Diagnosis of brain death in wistar rats at different levels of death induction

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Methods Fifteen adult male Wistar rats (Rattus norvergicus; HanUnib: WH) were randomly distributed into three groups of five animals: the control group (G0) with evaluation performed before brain death, and two groups (G1 and G2) with brain death induced at different times: immediately after induction (G1) and one hour after induction (G2). Venous and arterial blood samples were taken to perform complete blood count, biochemical, and blood gas assays. Samples were taken at specific times based on the group each rat belonged to. Results Statistically significant mean values were observed (PG2 and G0>G2), monocytes (G2>G1 and G0>G1), creatinine (G2>G0), aspartate aminotransferase (G1>G0), potassium (G2>G0), and bicarbonate (G0>G1). Discussion Furthermore, brain death showed a unique response in each organism, complicating its precise determination even more. 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F1000Research 2025, 13 :1490 ( https://doi.org/10.12688/f1000research.157233.2 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Brief Report Revised Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] Nayara Maria Gil Mazzante 1 , Fernanda Zuliani 1 , Rogerio Antonio de Oliveira 2 , [...] Júlia Soares Bodaneze 1 , Giovanna Farina Panebianco 1 , Natália Freitas de Souza 1 , Fernando Carmona Dinau 1 , Paola Alejandra Montenegro Cuellar 1 , Nadia Yumi Yamamoto dos Santos 1 , Ana Beatriz de Souza da Silva https://orcid.org/0000-0002-3300-6025 1 , Fernanda de Freitas Alves Vieira 1 , Natália Camargo Faraldo 3 , Gabriela Abreu Botelho 1 , Fernanda Barthelson Carvalho de Moura https://orcid.org/0000-0002-8153-2038 1 , Noeme Sousa Rocha 1 Nayara Maria Gil Mazzante 1 , Fernanda Zuliani 1 , [...] Rogerio Antonio de Oliveira 2 , Júlia Soares Bodaneze 1 , Giovanna Farina Panebianco 1 , Natália Freitas de Souza 1 , Fernando Carmona Dinau 1 , Paola Alejandra Montenegro Cuellar 1 , Nadia Yumi Yamamoto dos Santos 1 , Ana Beatriz de Souza da Silva https://orcid.org/0000-0002-3300-6025 1 , Fernanda de Freitas Alves Vieira 1 , Natália Camargo Faraldo 3 , Gabriela Abreu Botelho 1 , Fernanda Barthelson Carvalho de Moura https://orcid.org/0000-0002-8153-2038 1 , Noeme Sousa Rocha 1 PUBLISHED 06 May 2025 Author details Author details 1 Department of Veterinary Clinics, School of Veterinary Medicine and Animal Science, São Paulo State University - Botucatu Campus, Botucatu, State of São Paulo, Brazil 2 Department of Biostatistics, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, State of São Paulo, Brazil 3 Department of Veterinary Surgery and Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University, Botucatu, State of São Paulo, Brazil Nayara Maria Gil Mazzante Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Validation, Writing – Review & Editing Fernanda Zuliani Roles: Conceptualization, Data Curation, Investigation, Writing – Review & Editing Rogerio Antonio de Oliveira Roles: Investigation, Methodology, Writing – Review & Editing Júlia Soares Bodaneze Roles: Investigation, Methodology, Writing – Review & Editing Giovanna Farina Panebianco Roles: Investigation, Methodology, Writing – Review & Editing Natália Freitas de Souza Roles: Investigation, Methodology, Writing – Review & Editing Fernando Carmona Dinau Roles: Investigation, Writing – Original Draft Preparation Paola Alejandra Montenegro Cuellar Roles: Investigation, Writing – Original Draft Preparation Nadia Yumi Yamamoto dos Santos Roles: Investigation, Writing – Original Draft Preparation Ana Beatriz de Souza da Silva Roles: Visualization, Writing – Review & Editing Fernanda de Freitas Alves Vieira Roles: Visualization, Writing – Review & Editing Natália Camargo Faraldo Roles: Visualization, Writing – Review & Editing Gabriela Abreu Botelho Roles: Visualization, Writing – Review & Editing Fernanda Barthelson Carvalho de Moura Roles: Writing – Original Draft Preparation Noeme Sousa Rocha Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Project Administration, Resources, Supervision, Validation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS Abstract Objective This study aimed to evaluate hematologic, biochemical, and gasometric parameters in Wistar rats to better understand brain death parameters and reduce early misdiagnoses. Methods Fifteen adult male Wistar rats (Rattus norvergicus; HanUnib: WH) were randomly distributed into three groups of five animals: the control group (G0) with evaluation performed before brain death, and two groups (G1 and G2) with brain death induced at different times: immediately after induction (G1) and one hour after induction (G2). Venous and arterial blood samples were taken to perform complete blood count, biochemical, and blood gas assays. Samples were taken at specific times based on the group each rat belonged to. Results Statistically significant mean values were observed (PG2 and G0>G2), monocytes (G2>G1 and G0>G1), creatinine (G2>G0), aspartate aminotransferase (G1>G0), potassium (G2>G0), and bicarbonate (G0>G1). Discussion Furthermore, brain death showed a unique response in each organism, complicating its precise determination even more. READ ALL READ LESS Keywords brain death; hematologic analysis; Wistar rats Corresponding Author(s) Fernanda Barthelson Carvalho de Moura ( [email protected] ) Close Corresponding author: Fernanda Barthelson Carvalho de Moura Competing interests: No competing interests were disclosed. Grant information: This research was funded by “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES), grant number 25/2014 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Gil Mazzante NM et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Gil Mazzante NM, Zuliani F, Oliveira RAd et al. Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.12688/f1000research.157233.2 ) First published: 06 Dec 2024, 13 :1490 ( https://doi.org/10.12688/f1000research.157233.1 ) Latest published: 06 May 2025, 13 :1490 ( https://doi.org/10.12688/f1000research.157233.2 ) Revised Amendments from Version 1 This study aimed to evaluate hematologic, biochemical, and gasometric parameters in Wistar rats to better understand brain death parameters and reduce early misdiagnoses. Brain death (BD), the complete and irreversible loss of brain function, is a complex process characterized by an inflammatory state leading to cellular and molecular disturbances that alter the physiology and biochemistry of the organic system. Wistar rats were randomly distributed into groups, and the time criteria used in the research followed the main goal of this research, as we aim to evaluate the brain death in different levels of death induction. We were able to evaluate the progression of the parameters at different times and understand how the brain death progresses, since the acute changes manifest immediately after injury and can last for a few days. The laboratory parameters play a fundamental role in understanding the progression of brain death, as death results in leakage of cytokines, proteins, and other components, mainly in the acute phase. Also, several changes in brain death may not be detectable in macroscopical examination, so the study primarily focused on laboratory parameters, which could provide early indications of physiological changes. Our results, even in a short interval time evaluated and with a short number of animal models, demonstrate the tendency of those changes. This type of study, in which brain death is evaluated by animal laboratory changes, is not well studied and explored. We hope our study stimulates further research. We wish we could have explored these points more in our response and paper. In closing, we sincerely thank the reviewers for providing an opportunity to clarify our perspective and initiating a professional, productive, and interesting discussion. This study aimed to evaluate hematologic, biochemical, and gasometric parameters in Wistar rats to better understand brain death parameters and reduce early misdiagnoses. Brain death (BD), the complete and irreversible loss of brain function, is a complex process characterized by an inflammatory state leading to cellular and molecular disturbances that alter the physiology and biochemistry of the organic system. Wistar rats were randomly distributed into groups, and the time criteria used in the research followed the main goal of this research, as we aim to evaluate the brain death in different levels of death induction. We were able to evaluate the progression of the parameters at different times and understand how the brain death progresses, since the acute changes manifest immediately after injury and can last for a few days. The laboratory parameters play a fundamental role in understanding the progression of brain death, as death results in leakage of cytokines, proteins, and other components, mainly in the acute phase. Also, several changes in brain death may not be detectable in macroscopical examination, so the study primarily focused on laboratory parameters, which could provide early indications of physiological changes. Our results, even in a short interval time evaluated and with a short number of animal models, demonstrate the tendency of those changes. This type of study, in which brain death is evaluated by animal laboratory changes, is not well studied and explored. We hope our study stimulates further research. We wish we could have explored these points more in our response and paper. In closing, we sincerely thank the reviewers for providing an opportunity to clarify our perspective and initiating a professional, productive, and interesting discussion. See the authors' detailed response to the review by Mario Abbud-Filho and Ludmila Marzochi See the authors' detailed response to the review by Chiara Lazzeri READ REVIEWER RESPONSES Introduction Brain death (BD), the complete and irreversible loss of brain function, is a complex process characterized by an inflammatory state leading to cellular and molecular disturbances that alter the physiology and biochemistry of the organic system. 1 However, criteria for determining BD vary among countries, each having national laws or guidelines, resulting in a lack of standardization. In 50% of countries, clinical examination is sufficient to determine BD, whereas the other 50% require complementary tests. 2 Currently, the American Association of Neurology (AAN) defines BD with three cardinal signs: interruption of brain functions, including the brainstem, coma or unresponsiveness, and apnea. 3 In Brazil, social changes and advancements in medicine led the Federal Council of Medicine (CFM) to update the criteria for diagnosing BD in 2017, with Resolution No. 2,173 of November 23, replacing No. 1,408/97. The changes included specific physiological prerequisites for patients, requirements for doctors to provide care before diagnosing BD, the necessity of complementary tests, and specific training for doctors making this diagnosis. 4 , 5 Determining the moment of BD is complicated by significant complex pathophysiological changes involving excitation of the sympathetic nerves, hormonal imbalance, hemodynamic instability, and metabolic disorders with the release of cytokines, 6 which peak at alternating times due to the type of reaction and injury severity. 7 , 8 During BD progression, a systemic inflammatory response can worsen, leading to disseminated intravascular coagulation mediated by inflammatory mediators from the ischemic brain, ischemic reperfusion injury, metabolic changes during the catecholamine storm, and an inadequately restored cardiovascular state. This also produces sudden changes in blood pressure, hypoxemia, hypothermia, coagulopathy, and electrolyte and hormonal disorders. 9 , 10 More studies are needed to accurately determine the moment of BD, especially due to the scarcity of data. The literature on BD in animals is even more limited. Given the social importance of animals and the growing demand for scientific data to determine and establish death in animals, this has become an important issue for veterinarians. This importance is underscored by the increase in the animal market (farming, pet market, etc.) and the evolution of veterinary medicine (freelancers, clinics, hospitals, and research centers). Therefore, this study aimed to evaluate the hematological, biochemical, and gasometric parameters in rats, as these can be determinants of BD and help elucidate its concept, reducing errors in early diagnosis. Methods Fifteen Wistar adult male rats (Rattus norvegicus) of the HanUnib: WH line were acquired from the Multidisciplinary Center for Biological Research at the State University of Campinas (CEMIB - UNICAMP). They were housed in the Central Experimental Vivarium of the Experimental Research Unit (UNIPEX) at the Medical School (FMB) of São Paulo State University (UNESP). The rats were kept in polypropylene cages with metal grid covers in a room maintained at 22 °C and 55% humidity under a light-dark cycle. The protocols used in this study are in accordance with the Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation (COBEA) and were approved by the Ethics Committee on the Use of Animals (CEUA) of the Faculty of Medicine (FMB) of UNESP, Botucatu Campus, protocol No. 0259/2018 on 16 th January of 2019. Food and water were provided ad libitum, and environmental enrichment was performed using polyvinyl chloride (PVC) pipes and paper balls during the experimental period. Individual body weights were recorded weekly to monitor hygiene, adjust the number of animals per cage, and ensure their welfare. After an acclimatization period of 2 weeks, the animals were randomly divided into three groups, each with five animals: control group (G0) (n=5), evaluated before BD, and two groups with BD induction, immediately after induction (G1) (n=5) and 1 h after induction (G2) (n=5). Before the induction of BD, the animals were anesthetized, initially in an induction box with isoflurane (9–5%) and then with an isoflurane mask (5–2.5%). Intubation was performed using a 14G or 16G catheter, and rats were maintained in the inhalation anesthesia circuit with isoflurane (3–1.5%). Trichotomy and asepsis were performed using chlorhexidine degermante and 70% alcohol in the area of access to the aorta and femoral vein. A 24G catheter was used to access the aorta and femoral vein, and a three-way tap circuit was set up to monitor blood pressure and collect blood for blood gas analysis, along with venous blood collection for blood count and biochemical tests. Animals with mean arterial pressure (MAP) >70 mmHg were used for hematological, biochemical, and hemogasometric parameters. Those without stable MAP were excluded from the experiment. Blood samples were collected from each animal according to their respective group timings: G0 at time 0 (M0, before BD), G1 at time 1 (M1, shortly after BD induction), and G2 at time 2 (M2, 1 h after BD induction). The time criteria used in the research follow the main goal of this research as we aim to evaluate the brain death in different levels of death induction. Using these criteria, we were able to evaluate de progression of the parameters in different times and understand how the brain death progress, from immediately after death, hour after death compared with our control group. According to Hannoodee and Nasuruddin the acute changes manifest immediately after injury and can last for a few days. 11 Venous blood samples were drawn from the femoral vein into 0.5 ml ethylenediaminetetraacetic acid (EDTA) tubes and refrigerated (6–10 °C) for subsequent erythrogram examination (including red blood cells [RBC], hemoglobin [Hb], hematocrit [HCT], mean corpuscular volume [MCV], mean corpuscular hemoglobin concentration [MCHC], mean corpuscular hemoglobin [MCH], total plasma protein [TPT], red cell distribution width [RDW], and platelets) and leukogram (covering total leukocytes, neutrophils, segmented, lymphocytes, eosinophils, and monocytes). Processing was performed using a Hemacounter 60-RT 7600 hematological analyzer (Hemogram, China). Hematocrit was determined by centrifuging microhematocrit samples at 12,000 rpm for 5 min. Platelet counts were performed manually by diluting 20 μL of blood in 2 mL of Brecher solution and counting in a Neubauer chamber. The differential leukocyte count was executed on panotype-stained blood smears (Laborclin) under an optical microscope with immersion (1,000× magnification). The biochemical examination was conducted using the BS200E Analyzer (Mindray, Brazil). Venous blood samples from the femoral vein were collected in dry tubes, refrigerated (6–10 °C), and centrifuged at 2,500 rpm for 5 min. This process was followed by analyzing urea, creatinine (Cr), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), serum total protein (TP), albumin, globulin, creatine kinase (CK), and CK-MB. Blood gas analysis was performed using the ABL80 Flex Analyzer - BASIC Version (RADIOMETER). Arterial blood samples were collected from the aortic artery in 1 mL heparinized syringes (volume collected 0.7 mL), immediately packed in a styrofoam container with crushed ice, and analyzed for blood gases (pH, pCO 2 , and pO 2 ), electrolytes, and metabolites (Na + , K + , Ca 2+ , Cl - , Lac), and derived values (HCO 3 and sO 2 ). BD induction followed the protocol described by Esmaeilzadeh et al. 10 A Fogarty 14G catheter (Baxter Health Corp., CA, USA) was inserted via frontolateral trepanation into the skull using a surgical drill. Intracranial pressure (ICP) was increased by slowly inflating with 400–700 μL of saline. The catheter balloon inflation began with 100 μL of saline solution, and after 1 min, an additional 100 μL of solution was added. This procedure was repeated until the absence of corneal reflexes confirmed the BD, maximally dilated fixed pupils and 60 s apnea. Following BD confirmation, all animals had their body temperature maintained using a thermal mattress, and blood pressure (BP) and heart rate (HR) were continuously monitored with a multiparameter monitor, adjusted according to the respective group. Animals in the BD-induced groups were supported with mechanical ventilation after anesthesia withdrawal. After BD, the animals received a heated infusion of sodium chloride 0.9% supplemented with potassium chloride at a rate of 3–5 mL/kg/h, along with dopamine infusion (5–15 mcg/kg/min), with bolus administration as needed, to conclude animals’ euthanasia. Results In the comparison among G0, G1, and G2, no statistically significant differences (P<0.05) were observed in the mean values of RBC, Hb, HCT, MCV, MCHC, MCH, TPP, RDW, and platelets among the experimental animals ( Table 1 , Figures 1 and 2 ). Table 1. Comparisons between groups 0, 1 and 2 of the values for Red Blood Cells (RBC), Hemoglobin (Hb), Hematocrit (HCT), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin Concentration (MCHC), Mean Corpuscular Hemoglobin (MCH), Total Plasma Protein (TPP), Red Cell Distribution Width (RDW) and Erythrogram Platelets obtained from the experimental animals. Differences in group least squares means, adjusted for multiple comparisons - Tukey-Kramer of the variables. Variable Group _Group Estimate Standart Error Value z Pr˃│z│ Adj p RBC 1 2 -0.2860 0.1385 -2.06 0.0390 0.0973 RBC 1 0 -0.2953 0.1469 -2.01 0.0445 0.1098 RBC 2 0 -0.00930 0.1469 -0.06 0.9495 0.9978 Hb 1 2 -0.2579 0.1354 -1.91 0.0568 0.1372 Hb 1 0 -0.2765 0.1436 -1.93 0.0542 0.1315 Hb 2 0 -0.01857 0.1436 -0.13 0.8971 0.9908 HCT 1 2 -0.2850 0.1295 -2.20 0.0278 0.0710 HCT 1 0 -0.2706 0.1373 -1.97 0.0488 0.1195 HCT 2 0 0.01433 0.1373 -0.10 0.9169 0.9940 MCV 1 2 0.006614 0.03373 0.20 0.8445 0.9790 MCV 1 0 0.02887 0.03578 0.81 0.4198 0.6988 MCV 2 0 0.02225 0.03578 0.62 0.5340 0.8081 MCHC 1 2 0.02000 0.01799 1.11 0.2662 0.5067 MCHC 1 0 -0.01331 0.01908 -0.70 0.4855 0.7650 MCHC 2 0 -0.03331 0.01908 -1.75 0.0808 0.1883 MCH 1 2 0.02765 0.03999 0.69 0.4893 0.7685 MCH 1 0 0.01527 0.04242 0.36 0.7189 0.9311 MCH 2 0 -0.01238 0.04242 -0.29 0.7703 0.9541 TPP 1 2 -0.2016 0.2363 -0.85 0.3037 0.6700 TPP 1 0 -0.3842 0.2507 -1.53 0.1253 0.2755 TPP 2 0 -0.1827 0.2507 -0.73 0.4662 0.7465 RDW 1 2 -0.03441 0.02608 -1.32 0.1870 0.3843 RDW 1 0 -0.01010 0.02767 -0.37 0.7150 0.9291 RDW 2 0 0.02431 0.02767 0.88 0.3795 0.6537 Plaquets 1 2 -0.1220 0.2091 -0.58 0.5594 0.8288 Plaquets 1 0 -0.3171 0.2218 -1.43 0.1527 0.3254 Plaquets 2 0 -0.1951 0.2218 -0.88 0.3790 0.6531 Figure 1. Comparison of mean erythrogram values among G0, G1, and G2, with no statistical differences observed (p<0.05). Figure 2. Comparison of mean platelet values among G0, G1, and G2, with no statistical differences observed (p<0.05). Notably, animals in G1 exhibited thrombocytopenia. In the leukogram ( Table 2 ), comparisons among G0, G1, and G2 regarding total leukocytes, segmented leukocytes, lymphocytes, eosinophils, and monocytes from the experimental animals revealed variations in segmented leukocytes and monocytes (p<0.05) ( Figure 3 ). Table 2. Comparisons between groups 0, 1 and 2 of the values of Total Leukocytes, Segmented Leukocytes, Lymphocytes, Eosinophils and Monocytes of the Leukogram obtained from the experimental animals. Differences in group least squares means, adjusted for multiple comparisons - Tukey-Kramer of the variables. Variable Group _Group Estimate Standart Error Value z Pr˃│z│ Adj p Leukocytes 1 2 -0.4788 0.3086 -1.55 0.1208 0.2670 Leukocytes 1 0 0.02027 0.3273 0.06 0.9506 0.9979 Leukocytes 2 0 0.4991 0.3273 1.52 0.1273 0.2793 Segmented 1 2 4.8179 0.8869 5.43 <.0001 <.0001 * Segmented 1 0 0.9925 0.9407 1.06 0.2913 0.5420 Segmented 2 0 -3.8245 0.9407 -4.07 <.0001 0.0001 * Lymphocytes 1 2 -0.1416 0.3453 -0.41 0.6816 0.9114 Lymphocytes 1 0 0.06694 0.3662 0.18 0.8550 0.9817 Lymphocytes 2 0 0.2086 0.3662 0.57 0.5690 0.8363 Eosinophils 1 2 0.2800 0.3098 0.90 0.3660 0.6378 Eosinophils 1 0 -0.4601 0.3463 -1.33 0.1841 0.3794 Eosinophils 2 0 -0.7401 0.3463 -2.14 0.0326 0.0825 Monocytes 1 2 -0.9122 0.3619 -2.52 0.0117 0.0314 * Monocytes 1 0 -1.0284 0.3838 -2.68 0.0074 0.0202 * Monocytes 2 0 -0.1162 0.3838 -0.30 0.7621 0.9607 * Substantial statistical difference (when p<0.05). Figure 3. Comparison of mean leukogram values among G0, G1, and G2, showing variations in segmented leukocytes and monocytes. The biochemical tests ( Table 3 ) compared G0, G1, and G2 regarding urea, Cr, ALT, AST, ALP, GGT, serum TP, albumin, globulin, CK, and CK-MB from the experimental animals ( Figure 4 ). Only Cr and AST exhibited variations in their values. Table 3. Comparison between groups 0, 1 and 2 in relation to the values of Urea, Creatinine, Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Alkaline Phosphatase (AF), Gamma Glutamyl Transferase (GGT), Total Serum Protein (TSP), Albumin, Globulin, Creatine Kinase (CK) and Creatine Kinase - MB (CK-MB) of the Biochemical Examination obtained from the experimental animals. Differences in group least squares means, adjusted for multiple comparisons - Tukey-Kramer of the variables. Variable Group _Group Estimate Standart Error Value z Pr˃│z│ Adj p Urea 1 2 -0.05071 0.1513 -0.34 0.7375 0.9400 Urea 1 0 0.07189 0.1513 0.48 0.6348 0.8831 Urea 2 0 0.1226 0.1513 0.81 0.4179 0.6967 Creatinine 1 2 -0.1210 0.1460 -0.83 0.4071 0.6850 Creatinine 1 0 0.2935 0.1460 2.01 0.0444 0.1096 Creatinine 2 0 0.4146 0.1460 2.84 0.0045 0.0126 * ALT 1 2 -0.3916 0.2849 -1.37 0.1692 0.3542 ALT 1 0 0.2354 0.2849 0.83 0.4087 0.6867 ALT 2 0 0.6270 0.2849 2.20 0.0277 0.0710 AST 1 2 0.7952 0.3469 2.29 0.0219 0.0568 AST 1 0 0.9538 0.3469 2.75 0.0060 0.0164 * AST 2 0 0.1586 0.3469 0.46 0.6474 0.8911 AF 1 2 -0.5406 0.2725 -1.98 0.0472 0.1161 AF 1 0 0.08733 0.2725 0.32 0.7486 0.9449 AF 2 0 0.6280 0.2725 2.30 0.0212 0.0551 GGT 1 2 -0.06188 0.05020 -1.23 0.2177 0.4339 GGT 1 0 -0.06188 0.05020 -1.23 0.2177 0.4339 GGT 2 0 1.03E-17 0.05020 0.00 1.0000 1.0000 TSP 1 2 -0.06744 0.1127 -0.60 0.5497 0.8210 TSP 1 0 -0.1588 0.1127 -1.41 0.1589 0.3364 TSP 2 0 -0.09135 0.1127 -0.81 0.4177 0.6966 Albumin 1 2 -0.01026 0.1774 -0.06 0.9539 0.9982 Albumin 1 0 -0.2044 0.1774 -1.15 0.2493 0.4821 Albumin 2 0 -0.1942 0.1774 -1.09 0.2739 0.5177 Globulin 1 2 -0.1121 0.08407 -1.33 0.1824 0.3765 Globulin 1 0 -0.1197 0.08407 -1.42 0.1546 0.3288 Globulin 2 0 -0.00755 0.08407 -0.09 0.9285 0.9956 CK 1 2 0.8398 0.5325 1.58 0.1148 0.2556 CK 1 0 0.5379 0.5325 1.01 0.3125 0.5705 CK 2 0 -0.3019 0.5325 -0.57 0.5707 0.8377 CK-MB 1 2 0.2818 0.5311 0.53 0.5956 0.8563 CK-MB 1 0 -0.1141 0.5311 -0.21 0.8299 0.9749 CK-MB 2 0 -0.3959 0.5311 -0.75 0.4559 0.7363 * Substantial statistical difference (when p<0.05). Figure 4. Comparison of mean values of biochemical tests among G0, G1, and G2. Overall, blood gas analysis ( Table 4 ) showed that most animals in all groups had low hydrogen potential (pH) levels, increased oxygen partial pressures (PaO 2 ), and carbon dioxide partial pressures (PaCO 2 ). Table 4. Analysis of values of the Gasometric test variables of the animals in each group. Gasometric A G pH pCO 2 pO 2 Na + K + Ca 2+ Cl - Lac HCO 3 sO 2 1 0 6,97 132,7 142 152 4,16 0,93 108 1,2 29,1 96,7 2 0 7,09 82,0 99 153 4,63 0,43 110 2,9 24,0 93,9 3 0 7,24 63,2 170 147 4,74 0,72 117 1,3 26,4 99,2 4 0 7,10 78,9 180 149 4,81 0,85 109 5,2 23,1 98,9 5 0 7,16 86,1 168 137 4,89 0,90 108 0,6 29,7 98,9 6 1 6,92 91,0 157 154 5,16 1,07 103 5,3 17,8 97,3 7 1 7,24 41,0 63 145 5,43 0,93 110 7,4 17,0 87,7 8 1 7,12 69,7 208 149 4,65 0,79 111 4,7 17,7 99,4 9 1 6,97 70,9 187 151 4,82 0,95 104 6,9 15,6 98,7 10 1 7,23 40,8 45 127 6,22 0,49 101 4,6 16,4 73,0 11 2 6,74 75,9 143 154 8,23 0,72 94 9,5 9,8 94,3 12 2 7,08 70,4 157 156 4,61 0,58 112 0,5 20,1 98,4 13 2 7,03 56,6 134 151 6,11 1,13 104 6,2 14,4 97,2 14 2 7,16 87,4 124 144 5,69 1,03 108 1,2 30,0 97,3 15 2 7,12 77,7 64 150 4,75 1,14 110 0,8 24,3 82,8 In some animals, bicarbonate (HCO 3 ) levels were decreased, and the oxygen saturation index (sO 2 ) and lactate values tended to increase. Moreover, sodium levels showed a tendency to increase in most animals, potassium increased in only one, calcium decreased uniformly across all, and chloride tended to rise in the majority. In the bivariate analysis, the initial analysis of the nine erythrogram variables in group 0 using the Spearman test showed positive correlations with hemoglobin (Hb), platelets, mean corpuscular volum (MCV), and mean corpuscular hemoglobin (MCH) ( Tables 5 and 6 ). Table 5. Descriptive statistics of the Erythrogram variables of Group 0. Simple statistics Variable N Mean Standard Deviation Median Minimum Maximum RBC 4 8.69250 0.86904 8.69000 7.86000 9.53000 Hb 4 16.85000 0.69522 16.85000 16.00000 17.70000 HCT 4 48.50000 3.10913 47.50000 46.00000 53.00000 MCV 4 56.00000 3.57678 56.45000 51.30000 59.80000 MCHC 4 34.80000 1.07083 34.90000 33.40000 36.00000 MCH 4 19.50000 1.55134 19.25000 18.00000 21.50000 TPP 4 6.05000 0.30000 6.20000 5.60000 6.20000 RDW 4 14.42500 0.76757 14.40000 13.60000 15.30000 Platelets 4 865.44375 136.30908 879.96250 707.00000 994.85000 Table 6. Spearman's Correlation between the Erythrogram variables in Group 0. Spearman's Correlation Coefficients (N = 4) Prob >|r|sob H0: Rho=0 RBC Hb HCT MCV MCHC MCH TPP RDW Platelets RBC 1.00000 0.40000 0.80000 -0.80000 -0.80000 -0.80000 0.25820 0.20000 0.40000 0.6000 0.2000 0.2000 0.2000 0.2000 0.7418 0.8000 0.6000 Hb 0.40000 1.00000 0.80000 0.00000 -0.20000 0.00000 0.77460 -0.80000 1.00000 0.6000 0.2000 1.0000 0.8000 1.0000 0.2254 0.2000 <.0001 * HCT 0.80000 0.80000 1.00000 -0.60000 -0.40000 -0.60000 0.77460 -0.40000 0.80000 0.2000 0.2000 0.4000 0.6000 0.4000 0.2254 0.6000 0.2000 MCV -0.80000 0.00000 -0.60000 1.00000 0.40000 1.00000 -0.25820 -0.40000 0.00000 0.2000 1.0000 0.4000 0.6000 <.0001 * 0.7418 0.6000 1.0000 MCHC -0.80000 -0.20000 -0.40000 0.40000 1.00000 0.40000 0.25820 -0.40000 -0.20000 0.2000 0.8000 0.6000 0.6000 0.6000 0.7418 0.6000 0.8000 MCH -0.80000 0.00000 -0.60000 1.00000 0.40000 1.00000 -0.25820 -0.40000 0.00000 0.2000 1.0000 0.4000 <.0001 * 0.6000 0.7418 0.6000 1.0000 TPP 0.25820 0.77460 0.77460 -0.25820 0.25820 -0.25820 1.00000 -0.77460 0.77460 0.7418 0.2254 0.2254 0.7418 0.7418 0.7418 0.2254 0.2254 RDW 0.20000 -0.80000 -0.40000 -0.40000 -0.40000 -0.40000 -0.77460 1.00000 -0.80000 0.8000 0.2000 0.6000 0.6000 0.6000 0.6000 0.2254 0.2000 Platelets 0.40000 1.00000 0.80000 0.00000 -0.20000 0.00000 0.77460 -0.80000 1.00000 0.6000 <.0001 * 0.2000 1.0000 0.8000 1.0000 0.2254 0.2000 * Substantial statistical difference (when p<0.05). Discussion The meticulous control and interpretation of laboratory test changes are intrinsic to diagnosing BD. 12 , 13 Individual examination within each group revealed a tendency towards polycythemia in most animals, particularly notable in G2, consistent with findings from Fiocruz. 14 This can be explained by hypoxemia resulting from BD, leading to secondary absolute polycythemia. One subject in G1 showed anemia and hypoproteinemia. One study found hemoglobin and hematocrit concentrations also decreased after BD induction. 7 There were also animals in this group that showed thrombocytopenia. Another research revealed 38.5% of organ donors exhibited anemia and 30.8% had thrombocytopenia. 29 This phenomenon is attributed to common coagulation disorders following BD, driven by factors such as thromboplastin release, damaged brain tissue fibrinogen release, disseminated intravascular coagulation (DIC), and platelet and coagulation factor consumption due to fluid resuscitation volumes. 13 Regarding the analysis of variables in the leukogram examination, the reduction in segmented neutrophils in G2 may be related to increased bone marrow immunosuppression 1 h after BD. An increase in the number of circulating hematopoietic precursor cells, followed by bone marrow dysfunction, has been observed in patients with severe trauma, hemorrhagic shock, or burns. Stroke induces systemic inflammatory response and subsequent immunosuppression. 15 , 16 Evaluating the animals in each group individually, two animals (A6 and A11) in G1 and G2, respectively, presented leukocytosis. This change is justified by the inflammatory state resulting from the BD process, which activates inflammatory mediators like thromboxanes and leukocyte factors. Among the potential human donors, 66.2% had leukocytosis. 17 , 18 Conversely, Menegat and Sannomiya observed BD-induced leukopenia in rats, resulting in a reduction in lymphocytes, monocytes, and granulocytes. 19 The difference in Cr values between G0 and G2 in this study may be associated with renal function failure due to hemodynamic instability and hypotension, leading to decreased perfusion. 20 Another factor that can affect the kidneys and cause a sudden elevation of plasma Cr, which is widely used as an indicator of acute renal failure and changes in diuresis volume, is rhabdomyolysis. 21 In this study, most of the gas tests presented an acid-base imbalance. Most animals tended towards respiratory acidosis, and some presented metabolic acidosis. These disorders are associated with an increased risk of organ and system dysfunction, and metabolic acidosis is an indicator of unfavorable clinical outcomes. 22 , 23 In BD, gasometry values vary considerably, as the lungs are among the first organs to suffer changes in the body. The lungs seek to compensate and preserve a physiologically acceptable pH when the renal ability to maintain homeostasis is lost. 24 However, the lung tissue can be affected by pathophysiological and endocrine changes and the inflammatory reactions caused by BD. 25 Additionally, perfusion-ventilation imbalance and hypoxemia are the main manifestations of pulmonary changes due to intense adrenergic discharge, leading to increased venous return to the right ventricle consequently increased pulmonary flow. Along with elevated left atrial pressure from intense peripheral vasoconstriction, this results in increased capillary hydrostatic pressure, promoting capillary rupture, interstitial edema, and alveolar hemorrhage. 26 Abnormal partial oxygen pressure promotes the release of tumor necrosis factor-alpha and IL-1 beta, which are inflammatory cytokines that mediate lung lesions. 21 According to Mascia et al.19 and Botha et al.,6 30–45% of potential donors develop lung injury, with acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) being the most frequent. Nogueira and Pereira25 observed that 69.2% of patients had hyperoxia due to oxidative stress in the clinical picture of BD. In this study, lactate levels tended to increase. This is mainly due to altered energy metabolism, where excessive prolongation of anaerobic metabolism overcomes the body’s ability to remove lactate, leading to metabolic acidosis, which can become severe and cause fatigue, even when buffering mechanisms are well-developed. 27 , 28 According to Hodgson and Rose, an anaerobic threshold is commonly reached when lactate. 29 concentration is between 2–4 mmol/L. In our study, higher lactate levels were observed. In most animals, hypernatremia was observed, and an increase in potassium was seen in only one animal, contradicting Westphal et al., 20 who observed hyperkalemia and hypomagnesemia as common disorders associated with BD, potentially leading to arrhythmia. Additionally, calcium decreased in all animals, whereas chlorine levels tended to increase in most cases. Hypernatremia was observed in most animals, and an increase in potassium was seen in only one animal. This contradicts Westphal et al., 20 be related to progressive dysfunction of the hypothalamic-pituitary axis after BD and, in 80% of cases, the development of diabetes insipidus, which results in a decline in circulating antidiuretic hormone. 30 The polyuria resulting from this endocrine/metabolic change can lead to serious electrolyte disorders, such as hypernatremia, hypokalemia, hypocalcemia, hypophosphatemia, and hypomagnesemia. 14 In a study by Vasconcelos et al. with possible human donors, 47.7% had hypernatremia, similar to the values obtained in this study. 31 Therefore, this study recognizes and highlights the deleterious effects caused by BD, noting that these effects are multiple and cause various complications in the body, so an evaluation using different combined analyses is important, as shown in this study, to also diagnose different critical diseases prior to the brain death itself. Early detection is important to avoid progressive somatic deterioration and ensure better organ function. 31 , 32 The experimental model used in this study, which is interesting for evaluating a short period of time, did not produce more significant results for many variables. The speed of diagnosing BD may not lead to major changes in tests within 1 h after BD. Additionally, the number of animals (n) used was a limiting factor. In conclusion, BD showed a unique response in each organism, complicating its precise determination even more. However, the time criteria used in the research follow the main goal of this research as we aim to evaluate the brain death in different levels of death induction. Using these criteria, we were able to evaluate the progression of the parameters at different times and understand how the brain death progresses, from immediately after death, hour after death, compared with our control group. And it is settled that the acute changes manifest immediately after injury and can last for a few days. 11 So maybe the results we have had reflect the reality of the changes that we can and cannot observe in brain death. The small sample size of our study was a limitation for establishing parameters to settle brain death characteristics. However, we have calculated the minimum number of animals to use and validate the data. The main problem was the inconsistency of the values found, which supports our belief that the brain death parameters have large intervals, so future research could be elaborated with a more significant number of animals to see if it would impact the results. We indeed believe that BD shows a unique response in each organism, so it is a challenge to determine its precise time. But our results support the hypothetical theories we found during the literature review with a pilot study conducted in laboratory animals. We believe, within the results we found in this research that the laboratory parameters play a fundamental role in understanding the progression of brain death, as death results in leakage of cytokines, proteins, and other components, mainly in the acute phase. Also, several changes in brain death may not be detectable in macroscopical examination, which is why this study primarily focused on laboratory parameters, which could provide early indications of physiological changes. the primary objective of this paper was to evaluate the hematologic, biochemical, and gasometrical parameters in brain death to understand the modifications and reduce misdiagnosis by using these parameters as an indication in early stages. Our research is experimental, and we performed an individual examination of the specific groups used in the research. The experimental nature of this paper should be considered, as well as the small number of animals used in this research. Our results, even in a short interval time evaluated and with a short number of animal models, demonstrate the tendency of those changes. We hope our study stimulates further research, which could provide a better discussion of the results. Further studies with more animals and extended observation periods are necessary to elaborate a more comprehensive discussion of the mechanisms involved. In conclusion we believe that this study highlights the complex physiological changes associated with brain death (BD) in Wistar rats. Significant differences were observed between groups in the laboratory analysis, such as: segmented cells, monocytes, creatinine, aspartate aminotransferase, potassium, and bicarbonate levels, demonstrating the varied impact of BD on these parameters. The study shows that early detection is important to avoid progressive somatic deterioration and ensure better organ function. Although the experimental model and sample size in this work were not ideal, it has the potential to contribute to the understanding that individual responses to BD oscillate, complicating its diagnosis and emphasizing the need for further investigation to improve clinical diagnostic precision. Ethics We confirm that all efforts were made to ameliorate suffering of animals applying the rules settled to animal pain management and suffering in the “Brazilian Guide to Good Animal Euthanasia Practices” (available online in: https://www.cfmv.gov.br/guia-brasileiro-de-boas-praticas-para-a-eutanasia-em-animais/comunicacao/publicacoes/2020/08/03/#1 ) dictated by the Brazilian Federal Council of Veterinary Medicine (CFMV). Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation (COBEA) and were approved by the Ethics Committee on the Use of Animals (CEUA) of the Faculty of Medicine (FMB) of UNESP, Botucatu Campus, protocol No. 0259/2018 on 16 th January 2019. Data availability Underlying data Figshare: Parâmetros hematológicos, gasométricos e bioquímicos na indução da morte encefálica em ratos Wistar, DOI: https://doi.org/10.6084/m9.figshare.27586602.v6 33 This project contains the following underlying data: • PARÂMETROS HEMATOLÓGICOS, GASOMÉTRICOS E.docx • Underlying data spreadsheet.xlsx • IACUC • mazzante_nmg_dr_bot_sub-english.docx • Underlyingdataspreadsheet-english.xlsx • ARRIVE Checklist Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Reporting checklist Figshare: Arrive checklist for ‘ Parâmetros hematológicos, gasométricos e bioquímicos na indução da morte encefálica em ratos Wistar’ , DOI: https://doi.org/10.6084/m9.figshare.27586602.v6 Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Acknowledgments We thank the School of Veterinary Medicine and Animal Science of São Paulo State University for their support and encouragement during this study. Disclosure on use generative AI and AI-assisted technologies The authors declare that the Chat GPT – 4 in the response writing process to improve readability and language. References 1. Abram MB, Fox RC, Garcia-Palmier M, et al. : Defining death: medical, legal and ethical issues in the determination of death. Washington, DC: Georgetown University; 1981. Accessed January 22, 2019. Reference Source 2. 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Lima CM, Lima AK, Melo MGD, et al. : Valores de referência hematológicos e bioquímicos de ratos (Rattus novergicus linhagem Wistar) provenientes do biotério da Universidade Tiradentes. Sci. Plena. 2014; 10 : 1–9. 13. Fiocruz: Centro de Criação de Animais de Laboratório da Fundação Oswaldo Cruz. Resultado de exames hematológicos para ratos Wistar - Laudo Clínico. Portal da Fundação Oswaldo Cruz; 2004. Accessed January 22, 2019. Reference Source 14. Nogueira EC: Captação de órgãos em Sergipe e fatores associados à efetivação de potenciais doadores; 2008. 103f. Dissertação (Mestrado) - Universidade Federal de Sergipe. Aracaju. 15. Badami CD, Livingston DH, Sifri ZC, et al. : Hematopoietic progenitor cells mobilize to the site of injury after trauma and hemorrhagic shock in rats. J. Trauma. 2007; 63 : 596–602. discussion 600. PubMed Abstract | Publisher Full Text 16. 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Ventilação mecânica, controle endócrino metabólico e aspectos hematológicos e infecciosos. Rev. Bras. Ter. Intensiva. 2011b; 23 : 269–282. PubMed Abstract | Publisher Full Text 21. Nogueira EC, Pereira CU: Potencial para obtenção de órgãos em um hospital de urgência de Sergipe. Braz. J. Transplant. 2007; 10 : 756–761. Publisher Full Text 22. Barbosa MBG, Alves CAD, Filho HQ: Avaliação da acidose metabólica em pacientes Graves: método de Stewart-Fencl-Figge versus a abordagem tradicional de Henderson-Hasselbalch. Rev. Bras. Ter. Intensiva. 2006; 18 : 380–384. Publisher Full Text 23. Rocco JR: Diagnóstico dos distúrbios do metabolismo ácido-base. Rev. Bras. Ter. Intensiva. 2003; 15 : 184–192. 24. Viegas CA: Gasometria arterial. J. Pneumol. 2012; 28 : 233–238. 25. Mascia L, Sakr Y, Pasero D, et al. : Extracranial complications in patients with acute brain injury: a post-hoc analysis of the SOAP study. Intensive Care Med. 2008; 34 : 720–727. PubMed Abstract | Publisher Full Text 26. Guetti NR, Marques IR: Assistência de enfermagem ao potencial doador de órgãos em morte encefálica. Rev. Bras. Enferm. 2008; 61 : 91–97. PubMed Abstract | Publisher Full Text 27. Beard LA, Hinchcliff KW: Effect of NaCl and NaHCO3 on serum ionised calcium and blood gas status during sprinting. Equine Vet. J. 2002; 34 : 519–523. Publisher Full Text 28. Westphal GA, Caldeira Filho M, Vieira KD, et al. : Diretrizes para manutenção de múltiplos órgãos no potencial doador adulto falecido: Parte I. Aspectos gerais e suporte hemodinâmico. Rev. Bras. Ter. Intensiva. 2011; 23 : 255–268. PubMed Abstract | Publisher Full Text 29. Hodgson DR, Rose RJ: The athletic horse: principles and practice of equine sports medicine. 1st ed.Philadelphia: W. B. Saunders Company; 1994; 245–258. 30. Howlett TA, Keogh AM, Perry L, et al. : Anterior and posterior pituitary function in brain-stem-dead donors. A possible role for hormonal replacement therapy. Transplantation. 1989; 47 : 828–833. PubMed Abstract | Publisher Full Text 31. Vasconcelos Q, Freire ILS, Araújo RO, et al. : Avaliação laboratorial de potenciais doadores de órgãos e tecidos para transplantes. Rev. Rene. 2014; 15 : 273–281. 32. Da Silva JRF, Silva MHM, Ramos VP: Familiaridade dos profissionais de saúde sobre os critérios de diagnósticos de morte encefálica. Enferm. Foco. 2010; 1 : 98–103. Publisher Full Text 33. Barthelson Carvalho de Moura F, Maria Gil Mazzante N, Rocha NS: Parâmetros hematológicos, gasométricos e bioquímicos na indução da morte encefálica em ratos Wistar. figshare. 2024 [cited 2024 Nov 22]. Reference Source Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 06 Dec 2024 ADD YOUR COMMENT Comment Author details Author details 1 Department of Veterinary Clinics, School of Veterinary Medicine and Animal Science, São Paulo State University - Botucatu Campus, Botucatu, State of São Paulo, Brazil 2 Department of Biostatistics, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, State of São Paulo, Brazil 3 Department of Veterinary Surgery and Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University, Botucatu, State of São Paulo, Brazil Nayara Maria Gil Mazzante Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Validation, Writing – Review & Editing Fernanda Zuliani Roles: Conceptualization, Data Curation, Investigation, Writing – Review & Editing Rogerio Antonio de Oliveira Roles: Investigation, Methodology, Writing – Review & Editing Júlia Soares Bodaneze Roles: Investigation, Methodology, Writing – Review & Editing Giovanna Farina Panebianco Roles: Investigation, Methodology, Writing – Review & Editing Natália Freitas de Souza Roles: Investigation, Methodology, Writing – Review & Editing Fernando Carmona Dinau Roles: Investigation, Writing – Original Draft Preparation Paola Alejandra Montenegro Cuellar Roles: Investigation, Writing – Original Draft Preparation Nadia Yumi Yamamoto dos Santos Roles: Investigation, Writing – Original Draft Preparation Ana Beatriz de Souza da Silva Roles: Visualization, Writing – Review & Editing Fernanda de Freitas Alves Vieira Roles: Visualization, Writing – Review & Editing Natália Camargo Faraldo Roles: Visualization, Writing – Review & Editing Gabriela Abreu Botelho Roles: Visualization, Writing – Review & Editing Fernanda Barthelson Carvalho de Moura Roles: Writing – Original Draft Preparation Noeme Sousa Rocha Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Project Administration, Resources, Supervision, Validation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information This research was funded by “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES), grant number 25/2014 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (2) version 2 Revised Published: 06 May 2025, 13:1490 https://doi.org/10.12688/f1000research.157233.2 version 1 Published: 06 Dec 2024, 13:1490 https://doi.org/10.12688/f1000research.157233.1 Copyright © 2025 Gil Mazzante NM et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Gil Mazzante NM, Zuliani F, Oliveira RAd et al. Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.12688/f1000research.157233.2 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 2 VERSION 2 PUBLISHED 06 May 2025 Revised Views 0 Cite How to cite this report: Machado C. Reviewer Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.181285.r383475 ) The direct URL for this report is: https://f1000research.com/articles/13-1490/v2#referee-response-383475 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 24 May 2025 Calixto Machado , Institute of Neurology and Neurosurgery, Havana, Cuba Approved VIEWS 0 https://doi.org/10.5256/f1000research.181285.r383475 General Assessment This manuscript presents an exploratory study on the physiological and biochemical changes associated with brain death (BD) in Wistar rats. It aims to deepen our understanding of the variable responses to BD induction and to identify laboratory ... Continue reading READ ALL General Assessment This manuscript presents an exploratory study on the physiological and biochemical changes associated with brain death (BD) in Wistar rats. It aims to deepen our understanding of the variable responses to BD induction and to identify laboratory markers that may help refine early diagnostic criteria. The subject is highly relevant, particularly given the limited availability of experimental data addressing the pathophysiological dynamics of brain death in preclinical models. Despite a modest sample size, the study’s design is clear and methodologically sound. It includes three well-defined groups with varying durations after BD induction and offers statistically supported comparisons of hematologic, biochemical, and gasometric parameters. The observed variations, especially in segmented leukocytes, monocytes, creatinine, AST, potassium, and bicarbonate, contribute to our growing understanding of the systemic impact of BD. Ethical and Animal Welfare Considerations Of particular importance is the authors’ adherence to established ethical protocols in animal research. The study was approved by the Ethics Committee on the Use of Animals (CEUA – protocol 0259/2018) and followed both the Brazilian College of Animal Experimentation (COBEA) and the Brazilian Guide to Good Euthanasia Practices . This adherence ensures that all efforts were made to minimize suffering and prioritize humane treatment throughout the experimental process. The inclusion of environmental enrichment and diligent monitoring further exemplifies best practices in animal care. By explicitly detailing the anesthesia protocols, surgical preparation, and post-procedural monitoring, the authors demonstrate a commendable commitment to animal welfare and the 3Rs (Replacement, Reduction, and Refinement) in experimental design. Scientific Significance and Recommendations The research contributes novel data on how acute physiological changes manifest post-BD and reinforces the importance of laboratory parameters as early indicators of systemic decline. While the sample size limits generalizability, the study effectively functions as a pilot investigation that could inspire more expansive, statistically powered future research. I recommend the acceptance of this manuscript with minor revisions aimed at improving the clarity of conclusions and further contextualizing results within existing literature. Expanding on the potential clinical translational value of such findings in veterinary and human medicine would be particularly beneficial. Final Recommendation: ✅ Accept with minor revisions This study is ethically sound, scientifically relevant, and presents well-supported preliminary findings that merit dissemination. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Brain death, disorders of consciousness, neuroimaging, general neurology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Machado C. Reviewer Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.181285.r383475 ) The direct URL for this report is: https://f1000research.com/articles/13-1490/v2#referee-response-383475 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Lazzeri C. Reviewer Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.181285.r383095 ) The direct URL for this report is: https://f1000research.com/articles/13-1490/v2#referee-response-383095 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 14 May 2025 Chiara Lazzeri , Careggi Hospital, Florence, Italy Approved VIEWS 0 https://doi.org/10.5256/f1000research.181285.r383095 I read the revised version of the manuscript. The Authors provided ... Continue reading READ ALL I read the revised version of the manuscript. The Authors provided a point to point reply to the reviewers' comments. The manuscript improved. Competing Interests: No competing interests were disclosed. Reviewer Expertise: organ procurement and transplantation, cardiac arrest, ECMO, respiratory failure, critical care echocardiography I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Lazzeri C. Reviewer Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.181285.r383095 ) The direct URL for this report is: https://f1000research.com/articles/13-1490/v2#referee-response-383095 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Abbud-Filho M. Reviewer Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.181285.r383096 ) The direct URL for this report is: https://f1000research.com/articles/13-1490/v2#referee-response-383096 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 13 May 2025 Mario Abbud-Filho , Medicine, Hospital de Base (FUNFARME) and Medical School (FAMERP), São José do Rio Preto, São Paulo, Brazil Approved VIEWS 0 https://doi.org/10.5256/f1000research.181285.r383096 The authors answered the questions we addressed and improved the revised version. In the interest of the manuscript's clarity, they should include a visual representation, such as a diagram or timeline, summarizing the experimental design, particularly the timing of sample ... Continue reading READ ALL The authors answered the questions we addressed and improved the revised version. In the interest of the manuscript's clarity, they should include a visual representation, such as a diagram or timeline, summarizing the experimental design, particularly the timing of sample collection across the different groups. The illustration could make the description clearer and more concise. We thank you for the opportunity to revise the manuscript. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Renal transplantation, donor preconditioning, molecular aspects of marginal kidneys I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Abbud-Filho M. Reviewer Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.181285.r383096 ) The direct URL for this report is: https://f1000research.com/articles/13-1490/v2#referee-response-383096 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 1 VERSION 1 PUBLISHED 06 Dec 2024 Views 0 Cite How to cite this report: Abbud-Filho M and Marzochi L. Reviewer Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.172655.r366145 ) The direct URL for this report is: https://f1000research.com/articles/13-1490/v1#referee-response-366145 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 04 Mar 2025 Mario Abbud-Filho , Medicine, Hospital de Base (FUNFARME) and Medical School (FAMERP), São José do Rio Preto, São Paulo, Brazil Ludmila Marzochi , Medicine, Medical School Famerp Laboratory of Immunology & Experimental Transplantation, São José do Rio Preto, São Paulo, Brazil Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.172655.r366145 I was asked to review the manuscript entitled “Diagnosis of Brain Death in Wistar Rats at Different Levels of Death Induction” authored by Mazzante et al. Although there is indeed a debate—more philosophical than medical—about the definition of brain ... Continue reading READ ALL I was asked to review the manuscript entitled “Diagnosis of Brain Death in Wistar Rats at Different Levels of Death Induction” authored by Mazzante et al. Although there is indeed a debate—more philosophical than medical—about the definition of brain death, in the field of transplantation, there is no doubt regarding the selection of potential organ donors. From a biochemical and molecular perspective, brain death is of particular interest to researchers in this field, making the authors' work relevant. However, several issues raise major concerns: The small sample size limits the generalizability of the findings. Although the authors acknowledge this limitation, it remains a significant constraint. The selected time points for collecting laboratory parameters (M1 and M2) are difficult to interpret, as they primarily reflect acute changes associated with brain death. It seems overly simplistic to conclude the highly complex process of brain death based solely on the chosen laboratory parameters (hematological, renal, and hepatic markers). While the study identifies significant changes in various parameters, it does not sufficiently explore the underlying mechanisms driving these changes. For instance, the discussion could further elaborate on how brain death-induced inflammatory responses or hormonal imbalances contribute to the observed alterations in hematologic and biochemical markers. The discussion section could be strengthened by addressing conflicting findings in the literature more thoroughly. For example, while the study reports hypernatremia and hypokalemia, potential reasons for these discrepancies could have been explored more deeply. The conclusion is vague and does not fully summarize the key findings or their implications. A more concise and impactful conclusion would enhance the manuscript. In summary, the study is scientifically sound and addresses a relevant topic. However, significant revisions are necessary to improve its clarity, depth, and overall impact. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Renal transplantation, donor preconditioning, molecular aspects of marginal kidneys We confirm that we have read this submission and believe that we have an appropriate level of expertise to state that we do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Abbud-Filho M and Marzochi L. Reviewer Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.172655.r366145 ) The direct URL for this report is: https://f1000research.com/articles/13-1490/v1#referee-response-366145 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 10 May 2025 Fernanda Barthelson Carvalho de Moura , Department of Veterinary Clinics, School of Veterinary Medicine and Animal Science, São Paulo State University - Botucatu Campus, Botucatu, Brazil 10 May 2025 Author Response It is with great enthusiasm that we respond to Dr. Mario Abbud-Filho Dr. Ludmila Marzochi and Dr. Chiara Lazzeri comments concerning the study “Diagnosis of brain death in wistar rats at ... Continue reading It is with great enthusiasm that we respond to Dr. Mario Abbud-Filho Dr. Ludmila Marzochi and Dr. Chiara Lazzeri comments concerning the study “Diagnosis of brain death in wistar rats at different levels of death induction” 1 . The authors genuinely appreciate this consideration and interest in our work. In response to reviewers’ comments and requests, the time criteria used in the research follow the main goal of this research as we aim to evaluate the brain death in different levels of death induction. Using these criteria, we were able to evaluate de progression of the parameters in different times and understand how the brain death progress, from immediately after death, hour after death compared with our control group. According to Hannodee and Nasuruddin, the acute changes manifest immediately after injury and can last for a few days 2 . Additionally, the laboratory parameters play a fundamental role in understanding the progression of brain death, as death results in leakage of cytokines, proteins, and other components, mainly in the acute phase. Also, several changes in the brain death may not be detectable in macroscopical examination, which is the reason why the study primarily focused on laboratory parameters, which could provide early indications of physiological changes. We want to enhance that the primary objective of this paper was to evaluate the hematologic, biochemical, and gasometrical parameters in brain death to understand the modifications and reduce misdiagnosis by using these parameters as an indication in early stages. We acknowledge that discussing those changes could be explored in more detail. However, the experimental nature of this paper should be considered, as well as the small number of animals used in this research. Our results, even in a short interval time evaluated and with a short number of animal models, demonstrate the tendency of those changes. Further studies with more animals and extended observation periods are necessary to elaborate a more comprehensive discussion of the mechanisms involved. The authors agree that the discussion could be more detailed and deeper. However, our research is experimental, and we performed an individual examination of the specific groups used in the research. We provided a discussion comparing our findings with other results in the literature (ie, Fiocruz, Hodgson, and Rose) 3,4 ; however, this type of study, in which brain death is evaluated by animal laboratory changes, is not well studied and explored. We hope our study stimulates further research, which could provide a better discussion of the results. In conclusion we believe that this study highlights the complex physiological changes associated with brain death (BD) in Wistar rats. Significant differences were observed between groups in the laboratory analysis, such as: segmented cells, monocytes, creatinine, aspartate aminotransferase, potassium, and bicarbonate levels, demonstrating the varied impact of BD on these parameters. The study shows that early detection is important to avoid progressive somatic deterioration and ensure better organ function. Although the experimental model and sample size in this work were not ideal, it has the potential to contribute to the understanding that individual responses to BD oscillate, complicating its diagnosis and emphasizing the need for further investigation to improve clinical diagnostic precision. We sincerely wish we could now have explored these points more in our response and paper. In closing, we sincerely thank Dr. Mario Abbud-Filho, Dr. Ludmila Marzochi, and Dr. Chiara Lazzeri for providing an opportunity to clarify our perspective and initiating a professional, productive, and interesting discussion. References Gil Mazzante NM, Zuliani F, Oliveira RAd et al. Diagnosis of brain death in wistar rats at different levels of death induction [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2024, 13:1490 ( https://doi.org/10.12688/f1000research.157233.1 Hannoodee S, Nasuruddin DN. Acute Inflammatory Response. 2024 Jun 8. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan–. PMID: 32310543. Nogueira EC: Captação de órgãos em Sergipe e fatores associados à efetivação de potenciais doadores; 2008. 103f. Dissertação (Mestrado) - Universidade Federal de Sergipe. Aracaju. Hodgson DR, Rose RJ: The athletic horse: principles and practice of equine sports medicine. 1st ed.Philadelphia: W. B. Saunders Company; 1994; 245–258. It is with great enthusiasm that we respond to Dr. Mario Abbud-Filho Dr. Ludmila Marzochi and Dr. Chiara Lazzeri comments concerning the study “Diagnosis of brain death in wistar rats at different levels of death induction” 1 . The authors genuinely appreciate this consideration and interest in our work. In response to reviewers’ comments and requests, the time criteria used in the research follow the main goal of this research as we aim to evaluate the brain death in different levels of death induction. Using these criteria, we were able to evaluate de progression of the parameters in different times and understand how the brain death progress, from immediately after death, hour after death compared with our control group. According to Hannodee and Nasuruddin, the acute changes manifest immediately after injury and can last for a few days 2 . Additionally, the laboratory parameters play a fundamental role in understanding the progression of brain death, as death results in leakage of cytokines, proteins, and other components, mainly in the acute phase. Also, several changes in the brain death may not be detectable in macroscopical examination, which is the reason why the study primarily focused on laboratory parameters, which could provide early indications of physiological changes. We want to enhance that the primary objective of this paper was to evaluate the hematologic, biochemical, and gasometrical parameters in brain death to understand the modifications and reduce misdiagnosis by using these parameters as an indication in early stages. We acknowledge that discussing those changes could be explored in more detail. However, the experimental nature of this paper should be considered, as well as the small number of animals used in this research. Our results, even in a short interval time evaluated and with a short number of animal models, demonstrate the tendency of those changes. Further studies with more animals and extended observation periods are necessary to elaborate a more comprehensive discussion of the mechanisms involved. The authors agree that the discussion could be more detailed and deeper. However, our research is experimental, and we performed an individual examination of the specific groups used in the research. We provided a discussion comparing our findings with other results in the literature (ie, Fiocruz, Hodgson, and Rose) 3,4 ; however, this type of study, in which brain death is evaluated by animal laboratory changes, is not well studied and explored. We hope our study stimulates further research, which could provide a better discussion of the results. In conclusion we believe that this study highlights the complex physiological changes associated with brain death (BD) in Wistar rats. Significant differences were observed between groups in the laboratory analysis, such as: segmented cells, monocytes, creatinine, aspartate aminotransferase, potassium, and bicarbonate levels, demonstrating the varied impact of BD on these parameters. The study shows that early detection is important to avoid progressive somatic deterioration and ensure better organ function. Although the experimental model and sample size in this work were not ideal, it has the potential to contribute to the understanding that individual responses to BD oscillate, complicating its diagnosis and emphasizing the need for further investigation to improve clinical diagnostic precision. We sincerely wish we could now have explored these points more in our response and paper. In closing, we sincerely thank Dr. Mario Abbud-Filho, Dr. Ludmila Marzochi, and Dr. Chiara Lazzeri for providing an opportunity to clarify our perspective and initiating a professional, productive, and interesting discussion. References Gil Mazzante NM, Zuliani F, Oliveira RAd et al. Diagnosis of brain death in wistar rats at different levels of death induction [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2024, 13:1490 ( https://doi.org/10.12688/f1000research.157233.1 Hannoodee S, Nasuruddin DN. Acute Inflammatory Response. 2024 Jun 8. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan–. PMID: 32310543. Nogueira EC: Captação de órgãos em Sergipe e fatores associados à efetivação de potenciais doadores; 2008. 103f. Dissertação (Mestrado) - Universidade Federal de Sergipe. Aracaju. Hodgson DR, Rose RJ: The athletic horse: principles and practice of equine sports medicine. 1st ed.Philadelphia: W. B. Saunders Company; 1994; 245–258. Competing Interests: No competing interests to declare Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 10 May 2025 Fernanda Barthelson Carvalho de Moura , Department of Veterinary Clinics, School of Veterinary Medicine and Animal Science, São Paulo State University - Botucatu Campus, Botucatu, Brazil 10 May 2025 Author Response It is with great enthusiasm that we respond to Dr. Mario Abbud-Filho Dr. Ludmila Marzochi and Dr. Chiara Lazzeri comments concerning the study “Diagnosis of brain death in wistar rats at ... Continue reading It is with great enthusiasm that we respond to Dr. Mario Abbud-Filho Dr. Ludmila Marzochi and Dr. Chiara Lazzeri comments concerning the study “Diagnosis of brain death in wistar rats at different levels of death induction” 1 . The authors genuinely appreciate this consideration and interest in our work. In response to reviewers’ comments and requests, the time criteria used in the research follow the main goal of this research as we aim to evaluate the brain death in different levels of death induction. Using these criteria, we were able to evaluate de progression of the parameters in different times and understand how the brain death progress, from immediately after death, hour after death compared with our control group. According to Hannodee and Nasuruddin, the acute changes manifest immediately after injury and can last for a few days 2 . Additionally, the laboratory parameters play a fundamental role in understanding the progression of brain death, as death results in leakage of cytokines, proteins, and other components, mainly in the acute phase. Also, several changes in the brain death may not be detectable in macroscopical examination, which is the reason why the study primarily focused on laboratory parameters, which could provide early indications of physiological changes. We want to enhance that the primary objective of this paper was to evaluate the hematologic, biochemical, and gasometrical parameters in brain death to understand the modifications and reduce misdiagnosis by using these parameters as an indication in early stages. We acknowledge that discussing those changes could be explored in more detail. However, the experimental nature of this paper should be considered, as well as the small number of animals used in this research. Our results, even in a short interval time evaluated and with a short number of animal models, demonstrate the tendency of those changes. Further studies with more animals and extended observation periods are necessary to elaborate a more comprehensive discussion of the mechanisms involved. The authors agree that the discussion could be more detailed and deeper. However, our research is experimental, and we performed an individual examination of the specific groups used in the research. We provided a discussion comparing our findings with other results in the literature (ie, Fiocruz, Hodgson, and Rose) 3,4 ; however, this type of study, in which brain death is evaluated by animal laboratory changes, is not well studied and explored. We hope our study stimulates further research, which could provide a better discussion of the results. In conclusion we believe that this study highlights the complex physiological changes associated with brain death (BD) in Wistar rats. Significant differences were observed between groups in the laboratory analysis, such as: segmented cells, monocytes, creatinine, aspartate aminotransferase, potassium, and bicarbonate levels, demonstrating the varied impact of BD on these parameters. The study shows that early detection is important to avoid progressive somatic deterioration and ensure better organ function. Although the experimental model and sample size in this work were not ideal, it has the potential to contribute to the understanding that individual responses to BD oscillate, complicating its diagnosis and emphasizing the need for further investigation to improve clinical diagnostic precision. We sincerely wish we could now have explored these points more in our response and paper. In closing, we sincerely thank Dr. Mario Abbud-Filho, Dr. Ludmila Marzochi, and Dr. Chiara Lazzeri for providing an opportunity to clarify our perspective and initiating a professional, productive, and interesting discussion. References Gil Mazzante NM, Zuliani F, Oliveira RAd et al. Diagnosis of brain death in wistar rats at different levels of death induction [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2024, 13:1490 ( https://doi.org/10.12688/f1000research.157233.1 Hannoodee S, Nasuruddin DN. Acute Inflammatory Response. 2024 Jun 8. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan–. PMID: 32310543. Nogueira EC: Captação de órgãos em Sergipe e fatores associados à efetivação de potenciais doadores; 2008. 103f. Dissertação (Mestrado) - Universidade Federal de Sergipe. Aracaju. Hodgson DR, Rose RJ: The athletic horse: principles and practice of equine sports medicine. 1st ed.Philadelphia: W. B. Saunders Company; 1994; 245–258. It is with great enthusiasm that we respond to Dr. Mario Abbud-Filho Dr. Ludmila Marzochi and Dr. Chiara Lazzeri comments concerning the study “Diagnosis of brain death in wistar rats at different levels of death induction” 1 . The authors genuinely appreciate this consideration and interest in our work. In response to reviewers’ comments and requests, the time criteria used in the research follow the main goal of this research as we aim to evaluate the brain death in different levels of death induction. Using these criteria, we were able to evaluate de progression of the parameters in different times and understand how the brain death progress, from immediately after death, hour after death compared with our control group. According to Hannodee and Nasuruddin, the acute changes manifest immediately after injury and can last for a few days 2 . Additionally, the laboratory parameters play a fundamental role in understanding the progression of brain death, as death results in leakage of cytokines, proteins, and other components, mainly in the acute phase. Also, several changes in the brain death may not be detectable in macroscopical examination, which is the reason why the study primarily focused on laboratory parameters, which could provide early indications of physiological changes. We want to enhance that the primary objective of this paper was to evaluate the hematologic, biochemical, and gasometrical parameters in brain death to understand the modifications and reduce misdiagnosis by using these parameters as an indication in early stages. We acknowledge that discussing those changes could be explored in more detail. However, the experimental nature of this paper should be considered, as well as the small number of animals used in this research. Our results, even in a short interval time evaluated and with a short number of animal models, demonstrate the tendency of those changes. Further studies with more animals and extended observation periods are necessary to elaborate a more comprehensive discussion of the mechanisms involved. The authors agree that the discussion could be more detailed and deeper. However, our research is experimental, and we performed an individual examination of the specific groups used in the research. We provided a discussion comparing our findings with other results in the literature (ie, Fiocruz, Hodgson, and Rose) 3,4 ; however, this type of study, in which brain death is evaluated by animal laboratory changes, is not well studied and explored. We hope our study stimulates further research, which could provide a better discussion of the results. In conclusion we believe that this study highlights the complex physiological changes associated with brain death (BD) in Wistar rats. Significant differences were observed between groups in the laboratory analysis, such as: segmented cells, monocytes, creatinine, aspartate aminotransferase, potassium, and bicarbonate levels, demonstrating the varied impact of BD on these parameters. The study shows that early detection is important to avoid progressive somatic deterioration and ensure better organ function. Although the experimental model and sample size in this work were not ideal, it has the potential to contribute to the understanding that individual responses to BD oscillate, complicating its diagnosis and emphasizing the need for further investigation to improve clinical diagnostic precision. We sincerely wish we could now have explored these points more in our response and paper. In closing, we sincerely thank Dr. Mario Abbud-Filho, Dr. Ludmila Marzochi, and Dr. Chiara Lazzeri for providing an opportunity to clarify our perspective and initiating a professional, productive, and interesting discussion. References Gil Mazzante NM, Zuliani F, Oliveira RAd et al. Diagnosis of brain death in wistar rats at different levels of death induction [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2024, 13:1490 ( https://doi.org/10.12688/f1000research.157233.1 Hannoodee S, Nasuruddin DN. Acute Inflammatory Response. 2024 Jun 8. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan–. PMID: 32310543. Nogueira EC: Captação de órgãos em Sergipe e fatores associados à efetivação de potenciais doadores; 2008. 103f. Dissertação (Mestrado) - Universidade Federal de Sergipe. Aracaju. Hodgson DR, Rose RJ: The athletic horse: principles and practice of equine sports medicine. 1st ed.Philadelphia: W. B. Saunders Company; 1994; 245–258. Competing Interests: No competing interests to declare Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Lazzeri C. Reviewer Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.172655.r347400 ) The direct URL for this report is: https://f1000research.com/articles/13-1490/v1#referee-response-347400 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 17 Dec 2024 Chiara Lazzeri , Careggi Hospital, Florence, Italy Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.172655.r347400 The topic is quite interesting. The main limitation of the present study is the small number of animals included. This should be addressed in the discussion section. Major issues can be raised: a) in the discussion section each ... Continue reading READ ALL The topic is quite interesting. The main limitation of the present study is the small number of animals included. This should be addressed in the discussion section. Major issues can be raised: a) in the discussion section each altered parameter is described and discussed. Regarding polycytemia, other hypothesis should be considered, especially volemia b) in regard to coagulation disorders, Authors should add other hypothesized mechanism(s), including the cause of brain death (i.e. trauma) c) what about treatment of brain death d) the Authors should better elucidate the novelty of their investigation in respect to existing evidence on this topic Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: organ procurement and transplantation, cardiac arrest, ECMO, respiratory failure, critical care echocardiography I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Lazzeri C. Reviewer Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.172655.r347400 ) The direct URL for this report is: https://f1000research.com/articles/13-1490/v1#referee-response-347400 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 30 Apr 2025 Fernanda Barthelson Carvalho de Moura , Department of Veterinary Clinics, School of Veterinary Medicine and Animal Science, São Paulo State University - Botucatu Campus, Botucatu, Brazil 30 Apr 2025 Author Response It is with great enthusiasm that we respond to Dr. Mario Abbud-Filho Dr. Ludmila Marzochi and Dr. Chiara Lazzeri comments concerning the study “Diagnosis of brain death in wistar rats at ... Continue reading It is with great enthusiasm that we respond to Dr. Mario Abbud-Filho Dr. Ludmila Marzochi and Dr. Chiara Lazzeri comments concerning the study “Diagnosis of brain death in wistar rats at different levels of death induction” 1 . The authors genuinely appreciate this consideration and interest in our work. In response to reviewers’ comments and requests, the time criteria used in the research follow the main goal of this research as we aim to evaluate the brain death in different levels of death induction. Using these criteria, we were able to evaluate de progression of the parameters in different times and understand how the brain death progress, from immediately after death, hour after death compared with our control group. According to Hannodee and Nasuruddin, the acute changes manifest immediately after injury and can last for a few days 2 . Additionally, the laboratory parameters play a fundamental role in understanding the progression of brain death, as death results in leakage of cytokines, proteins, and other components, mainly in the acute phase. Also, several changes in the brain death may not be detectable in macroscopical examination, which is the reason why the study primarily focused on laboratory parameters, which could provide early indications of physiological changes. We want to enhance that the primary objective of this paper was to evaluate the hematologic, biochemical, and gasometrical parameters in brain death to understand the modifications and reduce misdiagnosis by using these parameters as an indication in early stages. We acknowledge that discussing those changes could be explored in more detail. However, the experimental nature of this paper should be considered, as well as the small number of animals used in this research. Our results, even in a short interval time evaluated and with a short number of animal models, demonstrate the tendency of those changes. Further studies with more animals and extended observation periods are necessary to elaborate a more comprehensive discussion of the mechanisms involved. The authors agree that the discussion could be more detailed and deeper. However, our research is experimental, and we performed an individual examination of the specific groups used in the research. We provided a discussion comparing our findings with other results in the literature (ie, Fiocruz, Hodgson, and Rose) 3,4 ; however, this type of study, in which brain death is evaluated by animal laboratory changes, is not well studied and explored. We hope our study stimulates further research, which could provide a better discussion of the results. In conclusion we believe that this study highlights the complex physiological changes associated with brain death (BD) in Wistar rats. Significant differences were observed between groups in the laboratory analysis, such as: segmented cells, monocytes, creatinine, aspartate aminotransferase, potassium, and bicarbonate levels, demonstrating the varied impact of BD on these parameters. The study shows that early detection is important to avoid progressive somatic deterioration and ensure better organ function. Although the experimental model and sample size in this work were not ideal, it has the potential to contribute to the understanding that individual responses to BD oscillate, complicating its diagnosis and emphasizing the need for further investigation to improve clinical diagnostic precision. We sincerely wish we could now have explored these points more in our response and paper. In closing, we sincerely thank Dr. Mario Abbud-Filho, Dr. Ludmila Marzochi, and Dr. Chiara Lazzeri for providing an opportunity to clarify our perspective and initiating a professional, productive, and interesting discussion. References Gil Mazzante NM, Zuliani F, Oliveira RAd et al. Diagnosis of brain death in wistar rats at different levels of death induction [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2024, 13:1490 ( https://doi.org/10.12688/f1000research.157233.1 Hannoodee S, Nasuruddin DN. Acute Inflammatory Response. 2024 Jun 8. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan–. PMID: 32310543. Nogueira EC: Captação de órgãos em Sergipe e fatores associados à efetivação de potenciais doadores; 2008. 103f. Dissertação (Mestrado) - Universidade Federal de Sergipe. Aracaju. Hodgson DR, Rose RJ: The athletic horse: principles and practice of equine sports medicine. 1st ed.Philadelphia: W. B. Saunders Company; 1994; 245–258. It is with great enthusiasm that we respond to Dr. Mario Abbud-Filho Dr. Ludmila Marzochi and Dr. Chiara Lazzeri comments concerning the study “Diagnosis of brain death in wistar rats at different levels of death induction” 1 . The authors genuinely appreciate this consideration and interest in our work. In response to reviewers’ comments and requests, the time criteria used in the research follow the main goal of this research as we aim to evaluate the brain death in different levels of death induction. Using these criteria, we were able to evaluate de progression of the parameters in different times and understand how the brain death progress, from immediately after death, hour after death compared with our control group. According to Hannodee and Nasuruddin, the acute changes manifest immediately after injury and can last for a few days 2 . Additionally, the laboratory parameters play a fundamental role in understanding the progression of brain death, as death results in leakage of cytokines, proteins, and other components, mainly in the acute phase. Also, several changes in the brain death may not be detectable in macroscopical examination, which is the reason why the study primarily focused on laboratory parameters, which could provide early indications of physiological changes. We want to enhance that the primary objective of this paper was to evaluate the hematologic, biochemical, and gasometrical parameters in brain death to understand the modifications and reduce misdiagnosis by using these parameters as an indication in early stages. We acknowledge that discussing those changes could be explored in more detail. However, the experimental nature of this paper should be considered, as well as the small number of animals used in this research. Our results, even in a short interval time evaluated and with a short number of animal models, demonstrate the tendency of those changes. Further studies with more animals and extended observation periods are necessary to elaborate a more comprehensive discussion of the mechanisms involved. The authors agree that the discussion could be more detailed and deeper. However, our research is experimental, and we performed an individual examination of the specific groups used in the research. We provided a discussion comparing our findings with other results in the literature (ie, Fiocruz, Hodgson, and Rose) 3,4 ; however, this type of study, in which brain death is evaluated by animal laboratory changes, is not well studied and explored. We hope our study stimulates further research, which could provide a better discussion of the results. In conclusion we believe that this study highlights the complex physiological changes associated with brain death (BD) in Wistar rats. Significant differences were observed between groups in the laboratory analysis, such as: segmented cells, monocytes, creatinine, aspartate aminotransferase, potassium, and bicarbonate levels, demonstrating the varied impact of BD on these parameters. The study shows that early detection is important to avoid progressive somatic deterioration and ensure better organ function. Although the experimental model and sample size in this work were not ideal, it has the potential to contribute to the understanding that individual responses to BD oscillate, complicating its diagnosis and emphasizing the need for further investigation to improve clinical diagnostic precision. We sincerely wish we could now have explored these points more in our response and paper. In closing, we sincerely thank Dr. Mario Abbud-Filho, Dr. Ludmila Marzochi, and Dr. Chiara Lazzeri for providing an opportunity to clarify our perspective and initiating a professional, productive, and interesting discussion. References Gil Mazzante NM, Zuliani F, Oliveira RAd et al. Diagnosis of brain death in wistar rats at different levels of death induction [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2024, 13:1490 ( https://doi.org/10.12688/f1000research.157233.1 Hannoodee S, Nasuruddin DN. Acute Inflammatory Response. 2024 Jun 8. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan–. PMID: 32310543. Nogueira EC: Captação de órgãos em Sergipe e fatores associados à efetivação de potenciais doadores; 2008. 103f. Dissertação (Mestrado) - Universidade Federal de Sergipe. Aracaju. Hodgson DR, Rose RJ: The athletic horse: principles and practice of equine sports medicine. 1st ed.Philadelphia: W. B. Saunders Company; 1994; 245–258. Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 30 Apr 2025 Fernanda Barthelson Carvalho de Moura , Department of Veterinary Clinics, School of Veterinary Medicine and Animal Science, São Paulo State University - Botucatu Campus, Botucatu, Brazil 30 Apr 2025 Author Response It is with great enthusiasm that we respond to Dr. Mario Abbud-Filho Dr. Ludmila Marzochi and Dr. Chiara Lazzeri comments concerning the study “Diagnosis of brain death in wistar rats at ... Continue reading It is with great enthusiasm that we respond to Dr. Mario Abbud-Filho Dr. Ludmila Marzochi and Dr. Chiara Lazzeri comments concerning the study “Diagnosis of brain death in wistar rats at different levels of death induction” 1 . The authors genuinely appreciate this consideration and interest in our work. In response to reviewers’ comments and requests, the time criteria used in the research follow the main goal of this research as we aim to evaluate the brain death in different levels of death induction. Using these criteria, we were able to evaluate de progression of the parameters in different times and understand how the brain death progress, from immediately after death, hour after death compared with our control group. According to Hannodee and Nasuruddin, the acute changes manifest immediately after injury and can last for a few days 2 . Additionally, the laboratory parameters play a fundamental role in understanding the progression of brain death, as death results in leakage of cytokines, proteins, and other components, mainly in the acute phase. Also, several changes in the brain death may not be detectable in macroscopical examination, which is the reason why the study primarily focused on laboratory parameters, which could provide early indications of physiological changes. We want to enhance that the primary objective of this paper was to evaluate the hematologic, biochemical, and gasometrical parameters in brain death to understand the modifications and reduce misdiagnosis by using these parameters as an indication in early stages. We acknowledge that discussing those changes could be explored in more detail. However, the experimental nature of this paper should be considered, as well as the small number of animals used in this research. Our results, even in a short interval time evaluated and with a short number of animal models, demonstrate the tendency of those changes. Further studies with more animals and extended observation periods are necessary to elaborate a more comprehensive discussion of the mechanisms involved. The authors agree that the discussion could be more detailed and deeper. However, our research is experimental, and we performed an individual examination of the specific groups used in the research. We provided a discussion comparing our findings with other results in the literature (ie, Fiocruz, Hodgson, and Rose) 3,4 ; however, this type of study, in which brain death is evaluated by animal laboratory changes, is not well studied and explored. We hope our study stimulates further research, which could provide a better discussion of the results. In conclusion we believe that this study highlights the complex physiological changes associated with brain death (BD) in Wistar rats. Significant differences were observed between groups in the laboratory analysis, such as: segmented cells, monocytes, creatinine, aspartate aminotransferase, potassium, and bicarbonate levels, demonstrating the varied impact of BD on these parameters. The study shows that early detection is important to avoid progressive somatic deterioration and ensure better organ function. Although the experimental model and sample size in this work were not ideal, it has the potential to contribute to the understanding that individual responses to BD oscillate, complicating its diagnosis and emphasizing the need for further investigation to improve clinical diagnostic precision. We sincerely wish we could now have explored these points more in our response and paper. In closing, we sincerely thank Dr. Mario Abbud-Filho, Dr. Ludmila Marzochi, and Dr. Chiara Lazzeri for providing an opportunity to clarify our perspective and initiating a professional, productive, and interesting discussion. References Gil Mazzante NM, Zuliani F, Oliveira RAd et al. Diagnosis of brain death in wistar rats at different levels of death induction [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2024, 13:1490 ( https://doi.org/10.12688/f1000research.157233.1 Hannoodee S, Nasuruddin DN. Acute Inflammatory Response. 2024 Jun 8. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan–. PMID: 32310543. Nogueira EC: Captação de órgãos em Sergipe e fatores associados à efetivação de potenciais doadores; 2008. 103f. Dissertação (Mestrado) - Universidade Federal de Sergipe. Aracaju. Hodgson DR, Rose RJ: The athletic horse: principles and practice of equine sports medicine. 1st ed.Philadelphia: W. B. Saunders Company; 1994; 245–258. It is with great enthusiasm that we respond to Dr. Mario Abbud-Filho Dr. Ludmila Marzochi and Dr. Chiara Lazzeri comments concerning the study “Diagnosis of brain death in wistar rats at different levels of death induction” 1 . The authors genuinely appreciate this consideration and interest in our work. In response to reviewers’ comments and requests, the time criteria used in the research follow the main goal of this research as we aim to evaluate the brain death in different levels of death induction. Using these criteria, we were able to evaluate de progression of the parameters in different times and understand how the brain death progress, from immediately after death, hour after death compared with our control group. According to Hannodee and Nasuruddin, the acute changes manifest immediately after injury and can last for a few days 2 . Additionally, the laboratory parameters play a fundamental role in understanding the progression of brain death, as death results in leakage of cytokines, proteins, and other components, mainly in the acute phase. Also, several changes in the brain death may not be detectable in macroscopical examination, which is the reason why the study primarily focused on laboratory parameters, which could provide early indications of physiological changes. We want to enhance that the primary objective of this paper was to evaluate the hematologic, biochemical, and gasometrical parameters in brain death to understand the modifications and reduce misdiagnosis by using these parameters as an indication in early stages. We acknowledge that discussing those changes could be explored in more detail. However, the experimental nature of this paper should be considered, as well as the small number of animals used in this research. Our results, even in a short interval time evaluated and with a short number of animal models, demonstrate the tendency of those changes. Further studies with more animals and extended observation periods are necessary to elaborate a more comprehensive discussion of the mechanisms involved. The authors agree that the discussion could be more detailed and deeper. However, our research is experimental, and we performed an individual examination of the specific groups used in the research. We provided a discussion comparing our findings with other results in the literature (ie, Fiocruz, Hodgson, and Rose) 3,4 ; however, this type of study, in which brain death is evaluated by animal laboratory changes, is not well studied and explored. We hope our study stimulates further research, which could provide a better discussion of the results. In conclusion we believe that this study highlights the complex physiological changes associated with brain death (BD) in Wistar rats. Significant differences were observed between groups in the laboratory analysis, such as: segmented cells, monocytes, creatinine, aspartate aminotransferase, potassium, and bicarbonate levels, demonstrating the varied impact of BD on these parameters. The study shows that early detection is important to avoid progressive somatic deterioration and ensure better organ function. Although the experimental model and sample size in this work were not ideal, it has the potential to contribute to the understanding that individual responses to BD oscillate, complicating its diagnosis and emphasizing the need for further investigation to improve clinical diagnostic precision. We sincerely wish we could now have explored these points more in our response and paper. In closing, we sincerely thank Dr. Mario Abbud-Filho, Dr. Ludmila Marzochi, and Dr. Chiara Lazzeri for providing an opportunity to clarify our perspective and initiating a professional, productive, and interesting discussion. References Gil Mazzante NM, Zuliani F, Oliveira RAd et al. Diagnosis of brain death in wistar rats at different levels of death induction [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2024, 13:1490 ( https://doi.org/10.12688/f1000research.157233.1 Hannoodee S, Nasuruddin DN. Acute Inflammatory Response. 2024 Jun 8. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan–. PMID: 32310543. Nogueira EC: Captação de órgãos em Sergipe e fatores associados à efetivação de potenciais doadores; 2008. 103f. Dissertação (Mestrado) - Universidade Federal de Sergipe. Aracaju. Hodgson DR, Rose RJ: The athletic horse: principles and practice of equine sports medicine. 1st ed.Philadelphia: W. B. Saunders Company; 1994; 245–258. Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 06 Dec 2024 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 Version 2 (revision) 06 May 25 read read read Version 1 06 Dec 24 read read Chiara Lazzeri , Careggi Hospital, Florence, Italy Mario Abbud-Filho , Hospital de Base (FUNFARME) and Medical School (FAMERP), São José do Rio Preto, Brazil Ludmila Marzochi , Medical School Famerp Laboratory of Immunology & Experimental Transplantation, São José do Rio Preto, Brazil Calixto Machado , Institute of Neurology and Neurosurgery, Havana, Cuba Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Machado C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 24 May 2025 | for Version 2 Calixto Machado , Institute of Neurology and Neurosurgery, Havana, Cuba 0 Views copyright © 2025 Machado C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions General Assessment This manuscript presents an exploratory study on the physiological and biochemical changes associated with brain death (BD) in Wistar rats. It aims to deepen our understanding of the variable responses to BD induction and to identify laboratory markers that may help refine early diagnostic criteria. The subject is highly relevant, particularly given the limited availability of experimental data addressing the pathophysiological dynamics of brain death in preclinical models. Despite a modest sample size, the study’s design is clear and methodologically sound. It includes three well-defined groups with varying durations after BD induction and offers statistically supported comparisons of hematologic, biochemical, and gasometric parameters. The observed variations, especially in segmented leukocytes, monocytes, creatinine, AST, potassium, and bicarbonate, contribute to our growing understanding of the systemic impact of BD. Ethical and Animal Welfare Considerations Of particular importance is the authors’ adherence to established ethical protocols in animal research. The study was approved by the Ethics Committee on the Use of Animals (CEUA – protocol 0259/2018) and followed both the Brazilian College of Animal Experimentation (COBEA) and the Brazilian Guide to Good Euthanasia Practices . This adherence ensures that all efforts were made to minimize suffering and prioritize humane treatment throughout the experimental process. The inclusion of environmental enrichment and diligent monitoring further exemplifies best practices in animal care. By explicitly detailing the anesthesia protocols, surgical preparation, and post-procedural monitoring, the authors demonstrate a commendable commitment to animal welfare and the 3Rs (Replacement, Reduction, and Refinement) in experimental design. Scientific Significance and Recommendations The research contributes novel data on how acute physiological changes manifest post-BD and reinforces the importance of laboratory parameters as early indicators of systemic decline. While the sample size limits generalizability, the study effectively functions as a pilot investigation that could inspire more expansive, statistically powered future research. I recommend the acceptance of this manuscript with minor revisions aimed at improving the clarity of conclusions and further contextualizing results within existing literature. Expanding on the potential clinical translational value of such findings in veterinary and human medicine would be particularly beneficial. Final Recommendation: ✅ Accept with minor revisions This study is ethically sound, scientifically relevant, and presents well-supported preliminary findings that merit dissemination. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Brain death, disorders of consciousness, neuroimaging, general neurology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Machado C. Peer Review Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.181285.r383475) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1490/v2#referee-response-383475 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Lazzeri C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 14 May 2025 | for Version 2 Chiara Lazzeri , Careggi Hospital, Florence, Italy 0 Views copyright © 2025 Lazzeri C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions I read the revised version of the manuscript. The Authors provided a point to point reply to the reviewers' comments. The manuscript improved. Competing Interests No competing interests were disclosed. Reviewer Expertise organ procurement and transplantation, cardiac arrest, ECMO, respiratory failure, critical care echocardiography I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Lazzeri C. Peer Review Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.181285.r383095) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1490/v2#referee-response-383095 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Abbud-Filho M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 13 May 2025 | for Version 2 Mario Abbud-Filho , Medicine, Hospital de Base (FUNFARME) and Medical School (FAMERP), São José do Rio Preto, São Paulo, Brazil 0 Views copyright © 2025 Abbud-Filho M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The authors answered the questions we addressed and improved the revised version. In the interest of the manuscript's clarity, they should include a visual representation, such as a diagram or timeline, summarizing the experimental design, particularly the timing of sample collection across the different groups. The illustration could make the description clearer and more concise. We thank you for the opportunity to revise the manuscript. Competing Interests No competing interests were disclosed. Reviewer Expertise Renal transplantation, donor preconditioning, molecular aspects of marginal kidneys I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Abbud-Filho M. Peer Review Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.181285.r383096) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1490/v2#referee-response-383096 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Abbud-Filho M et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 04 Mar 2025 | for Version 1 Mario Abbud-Filho , Medicine, Hospital de Base (FUNFARME) and Medical School (FAMERP), São José do Rio Preto, São Paulo, Brazil Ludmila Marzochi , Medicine, Medical School Famerp Laboratory of Immunology & Experimental Transplantation, São José do Rio Preto, São Paulo, Brazil 0 Views copyright © 2025 Abbud-Filho M et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions I was asked to review the manuscript entitled “Diagnosis of Brain Death in Wistar Rats at Different Levels of Death Induction” authored by Mazzante et al. Although there is indeed a debate—more philosophical than medical—about the definition of brain death, in the field of transplantation, there is no doubt regarding the selection of potential organ donors. From a biochemical and molecular perspective, brain death is of particular interest to researchers in this field, making the authors' work relevant. However, several issues raise major concerns: The small sample size limits the generalizability of the findings. Although the authors acknowledge this limitation, it remains a significant constraint. The selected time points for collecting laboratory parameters (M1 and M2) are difficult to interpret, as they primarily reflect acute changes associated with brain death. It seems overly simplistic to conclude the highly complex process of brain death based solely on the chosen laboratory parameters (hematological, renal, and hepatic markers). While the study identifies significant changes in various parameters, it does not sufficiently explore the underlying mechanisms driving these changes. For instance, the discussion could further elaborate on how brain death-induced inflammatory responses or hormonal imbalances contribute to the observed alterations in hematologic and biochemical markers. The discussion section could be strengthened by addressing conflicting findings in the literature more thoroughly. For example, while the study reports hypernatremia and hypokalemia, potential reasons for these discrepancies could have been explored more deeply. The conclusion is vague and does not fully summarize the key findings or their implications. A more concise and impactful conclusion would enhance the manuscript. In summary, the study is scientifically sound and addresses a relevant topic. However, significant revisions are necessary to improve its clarity, depth, and overall impact. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Renal transplantation, donor preconditioning, molecular aspects of marginal kidneys We confirm that we have read this submission and believe that we have an appropriate level of expertise to state that we do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (1) Author Response 10 May 2025 Fernanda Barthelson Carvalho de Moura, Department of Veterinary Clinics, School of Veterinary Medicine and Animal Science, São Paulo State University - Botucatu Campus, Botucatu, Brazil It is with great enthusiasm that we respond to Dr. Mario Abbud-Filho Dr. Ludmila Marzochi and Dr. Chiara Lazzeri comments concerning the study “Diagnosis of brain death in wistar rats at different levels of death induction” 1 . The authors genuinely appreciate this consideration and interest in our work. In response to reviewers’ comments and requests, the time criteria used in the research follow the main goal of this research as we aim to evaluate the brain death in different levels of death induction. Using these criteria, we were able to evaluate de progression of the parameters in different times and understand how the brain death progress, from immediately after death, hour after death compared with our control group. According to Hannodee and Nasuruddin, the acute changes manifest immediately after injury and can last for a few days 2 . Additionally, the laboratory parameters play a fundamental role in understanding the progression of brain death, as death results in leakage of cytokines, proteins, and other components, mainly in the acute phase. Also, several changes in the brain death may not be detectable in macroscopical examination, which is the reason why the study primarily focused on laboratory parameters, which could provide early indications of physiological changes. We want to enhance that the primary objective of this paper was to evaluate the hematologic, biochemical, and gasometrical parameters in brain death to understand the modifications and reduce misdiagnosis by using these parameters as an indication in early stages. We acknowledge that discussing those changes could be explored in more detail. However, the experimental nature of this paper should be considered, as well as the small number of animals used in this research. Our results, even in a short interval time evaluated and with a short number of animal models, demonstrate the tendency of those changes. Further studies with more animals and extended observation periods are necessary to elaborate a more comprehensive discussion of the mechanisms involved. The authors agree that the discussion could be more detailed and deeper. However, our research is experimental, and we performed an individual examination of the specific groups used in the research. We provided a discussion comparing our findings with other results in the literature (ie, Fiocruz, Hodgson, and Rose) 3,4 ; however, this type of study, in which brain death is evaluated by animal laboratory changes, is not well studied and explored. We hope our study stimulates further research, which could provide a better discussion of the results. In conclusion we believe that this study highlights the complex physiological changes associated with brain death (BD) in Wistar rats. Significant differences were observed between groups in the laboratory analysis, such as: segmented cells, monocytes, creatinine, aspartate aminotransferase, potassium, and bicarbonate levels, demonstrating the varied impact of BD on these parameters. The study shows that early detection is important to avoid progressive somatic deterioration and ensure better organ function. Although the experimental model and sample size in this work were not ideal, it has the potential to contribute to the understanding that individual responses to BD oscillate, complicating its diagnosis and emphasizing the need for further investigation to improve clinical diagnostic precision. We sincerely wish we could now have explored these points more in our response and paper. In closing, we sincerely thank Dr. Mario Abbud-Filho, Dr. Ludmila Marzochi, and Dr. Chiara Lazzeri for providing an opportunity to clarify our perspective and initiating a professional, productive, and interesting discussion. References Gil Mazzante NM, Zuliani F, Oliveira RAd et al. Diagnosis of brain death in wistar rats at different levels of death induction [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2024, 13:1490 ( https://doi.org/10.12688/f1000research.157233.1 Hannoodee S, Nasuruddin DN. Acute Inflammatory Response. 2024 Jun 8. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan–. PMID: 32310543. Nogueira EC: Captação de órgãos em Sergipe e fatores associados à efetivação de potenciais doadores; 2008. 103f. Dissertação (Mestrado) - Universidade Federal de Sergipe. Aracaju. Hodgson DR, Rose RJ: The athletic horse: principles and practice of equine sports medicine. 1st ed.Philadelphia: W. B. Saunders Company; 1994; 245–258. View more View less Competing Interests No competing interests to declare reply Respond Report a concern Abbud-Filho M and Marzochi L. Peer Review Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.172655.r366145) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1490/v1#referee-response-366145 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 Lazzeri C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 17 Dec 2024 | for Version 1 Chiara Lazzeri , Careggi Hospital, Florence, Italy 0 Views copyright © 2024 Lazzeri C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The topic is quite interesting. The main limitation of the present study is the small number of animals included. This should be addressed in the discussion section. Major issues can be raised: a) in the discussion section each altered parameter is described and discussed. Regarding polycytemia, other hypothesis should be considered, especially volemia b) in regard to coagulation disorders, Authors should add other hypothesized mechanism(s), including the cause of brain death (i.e. trauma) c) what about treatment of brain death d) the Authors should better elucidate the novelty of their investigation in respect to existing evidence on this topic Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise organ procurement and transplantation, cardiac arrest, ECMO, respiratory failure, critical care echocardiography I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 30 Apr 2025 Fernanda Barthelson Carvalho de Moura, Department of Veterinary Clinics, School of Veterinary Medicine and Animal Science, São Paulo State University - Botucatu Campus, Botucatu, Brazil It is with great enthusiasm that we respond to Dr. Mario Abbud-Filho Dr. Ludmila Marzochi and Dr. Chiara Lazzeri comments concerning the study “Diagnosis of brain death in wistar rats at different levels of death induction” 1 . The authors genuinely appreciate this consideration and interest in our work. In response to reviewers’ comments and requests, the time criteria used in the research follow the main goal of this research as we aim to evaluate the brain death in different levels of death induction. Using these criteria, we were able to evaluate de progression of the parameters in different times and understand how the brain death progress, from immediately after death, hour after death compared with our control group. According to Hannodee and Nasuruddin, the acute changes manifest immediately after injury and can last for a few days 2 . Additionally, the laboratory parameters play a fundamental role in understanding the progression of brain death, as death results in leakage of cytokines, proteins, and other components, mainly in the acute phase. Also, several changes in the brain death may not be detectable in macroscopical examination, which is the reason why the study primarily focused on laboratory parameters, which could provide early indications of physiological changes. We want to enhance that the primary objective of this paper was to evaluate the hematologic, biochemical, and gasometrical parameters in brain death to understand the modifications and reduce misdiagnosis by using these parameters as an indication in early stages. We acknowledge that discussing those changes could be explored in more detail. However, the experimental nature of this paper should be considered, as well as the small number of animals used in this research. Our results, even in a short interval time evaluated and with a short number of animal models, demonstrate the tendency of those changes. Further studies with more animals and extended observation periods are necessary to elaborate a more comprehensive discussion of the mechanisms involved. The authors agree that the discussion could be more detailed and deeper. However, our research is experimental, and we performed an individual examination of the specific groups used in the research. We provided a discussion comparing our findings with other results in the literature (ie, Fiocruz, Hodgson, and Rose) 3,4 ; however, this type of study, in which brain death is evaluated by animal laboratory changes, is not well studied and explored. We hope our study stimulates further research, which could provide a better discussion of the results. In conclusion we believe that this study highlights the complex physiological changes associated with brain death (BD) in Wistar rats. Significant differences were observed between groups in the laboratory analysis, such as: segmented cells, monocytes, creatinine, aspartate aminotransferase, potassium, and bicarbonate levels, demonstrating the varied impact of BD on these parameters. The study shows that early detection is important to avoid progressive somatic deterioration and ensure better organ function. Although the experimental model and sample size in this work were not ideal, it has the potential to contribute to the understanding that individual responses to BD oscillate, complicating its diagnosis and emphasizing the need for further investigation to improve clinical diagnostic precision. We sincerely wish we could now have explored these points more in our response and paper. In closing, we sincerely thank Dr. Mario Abbud-Filho, Dr. Ludmila Marzochi, and Dr. Chiara Lazzeri for providing an opportunity to clarify our perspective and initiating a professional, productive, and interesting discussion. References Gil Mazzante NM, Zuliani F, Oliveira RAd et al. Diagnosis of brain death in wistar rats at different levels of death induction [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2024, 13:1490 ( https://doi.org/10.12688/f1000research.157233.1 Hannoodee S, Nasuruddin DN. Acute Inflammatory Response. 2024 Jun 8. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan–. PMID: 32310543. Nogueira EC: Captação de órgãos em Sergipe e fatores associados à efetivação de potenciais doadores; 2008. 103f. Dissertação (Mestrado) - Universidade Federal de Sergipe. Aracaju. Hodgson DR, Rose RJ: The athletic horse: principles and practice of equine sports medicine. 1st ed.Philadelphia: W. B. Saunders Company; 1994; 245–258. View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Lazzeri C. Peer Review Report For: Diagnosis of brain death in wistar rats at different levels of death induction [version 2; peer review: 3 approved] . F1000Research 2025, 13 :1490 ( https://doi.org/10.5256/f1000research.172655.r347400) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1490/v1#referee-response-347400 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions Adjust parameters to alter display View on desktop for interactive features Includes Interactive Elements View on desktop for interactive features Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. 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