Selective head-and-neck cooling on biomarker levels and symptom rating following a boxing bout- protocol for an exploratory randomized trial

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Following impact, elevated brain temperature may lead to the development of exacerbated brain injury that can be monitored by blood biomarkers. We hypothesized that acute head-and-neck cooling, recently shown to shorten return-to-play in concussed ice hockey players, applied acutely following a boxing bout is associated with an attenuated release of blood biomarkers and improved symptom rating. Methods The trial is academically driven and funded by external and hospital research funds. Young, healthy elite boxers ≥ 18 years old are recruited. Prior to, and immediately after a competitive boxing bout over 3x2 or 3x3 minutes, blood samples are drawn. Boxers are randomized to intervention or control management by 1:1 allocation prior to baseline testing. After the initial post-fight blood sample is drawn and symptom rating using the sports concussion assessment tool-5 (SCAT-5) has been collected, the boxers receive either acute selective head-and-neck cooling for 45 min, or routine post-fight management. The number of head impacts are counted in all boxers on match video recordings. In both groups, blood samples are drawn 45 minutes after the initial post-bout blood sample, as well as 3 and 6 days post-fight. At all time points for blood sampling, the number of symptoms (NOS) and symptom severity score (SSS) are assessed using the symptom rating part of the SCAT-5. The primary endpoint is the difference in biomarker levels (GFAP, NF-L, tau, UCH-L1, neuronal-specific enolase) between immediately post-fight and pre-intervention levels, to those obtained at 6 days post-fight. The post-fight SCAT-5 NOS and SSS are secondary endpoints. Discussion There is no treatment available for boxing-induced brain injury. Biomarkers are surrogate yet objective marker of brain injury, and the head-and-neck cooling treatment may attenuate the release of brain injury-related biomarkers as well as reduce symptoms induced by head impacts attained during a boxing fight. Potentially, head-and-neck cooling is a treatment option acutely following a boxing bout. Trials registration ClinicalTrials.gov, ClinicalTrials.gov Identifier NCT06386484. Registered April 23, 2024. Sports concussion biomarker boxing glial fibrillary acidic protein (GFAP) neurofilament-light (Nf-L) Figures Figure 1 Figure 2 Introduction Background and rationale Trauma to the head is common in many contact sports, not least in boxing (1). If the trauma to the head, or to the body with energy transmitted to the head, is sufficient to inducea range of clinical symptoms and signs that may or may not involve loss of consciousness, a sport-related concussion (SRC) has occurred (2). In boxing, both participants receive, and deliver, numerous blows to the head during a typical bout. These repeated head impacts may not cause acute symptoms sufficient to reach the diagnostic criteria for an SRC, and are often named subconcussive impacts. These subconcussive head impacts may induce a brain injury and are considered a potential cause of impaired brain function at short- and long term (3). The neuropathological consequences of repeated SRC have been comprehensively investigated in previous studies, illustrating that repeated head impacts can cause structural and functional alterations to the brain (2)(4). Similarly, repeated head impacts may lead to, e.g. , cognitive impairment, mood disorders, and motor control problems as suggested not only in boxing but also in soccer heading (5)(6). The long-term consequences of SRC, which include, e.g. , an increased risk of neurodegenerative disorders such as Alzheimer´s disease, are increasingly recognized (2). Several studies have shown that retired athletes who have sustained multiple SRC during their career may develop chronic traumatic encephalopathy (CTE) , a progressive neurodegenerative disease associated with memory loss, depression, personality changes, and dementia (6)(7)(8)(1)(9)(10). At present, CTE can only be diagnosed post-mortem where key findings include irregular aggregation of phosphorylated tau protein at the depths of cortical sulci (6). In sports, the strenuous exercise leads to elevated core body- and brain- temperature (11), which may exacerbate brain injury induced by head impacts. Recently, we showed that selective head-and-neck cooling, aiming to rapidly normalize the elevated brain temperature, applied acutely post-SRC in elite ice hockey players resulted in earlier return-to-play and a smaller proportion of players with prolonged symptoms following the SRC (12). However, there is a lack of objective outcome measures for assessing the effects of head-and-neck cooling in athletes. There is a growing body of literature on the use of biomarkers of brain injury in athletes. In Olympic boxers, levels of cerebrospinal fluid biomarkers such as S100B, Tau, NFL and GFAP were collected at baseline, 1-6 days post-fight and after 14 days after repetitive head impacts attained in a boxing bout. The biomarkers showed both acute and cumulative effect of the impacts, with a lack of normalization of NFL and GFAP after the rest period, which may indicate an ongoing injury process (13). Inflammatory and muscle injury biomarkers were also increased in male elite boxers following a fight (14). When boxing and mixed martial art (MMA) were compared, retired boxers had a higher GFAP levels, and active boxers higher NFL than MMA fighters (15). In addition, serum NFL levels were elevated both at 7-10 days post bout and after 3 months of rest in boxers (16). These studies indicate that selected biomarkers may remain elevated during the 1 st post-injury week. In this randomized trial, we hypothesized that biomarkers may be a surrogate yet objective marker for the efficacy of selective head-and-neck cooling on attenuating brain injury in elite boxers, assessed by blood biomarkers immediately prior to and during the first 6 days after a competitive boxing fight. Objectives Research hypothesis: Acute and selective head-and-neck cooling immediately after a boxing bout attenuated brain injury as evident by a reduced release of brain injury-related biomarker levels during the 1 st post-fight week. Study objectives. Primary objective: To determine whether selective head-and-neck cooling attenuates the release of brain injury biomarkers. Secondary objectives: – To determine whether selective head-and-neck cooling improves symptom rating using the Sports Concussion Assessment tool- 5 (SCAT-5). – To determine whether any biomarker increase post-fight, compared to baseline levels, is associated with the number of head impacts To determine any adverse event associated with the head-and-neck cooling Trial design The head-and-neck Cooling of Brain temperature In BOXing (COBIBOX) trial is designed as a randomized, controlled, open, single-center superiority trial with two parallel groups and a primary endpoint of biomarker changes during the first 6 post-fight days. Clinical secondary outcome assessments of number of symptoms (NOS) and symptom severity score (SSS) using the symptom rating part of the of the SCAT-5 during the initial six post-injury days. Randomization will be performed in blocks with 1:1 allocation. Methods: participants, interventions, and outcomes Study setting: The study will take part predominately in southern parts of Sweden due to logistic reasons, aiming for feasibility of follow up with biomarkers at distinct post-fight time points. Blood samples will be collected at the site of the competitive boxing tournament or at the local boxing clubs and transferred to the biobank at Lund University Hospital. All data will be collected in Sweden. The trial is academically driven by local funding. The trial will be conducted at Lund University, Lund, Sweden. Eligibility criteria Inclusion criteria: Male and female elite boxers ≥18 years old Cleared for participation by the medical staff of the Swedish Boxing Association. Informed consent by each participant prior to the competitive event No boxing fights or match sparring during the initial 6 days after their last boxing fight Signed informed consent. Exclusion criteria: 1.> 40 years old or < 18 years old 2. Participation in competitive fights, match-sparring or any training causing additional head impacts during the first six post-fight days after being included in the study. 3. History of autoimmune neurological diseases or a neurodegenerative disorder. 4. History of previous traumatic brain injury resulting in an intracranial haemorrhage. Methods All boxing clubs are thoroughly informed of the protocol prior to study initiation and prior to each tournament. Boxers between the ages of 18 and 40 are included. Allocation to the treatment takes place the first morning of the tournament and the boxers are randomised by 1:1 by a randomly drawn paper slip with a sign if the boxer will receive either selective head-and-neck cooling or routine post-fight medical surveillance by an on-site physician. The selective head-and-neck cooling is to be initiated within 10 minutes post-fight with a duration of a minimum 45 minutes. After the boxer completes the fight (Figure 1-2), he or she follows the researcher to the cooling station and continues the head-and-neck cooling protocol. The PolarCap System consists of a high-powered portable cooling system, designed to reduce brain temperature by controlled cooling of the scalp and neck with a circulating coolant (PolarCap Coolant, PolarCool AB, Lund, Sweden). The coolant is maintained at 0°C and flows through a silicone-based head cap. An insulating neoprene cover is put on top of the cap for isolation. The players are allowed to relax (sitting or supine) as long as the cooling head cap and neoprene cover remained in the place on the head (17). Should the boxer experience discomfort and wish to discontinue the intervention, the cap is removed and the athlete excluded from the study. Blood samples are collected at the following time points: The morning of the first boxing fight at day 1 (baseline, at rest), immediately after the fight, 45 minutes after the fight in both groups, and after 3 and 6 days in all boxers. The blood samples obtained at 3- and 6-days post-fight will be drawn by a study nurse or a researcher (AaH) at the participant´s boxing club. The samples are transported to the hospital in a mobile refrigerator to maintain a temperature of 5-8° C. Thereafter the serum samples are centrifuged at 3000 RPM, at 4° C for 10 minutes, after which 800 -1200 µL is transferred using a pipet to 2ml tubes and stored at –80° C. At each time point for blood sample collection, boxers are also administered the SCAT-5 questionnaire, which is a self-report questionnaire with information on 22 different symptoms, each with a severity grading of 0-6, where 0 indicates no symptoms and 5-6 indicates severe symptoms. The maximum total score is 132. All boxing bouts are video recorded for later analysis. The total hits per bout, strikes to the head, strikes to the body and visible evidence of neurological disturbance (loss of consciousness, impaired balance and/or coordination, vacant expression etc .) will be assessed. After completion of the study, the boxers will be interviewed by phone to answer questions about their previous boxing, knock-out and concussion history. Ethics The study outlined in this article will be carried out in accordance with the Declaration of Helsinki and obtained regional ethics committee approval in Lund, Sweden (decision number Dnr 2022/06195). Prior to their enrolment in the trial, all study participants will provide written informed consent. Approval from the Swedish Boxing Association to include amateur and professional boxers registered in the Swedish Boxing Association (SBA) in the study will be received before the study is initiated. Forms describing the study are sent to all boxing clubs in southern Sweden that would participate in the tournaments and have enrolled boxer over 18 years old. All participants are informed of their rights to terminate their participation in the study at any given time without need to provide a reason for doing so. Study population Fourteen boxing clubs whose fighters enrolled in 5-7 boxing events in southern Sweden will be contacted and informed about the study and its inclusion criteria. Each participant signs the form containing comprehensive study details. Allocation After all boxers approve participation in the study during the tournament, and sign the informed consent, the study staff makes allocation on paper slips that equals the numbers of participants. Then the boxers randomly, by drawing a paper slip, receives either a sign for receiving head-and-neck cooling (C= cooled) or routine management (blank) (Fig1-2). All allocations are finalized a minimum of 3 hours prior to the start of the tournaments. The enrolment of participants will be done by a researcher (AaH) aided by boxing coaches. After the boxing bout, and after the boxer is evaluated for red flags mandating transport to nearest hospital, the boxer is brought to the intervention station to apply the Polarcap® system as previously described. Who will take informed consent? Informed consent documents will be sent for approval to the Swedish Boxing Association, after which it will be distributed to each club. A responsible researcher/physician (AaH) will inform the participants verbally and in written form either days before the competition or on the same day. Each participant will be informed about the study, informed of the possibility to withdraw from participation at any time, and provided time to ask questions. Prior to randomisation, each participants signs the informed consent form. Biomarker analysis Venous blood is collected in 3.5 ml serum and EDTA plasma tubes and stored in a mobile refrigerator at 4-8ºC until centrifuged at 4 ºC for 10 minutes at 3000 rpm. Remaining supernatant is transferred to 2 ml cryotubes and stored at –80 o C until analyzed. Once the study has been completed, blood biomarkers for brain injury will be measured in randomized samples using established immunoassays in the Clinical Neurochemistry Laboratory by board-certified laboratory technicians who will be blinded to the intervention. Participant timeline STUDY PERIOD Enrolment Pre-allocation Allocation Post-allocation Close-out TIMEPOINT -t 1 Baseline 0 Post-fight 45 min post-fight Day 3 Day 6 1 week post fight ENROLMENT: X Eligibility screen X Informed consent X Basic Medical examination X Allocation X INTERVENTIONS: X Selective head and neck cooling (A) X Return to fight protocol (A+B) X X X X Collecting blood sample X X X X X ASSESSMENTS (SCAT5): SCAT5 X SCAT5 - follow up X X X X Boxing history X A = intervention group. B = control group. Sport concussion assessment Tool 5 (SCAT5) Data collection The Sport Concussion Assessment Tool (SCAT5) form will be filled out at time of biomarker analysis and used as a secondary endpoint of the study. The analysis of symptom severity scores obtained by SCAT5 at the set timeline will facilitate the interpretation of outcome between the boxers independent of their previous boxing history after the completed 6-day follow up. Boxing history will be obtained by phone call interview at the 6-day follow-up or onsite at time of recruitment. The follow up for day 3 and 6 will be organized by the local clubs prior to the 1 st post-match training session, or at the study hospital. Sample size and statistical power Based on previous reports, an approximately 10% increase in key biomarkers is observed after a concussion in athletes (16). We hypothesized a 20% reduction of biomarker level by acute head-and-neck cooling. We assumed equal variance of the cooled and control groups. The selected power was set to 0.8. Power analysis was conducted using the following formula: (18). Based on the power calculations, a total number of 20 participants per group is expected to yield a sufficient statistical power. Since dropouts and missed sample times can be anticipated we aim for group sizes of 25 per group. Recruitment Participants are recruited by one researcher (AaH) by sending information about the study to the Swedish Boxing Association that approves participation, in written form or verbally. Thereafter the study must be approved by the organisers of the boxing tournament. Finally, recruitment of boxers who fulfil inclusion criteria and sign the written consent form after verbal and written information about the study is provided. Adverse events Any adverse events reported by the participant during the study period is noted and reported. Specifically, any deviation from protocol ( e.g. , sample time point, duration of head-and-neck cooling) will be noted. Symptoms, such as headache, nausea and dizziness, are reported to study researcher (AaH), although these symptoms may also be caused by the SRC per se (12). Discussion During a boxing fight, boxers typically attain a high number of blows to the head, which may lead to brain injuries. Due to the strenuous activity, these blows occur at a time of elevated core body temperature, which may exacerbate the brain injury. To date, there are no approved treatments for boxing-induced brain injuries. A blow to the head may lead to the release of blood biomarkers that reflect the degree of the brain injury caused by the impact. There is an increasing interest of these biomarkers that include markers for large caliber axon injury (neurofilament-light), neuronal injury (NSE) and glial injury (S100B, GFAP). Our present study may show whether the level of biomarkers is related to the number of hits or concussions received, the symptom development after the fight and most importantly the effects of the cooling intervention. Head-and-neck cooling was shown to decrease brain temperature measured by magnetic resonance imaging (MRI) (19), and improved cognitive outcome when applied acutely post-concussion (20). In a recent clinical trial including 132 concussed ice hockey players, a shorter recovery time was observed after acute selective head-and-neck cooling for at least 45 minutes (12). A shorter recovery was also observed when cooling was initiated up to 8 days post-concussion (21). Toprovide an objective outcome measure the cooling intervention in the present study, the primary outcome is blood biomarker levels, of which GFAP, NFL, S100B and tau may be the most important. While NF-L levels may remain elevated at longer time points post-fight, we had to use 6 days as our latest evaluation time point. This is based on the guidelines of the Swedish Boxing Association, which prohibit boxers from competing or having fights within 6 days of their most recent tournament. Thus, many boxers are assigned to match sparring or other tournaments after 6 days. The selection of biomarkers in the present trial is based on several previous studies (22)(23), mainly in traumatic brain injury (TBI) and neurodegenerative disorders (15). Currently, both tau isoforms and GFAP is used in prediction and diagnosis of neurodegenerative diseases, particularly Alzheimer’s disease, and S100B in mild traumatic brain injury guidelines (24)(25)(26). In numerous reports on mild, moderate or severe traumatic brain injury, blood and CSF biomarkers have been used in different time points post-injury (22). Following boxing bouts, P-tau, T-tau, NFL, GFAP, and S100B are all increased in the early phase after boxing. Of these, NFL, GAP and tau are increased several weeks- to months post-fight (27)(28)(13). In addition, blood S100B levels were significantly elevated in both competitive boxers and high levels exercised athletes such as runners (27) (29). These findings argue that boxing causese the release of several biomarkers reflecting the brain injury, and be associated with lead to long-term consequences. The role of brain temperature in acute brain injury has been evaluated in numerous trials. In particular, elevated brain temperature may negatively impact functional and neurological outcomes following traumatic brain injury (30)(31)(31). While systemic hypothermia has not been associated with improved outcomes in severe traumatic brain injury, we hypothesized that controlling body temperature may be an important factor in the recovery of boxing-induced brain injury. The primary aim of the head-and-neck cooling is not to provide cerebral hypothermia, it is to attenuate exercise-induced hyperthermia. In conclusion, the present trial may provide objective biomarker evidence for the role of attenuating cerebral hyperthermia by selective head-and-neck cooling acutely following a boxing bout. Trial status Recruitment started on November 4, 2021. Approximate completion of final recruitment is expected in the first weeks of January 2025. Protocol version 1.0, 2024–04-01. Declarations Acknowledgements Marit Bäckström for administrative assistance. The results of this trial will be presented in an appropriate scientific journal after the trial is completed. Authors’ contributions All authors have reviewed, edited, and approved the manuscripts final form. AaH and NM wrote the original draft. AaH, YT, HZ, KB and NM were responsible for conceptualization of the trial. Funding Open access funding provided by Lund University. Swedish Research Council for Sport Science, Hans-Gabriel and Alice Trolle-Wachtmeister (HGATW) Foundation for Medical Research, the Alborada Trust, Swedish Brain Foundation, and hospital ALF funds (all to NM). HZ is a Wallenberg Scholar and a Distinguished Professor at the Swedish Research Council supported by grants from the Swedish Research Council (#2023-00356; #2022-01018 and #2019-02397), the European Union’s Horizon Europe research and innovation programme under grant agreement No 101053962, and Swedish State Support for Clinical Research (#ALFGBG-71320). Availability of data and materials Data generated from this trial may be received from the authors upon reasonable request. Ethics approval and consent to participate. See ethics part. Consent for publication. When the participant/boxer is informed of the trial, information is given about the intent of publishing the results, and if consent is given for participation in the trial, consent for publications is given as well. Competing interests NM and YT are scientific advisors for PolarCool Inc. Lund, Sweden. Polar Cool provides the cooling helmet, although has not influenced the design of the study protocol. HZ has served at scientific advisory boards and/or as a consultant for Abbvie, Acumen, Alector, Alzinova, ALZPath, Amylyx, Annexon, Apellis, Artery Therapeutics, AZTherapies, Cognito Therapeutics, CogRx, Denali, Eisai, Merry Life, Nervgen, Novo Nordisk, Optoceutics, Passage Bio, Pinteon Therapeutics, Prothena, Red Abbey Labs, reMYND, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures in symposia sponsored by Alzecure, Biogen, Cellectricon, Fujirebio, Lilly, Novo Nordisk, and Roche, and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (outside submitted work). KB has served as a consultant, at advisory boards, or at data monitoring committees for Abcam, Axon, Biogen, JOMDD/Shimadzu. 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Neselius S, Zetterberg H, Blennow K, Marcusson J, Brisby H. Increased CSF levels of phosphorylated neurofilament heavy protein following bout in amateur boxers. PLoS One. 2013;8(11):e81249. Zetterberg H, Hietala MA, Jonsson M, Andreasen N, Styrud E, Karlsson I, et al. Neurochemical aftermath of amateur boxing. Arch Neurol. 2006;63(9):1277-80. Otto M, Holthusen S, Bahn E, Söhnchen N, Wiltfang J, Geese R, et al. Boxing and running lead to a rise in serum levels of S-100B protein. Int J Sports Med. 2000;21(8):551-5. Hinson HE, Rowell S, Morris C, Lin AL, Schreiber MA. Early fever after trauma: Does it matter? J Trauma Acute Care Surg. 2018;84(1):19-24. Pegoli M, Zurlo Z, Bilotta F. Temperature management in acute brain injury: A systematic review of clinical evidence. Clin Neurol Neurosurg. 2020;197:106165. 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Participants will be randomly allocated to either the control or intervention group, involving selective head-and-neck cooling. Baseline SCAT5 and blood samples will be collected. Subsequently, post-fight SCAT5 and blood samples will be collected for both groups. Additionally, the intervention group will undergo selective head-and-neck cooling for 45 minutes. Following this, both the control and intervention groups will follow the same protocol, undergoing SCAT5 and blood sample collection at 45 minutes post-fight, as well as on Day 3 and Day 6 for follow-up.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4343194/v1/6a69e09daa1d570f6abfbe3f.jpg"},{"id":61866525,"identity":"fccd1d60-d6ed-4b97-8457-0717651c7b15","added_by":"auto","created_at":"2024-08-06 12:15:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":101200,"visible":true,"origin":"","legend":"\u003cp\u003eBoxers who participate in tournaments can either fight one or two boxing bouts, depends on whether they win or lose. The selective head-and-neck cooling take part after last boxing bout (dark coloured boxes). For boxer who fights bout 2/2, new baseline SCAT 5 and blood samples will be collected, shown as baseline 2.0 in the figure.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4343194/v1/548333984918d3306230e103.jpg"},{"id":70237389,"identity":"4158d06a-9cb8-49f0-88a2-f6c94ee7ef6a","added_by":"auto","created_at":"2024-11-30 05:41:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":750110,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4343194/v1/6ba2f0c6-509d-4a4c-813d-975023d0d2b7.pdf"},{"id":61866527,"identity":"a59524bf-0080-408b-991d-277d62c33dda","added_by":"auto","created_at":"2024-08-06 12:15:42","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":58810,"visible":true,"origin":"","legend":"","description":"","filename":"SPIRITChecklistCOBIBOXsubmit.docx","url":"https://assets-eu.researchsquare.com/files/rs-4343194/v1/dc4573760b15e12c6cfe561c.docx"}],"financialInterests":"","formattedTitle":"Selective head-and-neck cooling on biomarker levels and symptom rating following a boxing bout- protocol for an exploratory randomized trial","fulltext":[{"header":"Introduction","content":"\u003ch3\u003e\u003cstrong\u003eBackground and rationale\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTrauma to the head is common in many contact sports, not least in boxing\u0026nbsp;(1). If the trauma to the head, or to the body with energy transmitted to the head, is sufficient to inducea range of clinical symptoms and signs that may or may not involve loss of consciousness, a sport-related concussion\u0026nbsp;(SRC) has occurred\u0026nbsp;(2). In boxing, both participants receive, and deliver, numerous blows to the head during a typical bout. These repeated head impacts may not cause acute symptoms sufficient to reach the diagnostic criteria for an SRC, and are often named subconcussive impacts. These subconcussive head impacts may induce a brain injury and are considered a potential cause of impaired brain function at short- and long term\u0026nbsp;(3). The neuropathological consequences of repeated SRC have been comprehensively investigated in previous studies, illustrating that repeated head impacts can cause structural and functional alterations to the brain\u0026nbsp;(2)(4).\u003c/p\u003e\n\u003cp\u003eSimilarly, repeated head impacts may lead to, \u003cem\u003ee.g.\u003c/em\u003e, cognitive impairment, mood disorders, and motor control problems\u0026nbsp;as suggested not only in boxing but also in soccer heading\u0026nbsp;(5)(6). The long-term consequences of SRC, which include, \u003cem\u003ee.g.\u003c/em\u003e, an increased risk of neurodegenerative disorders such as Alzheimer\u0026acute;s disease, are increasingly recognized\u0026nbsp;(2). Several studies have shown that retired athletes who have sustained multiple SRC during their career may develop chronic traumatic encephalopathy (CTE)\u003cs\u003e,\u003c/s\u003e a progressive neurodegenerative disease associated with memory loss, depression, personality changes, and dementia\u0026nbsp;(6)(7)(8)(1)(9)(10). At present, CTE can only be diagnosed\u0026nbsp;\u003cem\u003epost-mortem\u003c/em\u003e where key findings include irregular aggregation of phosphorylated tau protein at the depths of cortical sulci\u0026nbsp;(6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn sports, the strenuous exercise leads to\u0026nbsp;elevated core body- and brain- temperature\u0026nbsp;(11), which may exacerbate brain injury induced by\u0026nbsp;head impacts. Recently, we showed that selective head-and-neck cooling, aiming to rapidly normalize the elevated brain temperature, applied acutely post-SRC in elite ice hockey players resulted in earlier return-to-play and a smaller proportion of players with prolonged symptoms following the SRC\u0026nbsp;(12). However, there is a lack of objective outcome measures for assessing the effects of head-and-neck cooling in athletes.\u003c/p\u003e\n\u003cp\u003eThere is a growing body of literature on the use of biomarkers of brain injury in athletes. In Olympic boxers, levels of cerebrospinal fluid biomarkers such as S100B, Tau, NFL and GFAP were collected at baseline, 1-6 days post-fight and after 14 days after repetitive head impacts attained in a boxing bout. The biomarkers showed both acute and cumulative effect of the impacts, with a lack of normalization of NFL and GFAP after the rest period, which may indicate an ongoing injury process\u0026nbsp;(13). Inflammatory and muscle injury biomarkers were also increased in male elite boxers following a fight\u0026nbsp;(14). When boxing and mixed martial art (MMA) were compared, retired boxers had a higher GFAP levels, and active boxers higher NFL than MMA fighters\u0026nbsp;(15). In addition, serum NFL levels were elevated both at 7-10 days post bout and after 3 months of rest in boxers\u0026nbsp;(16). These studies indicate that selected biomarkers may remain elevated during the 1\u003csup\u003est\u003c/sup\u003e post-injury week. In this randomized trial, we hypothesized that biomarkers may be a surrogate yet objective marker for the efficacy of selective head-and-neck cooling on attenuating brain injury in elite boxers, assessed by blood biomarkers immediately prior to and during the first 6 days after a competitive boxing fight.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch hypothesis: Acute and selective head-and-neck cooling immediately after a boxing bout attenuated brain injury as evident by a reduced release of brain injury-related biomarker levels during the 1\u003csup\u003est\u003c/sup\u003e post-fight week.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStudy objectives.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary objective: To determine whether selective head-and-neck cooling attenuates the release of brain injury biomarkers.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSecondary objectives:\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ndash; To determine whether selective head-and-neck cooling improves symptom rating using the Sports Concussion Assessment tool- 5 (SCAT-5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026ndash; \u0026nbsp;To determine whether any biomarker increase post-fight, compared to baseline levels, is associated with the number of head impacts\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eTo determine any adverse event associated with the head-and-neck cooling\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTrial design\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe head-and-neck Cooling of Brain temperature In BOXing (COBIBOX) trial is designed as a randomized, controlled, open, single-center superiority trial with two parallel groups and a primary endpoint of biomarker changes during the first 6 post-fight days.\u003c/p\u003e\n\u003cp\u003eClinical secondary outcome assessments of number of symptoms (NOS) and symptom severity score (SSS) using the\u0026nbsp;symptom rating part of the of the SCAT-5 during the initial six post-injury days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRandomization will be performed in blocks with 1:1 allocation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: participants, interventions, and outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy setting:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study will take part predominately in southern parts of Sweden due to logistic reasons, aiming for feasibility of follow up with biomarkers at distinct post-fight time points. Blood samples will be collected at the site of the competitive boxing tournament or at the local boxing clubs and transferred to the biobank at Lund University Hospital. All data will be collected in Sweden.\u003c/p\u003e\n\u003cp\u003eThe trial is academically driven by local funding. The trial will be conducted at\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLund University, Lund, Sweden.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEligibility criteria\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInclusion criteria:\u003c/em\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eMale and female elite boxers \u0026ge;18 years old\u003c/li\u003e\n \u003cli\u003eCleared for participation by the medical staff of the Swedish Boxing Association.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eInformed consent by each participant prior to the competitive event\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eNo boxing fights or match sparring during the initial 6 days after their last boxing fight\u003c/li\u003e\n \u003cli\u003eSigned informed consent.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cem\u003eExclusion criteria:\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e1.\u0026gt; 40 years old or \u0026lt; 18 years old\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2. Participation in competitive fights, match-sparring or any training causing additional head impacts during the first six post-fight days after being included in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3. History of autoimmune neurological diseases or a neurodegenerative disorder.\u003c/p\u003e\n\u003cp\u003e4. History of previous traumatic brain injury resulting in an intracranial haemorrhage.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eAll boxing clubs are thoroughly informed of the protocol prior to study initiation and prior to each tournament. Boxers between the ages of 18 and 40 are included. Allocation to the treatment takes place the first morning of the tournament and the boxers are randomised by 1:1 by a randomly drawn paper slip with a sign if the boxer will receive either selective head-and-neck cooling or routine post-fight medical surveillance by an on-site physician. The selective head-and-neck cooling is to be initiated within 10 minutes post-fight with a duration of a minimum 45 minutes. After the boxer completes the fight (Figure 1-2), he or she follows the researcher to the cooling station and continues the head-and-neck cooling protocol. The PolarCap System consists of a high-powered portable cooling system, designed to reduce brain temperature by controlled cooling of the scalp and neck with a circulating coolant (PolarCap Coolant, PolarCool AB, Lund, Sweden). The coolant is maintained at 0°C and flows through a silicone-based head cap. An insulating neoprene cover is put on top of the cap for isolation. The players are allowed to relax (sitting or supine) as long as the cooling head cap and neoprene cover remained in the place on the head (17). Should the boxer experience discomfort and wish to discontinue the intervention, the cap is removed and the athlete excluded from the study. \u0026nbsp;Blood samples are collected at the following time points: The morning of the first boxing fight at day 1 (baseline, at rest), immediately after the fight, 45 minutes after the fight in both groups, and after 3 and 6 days in all boxers. The blood samples obtained at 3- and 6-days post-fight will be drawn by a study nurse or a researcher (AaH) at the participant´s boxing club. The samples are transported to the hospital in a mobile refrigerator to maintain a temperature of 5-8° C. Thereafter the serum samples are centrifuged at 3000 RPM, at 4° C for 10 minutes, after which 800 -1200 µL is transferred using a pipet to 2ml tubes and stored at –80° C.\u003c/p\u003e\n\u003cp\u003eAt each time point for blood sample collection, boxers are also administered the SCAT-5 questionnaire, which is a self-report questionnaire with information on 22 different symptoms, each with a severity grading of 0-6, where 0 indicates no symptoms and 5-6 indicates severe symptoms. The maximum total score is 132. All boxing bouts are video recorded for later analysis. The total hits per bout, strikes to the head, strikes to the body and visible evidence of neurological disturbance (loss of consciousness, impaired balance and/or coordination, vacant expression \u003cem\u003eetc\u003c/em\u003e.) will be assessed. After completion of the study, the boxers will be interviewed by phone to answer questions about their previous boxing, knock-out and concussion history.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study outlined in this article will be carried out in accordance with the Declaration of Helsinki and obtained regional ethics committee approval in Lund, Sweden (decision number Dnr 2022/06195). Prior to their enrolment in the trial, all study participants will provide written informed consent. Approval from the Swedish Boxing Association to include amateur and professional boxers registered in the Swedish Boxing Association (SBA) in the study will be received\u003cu\u003e\u0026nbsp;\u003c/u\u003ebefore the study is initiated.\u0026nbsp;Forms describing the study are sent to all boxing clubs in southern Sweden that would participate in the tournaments and have enrolled boxer over 18 years old. All participants are informed of their rights to terminate their participation in the study at any given time without need to provide a reason for doing so.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFourteen boxing clubs whose fighters enrolled in 5-7 boxing events in southern Sweden will be contacted and informed about the study and its inclusion criteria. Each participant signs the form containing comprehensive study details.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAllocation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter all boxers approve participation in the study during the tournament, and sign the informed consent, the study staff makes allocation on paper slips that equals the numbers of participants. Then the boxers randomly, by drawing a paper slip, receives either a sign for receiving head-and-neck cooling (C= cooled) or routine management (blank) (Fig1-2). All allocations are finalized a minimum of 3 hours prior to the start of the tournaments.\u003c/p\u003e\n\u003cp\u003eThe enrolment of participants will be done by a researcher (AaH) aided by boxing coaches.\u003c/p\u003e\n\u003cp\u003eAfter the boxing bout, and after the boxer is evaluated for red flags mandating transport to nearest hospital, the boxer is brought to the intervention station to apply the Polarcap® system as previously described.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWho will take informed consent?\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent documents will be sent for approval to the Swedish Boxing Association, after which it will be distributed to each club. A responsible researcher/physician (AaH) will inform the participants verbally and in written form either days before the competition or on the same day. Each participant will be informed about the study, informed of the possibility to withdraw from participation at any time, and provided time to ask questions. Prior to randomisation, each participants signs the informed consent form.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiomarker analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVenous blood is collected in 3.5 ml serum and EDTA plasma tubes and stored in a mobile refrigerator at 4-8ºC until centrifuged at 4 ºC for 10 minutes at 3000\u0026nbsp;rpm. Remaining supernatant is transferred to 2 ml cryotubes and stored at –80\u003csup\u003eo\u003c/sup\u003eC until analyzed. Once the study has been completed, blood biomarkers for brain injury will be measured in randomized samples using established immunoassays in the Clinical Neurochemistry Laboratory by board-certified laboratory technicians who will be blinded to the intervention.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eParticipant timeline\u003c/strong\u003e\u0026nbsp;\u003c/h4\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"690\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.08417997097243%\" colspan=\"8\"\u003e\n \u003cp\u003e\u003cstrong\u003eSTUDY PERIOD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u003cstrong\u003eEnrolment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePre-allocation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAllocation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.494920174165458%\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003ePost-allocation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\"\u003e\n \u003cp\u003e\u003cstrong\u003eClose-out\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTIMEPOINT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u003cstrong\u003e-t\u003csub\u003e1\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBaseline\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.272859216255442%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003csub\u003ePost-fight\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.57910014513788%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003csub\u003e45 min\u0026nbsp;\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003epost-fight\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDay 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDay 6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1 week post fight\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\"\u003e\n \u003cp\u003e\u003cstrong\u003eENROLMENT:\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\" valign=\"bottom\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.272859216255442%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.57910014513788%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\"\u003e\n \u003cp\u003e\u003cstrong\u003eEligibility screen\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.272859216255442%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.57910014513788%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\"\u003e\n \u003cp\u003e\u003cstrong\u003eInformed consent\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.272859216255442%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.57910014513788%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBasic Medical examination\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.272859216255442%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.57910014513788%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAllocation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.272859216255442%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.57910014513788%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\"\u003e\n \u003cp\u003e\u003cstrong\u003eINTERVENTIONS:\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.272859216255442%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.57910014513788%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSelective head and neck cooling (A)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.272859216255442%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.57910014513788%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\"\u003e\n \u003cp\u003e\u003cstrong\u003eReturn to fight protocol (A+B)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.272859216255442%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.57910014513788%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCollecting blood sample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.272859216255442%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.57910014513788%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\"\u003e\n \u003cp\u003e\u003cstrong\u003eASSESSMENTS (SCAT5):\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.272859216255442%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.57910014513788%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eSCAT5 \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.272859216255442%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.57910014513788%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eSCAT5 - follow up\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.272859216255442%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.57910014513788%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.915820029027575%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eBoxing history\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.611030478955007%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.030478955007258%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.272859216255442%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.57910014513788%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.821480406386066%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.336719883889696%\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cul\u003e\n \u003cli\u003eA = intervention group. B = control group.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSport concussion assessment Tool 5 (SCAT5)\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eData collection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Sport Concussion Assessment Tool (SCAT5) form will be filled out at time of biomarker analysis and used as a secondary endpoint of the study. The analysis of symptom severity scores obtained by SCAT5 at the set timeline will facilitate the interpretation of outcome between the boxers independent of their previous boxing history after the completed 6-day follow up. \u0026nbsp;Boxing history will be obtained by phone call interview at the 6-day follow-up or onsite at time of recruitment. The follow up for day 3 and 6 will be organized by the local clubs prior to the 1\u003csup\u003est\u003c/sup\u003e post-match training session, or at the study hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample size and statistical power\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on previous reports, an approximately 10% increase in key biomarkers is observed after a concussion in athletes (16). We hypothesized a 20% reduction of biomarker level by acute head-and-neck cooling. We assumed equal variance of the cooled and control groups. The selected power was set to 0.8. Power analysis was conducted using the following formula: \u0026nbsp;\u003cimg src=\"data:image/png;base64,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\"\u003e(18).\u003c/p\u003e\n\u003cp\u003eBased on the power calculations, a total number of 20 participants per group is expected to yield a sufficient statistical power. Since dropouts and missed sample times can be anticipated we aim for group sizes of 25 per group.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eRecruitment\u0026nbsp;\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eParticipants are recruited by one researcher (AaH) by sending information about the study to the Swedish Boxing Association that approves participation, in written form or verbally. Thereafter the study must be approved by the organisers of the boxing tournament. Finally, recruitment of boxers who fulfil inclusion criteria and sign the written consent form after verbal and written information about the study is provided.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdverse events\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAny adverse events reported by the participant during the study period is noted and reported. Specifically, any deviation from protocol (\u003cem\u003ee.g.\u003c/em\u003e, sample time point, duration of head-and-neck cooling) will be noted. Symptoms, such as headache, nausea and dizziness, are reported to study researcher (AaH), although these symptoms may also be caused by the SRC \u003cem\u003eper se\u0026nbsp;\u003c/em\u003e(12).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDuring a boxing fight, boxers typically attain a high number of blows to the head, which may lead to brain injuries. Due to the strenuous activity, these blows occur at a time of elevated core body temperature, which may exacerbate the brain injury. To date, there are no approved treatments for boxing-induced brain injuries. A blow to the head may lead to the release of blood biomarkers that reflect the degree of the brain injury caused by the impact. There is an increasing interest of these biomarkers that include markers for large caliber axon injury (neurofilament-light), neuronal injury (NSE) and glial injury (S100B, GFAP). Our present study may show whether the level of biomarkers is related to the number of hits or concussions received, the symptom development after the fight and most importantly the effects of the cooling intervention.\u003c/p\u003e\n\u003cp\u003eHead-and-neck cooling was shown to decrease brain temperature measured by magnetic resonance imaging (MRI)\u0026nbsp;(19), and improved cognitive outcome when applied acutely post-concussion\u0026nbsp;(20). In a recent clinical trial including 132 concussed ice hockey players, a shorter recovery time was observed after acute selective head-and-neck cooling for at least 45 minutes\u0026nbsp;(12). A shorter recovery was also observed when cooling was initiated up to 8 days post-concussion \u0026nbsp;(21). Toprovide an objective outcome measure the cooling intervention in the present study, the primary outcome is blood biomarker levels, of which GFAP, NFL, S100B and tau may be the most important. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile NF-L levels may remain elevated at longer time points post-fight, we had to use 6 days as our latest evaluation time point. This is based on the guidelines of the Swedish Boxing Association, which prohibit boxers from competing or having fights within 6 days of their most recent tournament. Thus, many boxers are assigned to match sparring or other tournaments after 6 days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe selection of biomarkers in the present trial is based on several previous studies\u0026nbsp;(22)(23), mainly in traumatic brain injury (TBI) and neurodegenerative disorders\u0026nbsp;(15). Currently, both tau isoforms and GFAP is used in prediction and diagnosis of neurodegenerative diseases, particularly Alzheimer\u0026rsquo;s disease, and S100B in mild traumatic brain injury guidelines\u0026nbsp;(24)(25)(26).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn numerous reports on mild, moderate or severe traumatic brain injury, blood and CSF biomarkers have been used in different time points post-injury\u0026nbsp;(22). Following boxing bouts, P-tau, T-tau, NFL, GFAP, and S100B are all increased in the early phase after boxing. Of these, NFL, GAP and tau are increased several weeks- to months post-fight\u0026nbsp;(27)(28)(13). In addition, blood S100B levels were significantly elevated in both competitive boxers and high levels exercised athletes such as runners\u0026nbsp;(27)\u0026nbsp;(29).\u0026nbsp;These findings argue that boxing causese the release of several biomarkers reflecting the brain injury, and be associated with lead to long-term consequences.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe role of brain temperature in acute brain injury has been evaluated in numerous trials. In particular, elevated brain temperature may negatively impact functional and neurological outcomes following traumatic brain injury\u0026nbsp;(30)(31)(31). While systemic hypothermia has not been associated with improved outcomes in severe traumatic brain injury, we hypothesized that controlling body temperature may be an important factor in the recovery of boxing-induced brain injury. The primary aim of the head-and-neck cooling is not to provide cerebral hypothermia, it is to attenuate exercise-induced hyperthermia. In conclusion, the present trial may provide objective biomarker evidence for the role of attenuating cerebral hyperthermia by selective head-and-neck cooling acutely following a boxing bout.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial status\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecruitment started on November 4, 2021. Approximate completion of final recruitment is expected in the first weeks of January 2025.\u003c/p\u003e\n\u003cp\u003eProtocol version 1.0, 2024\u0026ndash;04-01.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMarit B\u0026auml;ckstr\u0026ouml;m for administrative assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results of this trial will be presented in an appropriate scientific journal after the trial is completed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have reviewed, edited, and approved the manuscripts final form. AaH and NM wrote the original draft. AaH, YT, HZ, KB and NM were responsible for conceptualization of the trial. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOpen access funding provided by Lund University.\u0026nbsp;Swedish Research Council for Sport Science, Hans-Gabriel and Alice Trolle-Wachtmeister (HGATW) Foundation for Medical Research, the Alborada Trust, Swedish Brain Foundation, and hospital ALF funds (all to NM). HZ is a Wallenberg Scholar and a Distinguished Professor at the Swedish Research Council supported by grants from the Swedish Research Council (#2023-00356; #2022-01018 and #2019-02397), the European Union\u0026rsquo;s Horizon Europe research and innovation programme under grant agreement No 101053962, and Swedish State Support for Clinical Research (#ALFGBG-71320).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData generated from this trial may be received from the authors upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSee ethics part.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen the participant/boxer is informed of the trial, information is given about the intent of publishing the results, and if consent is given for participation in the trial, consent for publications is given as well.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNM and YT are scientific advisors for PolarCool Inc. Lund, Sweden. Polar Cool provides the cooling helmet, although has not influenced the design of the study protocol. HZ has served at scientific advisory boards and/or as a consultant for Abbvie, Acumen, Alector, Alzinova, ALZPath, Amylyx, Annexon, Apellis, Artery Therapeutics, AZTherapies, Cognito Therapeutics, CogRx, Denali, Eisai, Merry Life, Nervgen, Novo Nordisk, Optoceutics, Passage Bio, Pinteon Therapeutics, Prothena, Red Abbey Labs, reMYND, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures in symposia sponsored by Alzecure, Biogen, Cellectricon, Fujirebio, Lilly, Novo Nordisk, and Roche, and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (outside submitted work). KB has served as a consultant, at advisory boards, or at data monitoring committees for Abcam, Axon, Biogen, JOMDD/Shimadzu. Julius Clinical, Lilly, MagQu, Novartis, Prothena, Roche Diagnostics, and Siemens Healthineers, and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program. IR has served on advisory boards and given talks sponsored by Biogen, Bayer, Merck, Roche, GlaxoSmithKlein, TEVA, Sanofi and UCB.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBernick C, Banks S, Phillips M, Lowe M, Shin W, Obuchowski N, et al. Professional fighters brain health study: rationale and methods. Am J Epidemiol. 2013;178(2):280-6.\u003c/li\u003e\n\u003cli\u003ePatricios JS, Schneider KJ, Dvorak J, Ahmed OH, Blauwet C, Cantu RC, et al. Consensus statement on concussion in sport: the 6th International Conference on Concussion in Sport-Amsterdam, October 2022. Br J Sports Med. 2023;57(11):695-711.\u003c/li\u003e\n\u003cli\u003eLember LM, Ntikas M, Mondello S, Wilson L, Di Virgilio TG, Hunter AM, et al. 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Effects of Boxing Matches on Metabolic, Hormonal, and Inflammatory Parameters in Male Elite Boxers. Medicina (Kaunas). 2019;55(6).\u003c/li\u003e\n\u003cli\u003eBernick C, Shan G, Ritter A, Ashton NJ, Blennow K, Lantero-Rodriguez J, et al. Blood biomarkers and neurodegeneration in individuals exposed to repetitive head impacts. Alzheimers Res Ther. 15. England: \u0026copy; 2023. BioMed Central Ltd., part of Springer Nature.; 2023. p. 173.\u003c/li\u003e\n\u003cli\u003eShahim P, Zetterberg H, Tegner Y, Blennow K. Serum neurofilament light as a biomarker for mild traumatic brain injury in contact sports. Neurology. 2017;88(19):1788-94.\u003c/li\u003e\n\u003cli\u003eGard A, Tegner Y, Bakhsheshi MF, Marklund N. Selective head-neck cooling after concussion shortens return-to-play in ice hockey players. Concussion. 2021;6(2):CNC90.\u003c/li\u003e\n\u003cli\u003eSerdar CC, Cihan M, Yucel D, Serdar MA. Sample size, power and effect size revisited: simplified and practical approaches in pre-clinical, clinical and laboratory studies. Biochem Med (Zagreb). 2021;31(1):010502.\u003c/li\u003e\n\u003cli\u003eWalter A, Finelli K, Bai X, Johnson B, Neuberger T, Seidenberg P, et al. Neurobiological effect of selective brain cooling after concussive injury. Brain Imaging Behav. 2018;12(3):891-900.\u003c/li\u003e\n\u003cli\u003eSmith MA, McNinch NL, Chaney D, Shauver L, Murray T, Kline P, et al. Reduced Concussion Symptom Burden in Early Adolescent Athletes Using a Head-Neck Cooling Device. Clin J Sport Med. 2024.\u003c/li\u003e\n\u003cli\u003eCongeni J, Murray T, Kline P, Bouhenni R, Morgan D, Liebig C, et al. Preliminary Safety and Efficacy of Head and Neck Cooling Therapy After Concussion in Adolescent Athletes: A Randomized Pilot Trial. Clin J Sport Med. 2022;32(4):341-7.\u003c/li\u003e\n\u003cli\u003eHossain I, Marklund N, Czeiter E, Hutchinson P, Buki A. Blood biomarkers for traumatic brain injury: A narrative review of current evidence. Brain and Spine. 2024;4:102735.\u003c/li\u003e\n\u003cli\u003eMeier TB, Huber DL, Bohorquez-Montoya L, Nitta ME, Savitz J, Teague TK, et al. A Prospective Study of Acute Blood-Based Biomarkers for Sport-Related Concussion. Ann Neurol. 2020;87(6):907-20.\u003c/li\u003e\n\u003cli\u003eMattsson N, Zetterberg H, Janelidze S, Insel PS, Andreasson U, Stomrud E, et al. Plasma tau in Alzheimer disease. Neurology. 2016;87(17):1827-35.\u003c/li\u003e\n\u003cli\u003eGill J, Latour L, Diaz-Arrastia R, Motamedi V, Turtzo C, Shahim P, et al. Glial fibrillary acidic protein elevations relate to neuroimaging abnormalities after mild TBI. Neurology. 2018;91(15):e1385-e9.\u003c/li\u003e\n\u003cli\u003eBiberthaler P, Linsenmeier U, Pfeifer KJ, Kroetz M, Mussack T, Kanz KG, et al. Serum S-100B concentration provides additional information fot the indication of computed tomography in patients after minor head injury: a prospective multicenter study. Shock. 2006;25(5):446-53.\u003c/li\u003e\n\u003cli\u003eNeselius S, Zetterberg H, Blennow K, Marcusson J, Brisby H. Increased CSF levels of phosphorylated neurofilament heavy protein following bout in amateur boxers. PLoS One. 2013;8(11):e81249.\u003c/li\u003e\n\u003cli\u003eZetterberg H, Hietala MA, Jonsson M, Andreasen N, Styrud E, Karlsson I, et al. Neurochemical aftermath of amateur boxing. Arch Neurol. 2006;63(9):1277-80.\u003c/li\u003e\n\u003cli\u003eOtto M, Holthusen S, Bahn E, S\u0026ouml;hnchen N, Wiltfang J, Geese R, et al. Boxing and running lead to a rise in serum levels of S-100B protein. Int J Sports Med. 2000;21(8):551-5.\u003c/li\u003e\n\u003cli\u003eHinson HE, Rowell S, Morris C, Lin AL, Schreiber MA. Early fever after trauma: Does it matter? J Trauma Acute Care Surg. 2018;84(1):19-24.\u003c/li\u003e\n\u003cli\u003ePegoli M, Zurlo Z, Bilotta F. Temperature management in acute brain injury: A systematic review of clinical evidence. Clin Neurol Neurosurg. 2020;197:106165.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Sports concussion, biomarker, boxing, glial fibrillary acidic protein (GFAP), neurofilament-light (Nf-L)","lastPublishedDoi":"10.21203/rs.3.rs-4343194/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4343194/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHead impacts are common in contact sports such as boxing and occur at times of elevated core body and brain temperatures induced by the exercise. Following impact, elevated brain temperature may lead to the development of exacerbated brain injury that can be monitored by blood biomarkers. We hypothesized that acute head-and-neck cooling, recently shown to shorten return-to-play in concussed ice hockey players, applied acutely following a boxing bout is associated with an attenuated release of blood biomarkers and improved symptom rating.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe trial is academically driven and funded by external and hospital research funds. Young, healthy elite boxers ≥ 18 years old are recruited. Prior to, and immediately after a competitive boxing bout over 3x2 or 3x3 minutes, blood samples are drawn. Boxers are randomized to intervention or control management by 1:1 allocation prior to baseline testing. After the initial post-fight blood sample is drawn and symptom rating using the sports concussion assessment tool-5 (SCAT-5) has been collected, the boxers receive either acute selective head-and-neck cooling for 45 min, or routine post-fight management. The number of head impacts are counted in all boxers on match video recordings. In both groups, blood samples are drawn 45 minutes after the initial post-bout blood sample, as well as 3 and 6 days post-fight. At all time points for blood sampling, the number of symptoms (NOS) and symptom severity score (SSS) are assessed using the symptom rating part of the SCAT-5. The primary endpoint is the difference in biomarker levels (GFAP, NF-L, tau, UCH-L1, neuronal-specific enolase) between immediately post-fight and pre-intervention levels, to those obtained at 6 days post-fight. The post-fight SCAT-5 NOS and SSS are secondary endpoints.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no treatment available for boxing-induced brain injury. Biomarkers are surrogate yet objective marker of brain injury, and the head-and-neck cooling treatment may attenuate the release of brain injury-related biomarkers as well as reduce symptoms induced by head impacts attained during a boxing fight. Potentially, head-and-neck cooling is a treatment option acutely following a boxing bout.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrials registration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinicalTrials.gov, ClinicalTrials.gov Identifier NCT06386484. Registered April 23, 2024.\u003c/p\u003e","manuscriptTitle":"Selective head-and-neck cooling on biomarker levels and symptom rating following a boxing bout- protocol for an exploratory randomized trial","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-06 12:15:37","doi":"10.21203/rs.3.rs-4343194/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":"871237fd-2e13-46a1-91d8-0e5232243a85","owner":[],"postedDate":"August 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-30T05:33:51+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-06 12:15:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4343194","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4343194","identity":"rs-4343194","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}