 2021 with decision number of 59.\u003c/p\u003e\n\u003cp\u003eClinical trial number:not applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInformed Consent Statement: The consent form has been obtained\u003c/p\u003e\n\u003cp\u003eData Availability Statement: The datasets generated and analyzed for the study are available from the corresponding author upon a reasonable request.\u003c/p\u003e\n\u003cp\u003eConflicts of Interest: The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eZhao M, Zhu P, Fujino M, Zhuang J, Guo H, Sheikh IA et.al. Oxidative Stress in Hypoxic-Ischemic Encephalopathy: Molecular Mechanisms and Therapeutic Strategies. Int. J. Mol. Sci. 2016 ;17:2078.\u003c/li\u003e\n \u003cli\u003eAzzopardi D,Strohm B, Marlow N, Brocklehurst P,Dierly A! Eddama O! Goodwin J! Halliday H.L, Juszczak E, Kapeleu O. et al. Effects of hypothermia for perinatal asphyxia on childhood outcomes. N. Engl. J. Med. 2014;371: 140\u0026ndash;149.\u003c/li\u003e\n \u003cli\u003eThoresen M, Penrice J, Lorek A, Cady E.B, Wylezinska M, Kirkbride V, Cooper, C.E, Brown, G.C, Edwards A.D, Wyatt J.S.et al. Mild hypothermia after severe transient hypoxia-ischemia ameliorates delayed cerebral energy failure in the newborn piglet. Pediatr. Res. 1995, 37, 667\u0026ndash;670\u003c/li\u003e\n \u003cli\u003eTissier R, Pons S, Zini R, Darbera L, Lidouren F, Ghaleh B, Berdeaux A, Morin D. Mild hypothermia reduces per-ischemic reactive oxygen species production and preserves mitochondrial respiratory complexes. Resuscitation 2013, 84, 249\u0026ndash;255.\u003c/li\u003e\n \u003cli\u003eBayir H, Adelson P.D, Wisniewski S.R, Shore P, Lai, Y, Brown D, Janesko-Feldman K.L, Kagan V.E, Kochanek P.M. Therapeutic hypothermia preserves antioxidant defenses after severe traumatic brain injury in infants and children. Crit. Care Med. 2009, 37, 689\u0026ndash;695.\u003c/li\u003e\n \u003cli\u003eWei L, Han BH, Li Y, Keogh CL, Holtzman DM, Yu SP. Cell death mechanism and protective effect of erythropoietin after focal ischemia in the whisker-barrel cortex of neonatal rats. J Pharmacol Exp Ther 2006;317:109\u0026ndash;116.\u003c/li\u003e\n \u003cli\u003eZhu C, Kang W, Xu F, Cheng X, Zhang Z, Jia L, et.al. Erythropoietin improved neurologic outcomes in newborns with hypoxic-ischemicencephalopathy. Pediatrics 2009;124:e218\u0026ndash;e226.\u003c/li\u003e\n \u003cli\u003eMarro PJ, Mishra OP, Delivoria-Papadopoulos M. Effect of allopurinol on brain adenosine levels during hypoxia in newborn piglets. Brain Res 2006;1073-1074:444\u0026ndash;450.\u003c/li\u003e\n \u003cli\u003eKelen D, Robertson NJ. Experimental treatments for hypoxic ischaemic encephalopathy. Early Hum Dev 2010;86:369\u0026ndash;377.\u003c/li\u003e\n \u003cli\u003eCommittee on Fetus and Newborn, Papile LA, Baley JE, Benitz W, Cummings J, Carlo WA, Eichenwald E, Kumar P, Polin RA, Tan RC, Wang KS Hypothermia and neonatal encephalopathy. Pediatrics.2014;133(6):1146.\u003c/li\u003e\n \u003cli\u003eSarnat HB, Sarnat MS. Neonatal encephalopathy following fetal distress. A clinical and electroencephalographic study. Arch Neurol. 1976 ;33:696-705.\u003c/li\u003e\n \u003cli\u003eKaraarslan S, Alp H, Baysal T, Cimen D, Ors R, Oran B. Is myocardial performance index useful in differential diagnosis of moderate and severe hypoxicischaemic encephalopathy? A serial Doppler echocardiographic evaluation.Cardiol Young. 2012;22(3):335-340.\u003c/li\u003e\n \u003cli\u003eO. Erel A new automated colorimetric method for measuring total oxidant status. Clin Biochem 2005;38: 1103-1111.\u003c/li\u003e\n \u003cli\u003eO. Erel. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation Clin Biochem 2004; 37: 277-285\u003c/li\u003e\n \u003cli\u003eMcManus T, Sadgrove M, Pringle AK, Chad JE, Sunstrom LE. Intraischaemic hypothermia reducesfree radical production and protects against ischaemic insults in cultured hippocampal slices. J Neurochem 2004;91:327\u0026ndash;36.\u003c/li\u003e\n \u003cli\u003eLei B, Adachi N, Arai T. The effect of hypothermia on H2O2 production during ischemia and reperfusion: a microdialysis study in the gerbil hippocampus.Neurosci Lett 1997;222:91\u0026ndash;4\u003c/li\u003e\n \u003cli\u003eThoresen M, Satas S, Puka-Sundvall M, Whitelaw A, Hallstrorn A, Loberg EM et al. Post-hypoxic hypothermia reduces cerebrocortical release of NO and excitotoxins. Neuroreport 1997;8:3359\u0026ndash;62.\u003c/li\u003e\n \u003cli\u003eLei B, Tan X, Cai H, Xu Q, Guo Q.Effect of moderate hypothermia on lipid peroxidation incanine brain tissue after cardiac arrest and resuscitation. Stroke 1994;25:147\u0026ndash;52.\u003c/li\u003e\n \u003cli\u003eRallis D, Dermitzaki N, Baltogianni M, Kapetaniou K, Giapros V. Balance of Antioxidants vs. Oxidants in Perinatal Asphyxia. Appl. Sci. 2024, 14, 9651.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5692677/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5692677/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e To evaluate the effect of therapeutic hypothermia (TH) on oxidative stress in neonates with hypoxic-ischemic encephalopathy(HİE).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod:\u003c/strong\u003e This study was designed as a prospective study. Sixteen term neonates diagnosed with HİE who were admitted for TH in a neonatal intensive care unit within one year were included in the study. The oxidative stress markers (Total Antioxidant Capacity ( TAC) and Total Oxidative Stress (TOS) in venous blood were measured before and after TH. Oxidative stress index(OSI) was calculated as the percent ratio of TOS to TAS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e After TH, the TAC increased compared to before (p=0.001), while TOS (p=0.005) and OSI (p=0.001) decreased.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThis study demonstrates that therapeutic hypothermia applied to newborns with hypoxic-ischemic encephalopathy reduces oxidative stress. Adjuvant treatments that reduce oxidative stress may enhance the effect of hypothermia.\u003c/p\u003e","manuscriptTitle":"Therapeutic hypothermia reduces oxidative stress in newborns with hypoxic-ischemic encephalopathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-13 05:30:35","doi":"10.21203/rs.3.rs-5692677/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"53427c4b-14cc-49ee-a2ec-8aa8a80ce900","owner":[],"postedDate":"February 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-13T05:30:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-13 05:30:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5692677","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5692677","identity":"rs-5692677","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}