Acetaminophen, a new tool to refine experimental infectious processes: the case of murine toxoplasmosis

Acetaminophen, a new tool to refine experimental infectious processes: the case of murine toxoplasmosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Acetaminophen, a new tool to refine experimental infectious processes: the case of murine toxoplasmosis Nathalie Moiré, Axel Cauty, Christelle Rossignol, Corinne Beaugé, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5790171/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Over the past few years, animal welfare has taken more place in society and especially in scientific research. It has become necessary to refine the experimental procedures as much as possible in infectiology as in our reference model, toxoplasmosis, in agreement with the 3Rs rule and different ethical concerns. Thus, the establishment of a treatment using analgesics would relieve animals acutely infected with Toxoplasma gondii . However, the use of this analgesic should in no way alter the pathophysiology of the disease and the immune response of the host, so as not to interfere with the initial scientific study. Currently, little is known about the use of acetaminophen in an infectious model. In the present work, we studied the impact of acetaminophen at a reference dose of 30 mg/kg/day in a murine model of acute toxoplasmosis. To do this, zoonotic, telemetric, behavioral, histological and immune parameters were analyzed to better characterize the consequences of a treatment with acetaminophen either via gavage or via self-medication in Gel Water. Acetaminophen administered by gavage did not induce cellular or tissue toxicity and did not alter the physiological development of mice either. Moreover, the very nature of Gel Water, independently of APAP, has had an impact on the immune response. The acetaminophen improved the general well-being and slowed down the appearance of clinical signs without modifying the physiopathology or the immune responses induced by T. gondii . These first results in mice validated our initial hypothesis that acetaminophen appears to be a pharmacological tool to refine and improve animal welfare during the acute phase of toxoplasmosis. Therefore, our project has highlighted the combination of specific markers to contribute to animal welfare in mice. In the long term, the use of acetaminophen could be extended to other infectious models with other target animal species. Health sciences/Pathogenesis/Immunopathogenesis/Adaptive immunity Health sciences/Pathogenesis/Immunopathogenesis/Innate immunity Health sciences/Pathogenesis/Inflammation/Acute inflammation Health sciences/Pathogenesis/Inflammation/Chronic inflammation Health sciences/Health care/Medical ethics welfare acetaminophen toxoplasmosis mice behavior immune response Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction In recent years, animal welfare has become more important in society, especially in scientific research. Since 2013, a European decree has been established regarding the use of animals in experimental procedures, and the need for precise ethical evaluation of each project to be carried out. This involves strengthening ethics committees but also reminding and highlighting a rule that has existed since 1959: the 3Rs 1 , 2 . The objectives of this rule are multiple and therefore go through three main points: To R educe as much as possible the number of animals used during experiments and to R eplace animal models with in vitro or bioinformatics models when possible. A key point of this rule, to R efine, refers to methods that minimize the pain, suffering, distress that may be experienced by research animals, and which improve their welfare. Pain and suffering can alter animal’s behavior, physiology and immunology that can lead to variation in experimental results that may impair the reliability of studies 3 . Refinement can include an enrichment of the animals’ environment, for example a game or a reward which can improve the general well-being 4 , 5 and the introduction of endpoints that allow humans to estimate animal suffering. Finally, pain management using analgesic and/or anti-pyretic substances has also an impact on scientific results based on the choice on appropriate analgesic or the lack of pain treatment. The use of drugs is not so straightforward in experiments involving animals, especially in infectious disease models because of the influence that pharmacological substances could have on the results of the study. A dilemma then arises because symptoms related to an infection that can lead to the death of the animal could be limited with the appropriate medication. However, untreated pain can also affect, by example, the immune system. In all cases experimental bias may occur 6 . Before being able to generalize the use of drugs to relieve animals, it is necessary to know their mechanisms of action, the possible toxicity they could induce, but also their influence on the immune response and whether a cause-and-effect relationship to the infectious agent used can be observed. Opioids and nonsteroidal anti-inflammatory drugs (NSAIDs) are the two main groups of pain relievers available 7 . Buprenorphine and meloxicam are the most commonly used analgesics in rodents and extensive literature review of pharmacokinetics of these drugs are available. Although it is one of the most largely used analgesic antipyretic drug in the world for humans 8 , acetaminophen (N-acetyl-p-aminophenol or APAP) is rarely used to treat rodents. It is known to have no anti-inflammatory effect, unlike other nonsteroidal anti-inflammatory drugs 9 . The mechanism by which it produces its analgesic effect is largely unknown 10 . It is supposed that acetaminophen would inhibit the synthesis of prostaglandins comprising a COX site (active site of the majority of NSAIDs 11 ) in the central nervous system, prostaglandins having a pro-nociceptive role and potentially causing fever in the hypothalamus on which acetaminophen would act 12 . Similar to its analgesic action, the mechanism of antipyretic action of this drug remains poorly understood. However, its administration is easily done orally and its absorption by oral route is complete and rapid, as the maximum plasma concentration is reached within one hour of ingestion 13 . Following oral administration, its systemic bioavailability is dose-dependent and ranges from 70–90% with differences between animal species 14 . Acetaminophen is distributed rapidly in all tissues and is quickly eliminated by reaction with reduced glutathione and then excreted in the urine after conjugation with cysteine and mercapturic acid 15 . Nevertheless, literature studying acetaminophen use in experimental infectious rodent models is rare and most use concern pain relief after severe invasive procedures (in combination with opioids) or in cancer model. Here, we were more interested in the antipyretic effect of acetaminophen and how this could reduce overall stress and unease during the acute phase of an infectious disease. For this study, we focused on a murine toxoplasmosis model. The agent responsible for toxoplasmosis is Toxoplasma gondii ( T. gondii ), an obligate intracellular protist. It is present all over the world and in a wide diversity of hosts 16 . It exists in 3 stages: tachyzoites rapidly multiplying in the acute phase of infection, bradyzoites within latent cysts in tissue; and sporozoites within oocysts, a form of resistance in the environment 17 . Initial contact with the parasite triggers a protective immune response in immunocompetent animals or humans with no, or only few, symptoms. However, the infection can lead to clinical symptoms (weight loss, temperature drop, prostration…) and death in some mouse models, depending on the infectious dose. T. gondii induces a pro-inflammatory (TH1 type) immune response, mediated by IFN-γ and IL-12 production 18 , 19 by T lymphocytes and antigen presenting cells, respectively. This immune response induced the control of the parasite in the acute phase, that allows its persistence in different tissues as dormant cysts resulting in chronic infection. This transient inflammation characteristic of the acute phase of toxoplasmosis, can be responsible for clinical symptoms. The objective of the study was to find out whether treating infected mice with APAP could relieve the pain or discomfort induced during the acute phase of toxoplasmosis, without altering the dissemination of the parasite and the immune response while improving the living conditions of the animals. For this, the harmlessness of APAP in CBA/J mice has been checked at the dose used (30 mg/kg/day), by gavage or by self-medication. This dose is recommended by the supplier of the veterinary medicinal product already used in pigs 20 , 21 . The same treatment has been transposed to CBA/J mice infected with Toxoplasma gondii , to study the effects of APAP on the pathophysiology (parasite multiplication and installation) of the infectious model and the specific humoral and cellular immune responses. Zootechnical analyzes (temperature, weight, behavior) as well as immunological, biochemical and histological analyzes were also performed. The main objective of the entire study was to validate the proof of concept for the use of APAP as a pharmacological tool to refine animal experiments using the murine model of toxoplasmosis. Materials and Methods Animals and Ethics statement Experiments were carried out according to EU directives and French regulations (Directive 2010/63 / EU, 2010; Rural Code, 2018; Decree n ° 2013 − 118, 2013, https://www.legifrance.gouv.fr/loda/id/JORFTEXT000027038013/ ). All experimental procedures have been evaluated and approved by the Ministry of Higher Education and Research (APAFIS # 2018021917268751.V3–13634). The procedures involving mice were evaluated by the Val de Loire ethics committee (CEEA VdL, committee number 19) and took place at the INRAE Platform for Experimental Infection PFIE (UE-1277 PFIE, INRAE Centre-Val-de Loire) Valley research, Nouzilly, France, https://doi.org/10.15454/1.5535888072272498e12 ). A total of 90 female mice of the CBA/J line (Janvier Labs, Le Genest-Saint-Isle, France) aged 5 weeks at the start of the experiments were used. Mice were randomly identified using telemetric sensors (Biolog-Animal®, Paris) implanted subcutaneously in the dorsal region, under general anesthesia with 4% isoflurane (Vetflurane®, Virbac, France). At the implantation area, a small amount of Tronothane® 1% in Gel form (DELPHARM, L’Aigle) was applied to relieve the animal. After injection, a small massage at the injection site was performed to maintain the chip. These telemetric sensors allow individual monitoring and temperature measurement added to general condition, weight, and behavior throughout the protocols. Mice were housed in groups of 4 to allow social interaction in T2 type cages on bedding and in an enriched environment on a Techniplast ventilated rack (Techniplast, Louviers). Humidity (between 45% and 65%) and temperature (between 20°C and 24°C) were controlled daily. Mice were followed by daily zootechnical visits with a checklist of previously clinical signs allowing quotation of welfare (Table 1 ). Table 1 Description of clinical signs and quotation following infection with T. gondii during the acute phase for the assessment of well-being. Quotation 5 corresponds to the limit points reached, leading to the animal's death. Quotation Clinical signs 0 No symptom 1 Increase or drop in temperature or weight loss compared to D0 ( 38.2 ° C) 2 Weight loss and/or temperature drop compared to D0 and/or tousled hair 3 Weight loss + temperature drop + tousled hairs or almond-shaped eyes (beginning of facial tension) or low vibrissae or prostration 4 Weight loss + temperature drop + tousled hairs and/or low vibrissae and/or almond-shaped eyes (facial tension and/or ears back) and/or prostration 5 Weight loss + temperature drop + tousled hairs + low vibrissae + almond eyes (face tightness, ear back) + prostration Experimental designs Acetaminophen (N-acetyl-p-aminophenol or APAP) was administered to non-infected mice (first experiment) and to mice infected with T. gondii cysts (second experiment) for five consecutive days (Fig. 1 A and B). Blood samples were taken and zootechnical data collected throughout the experiments. Organs (spleen, liver, kidneys, stomach) were sampled at the end of the experiments for histological analyzes. Oral administration of acetaminophen (APAP) APAP solutions were prepared every morning for 5 days of treatment before their administration. For gavage, 75 µL of 40% Pracetam® (400 mg/mL, CEVA Santé Animal, Libourne, France) were mixed in 9925 µL of water to obtain the correct dose (30 mg/kg/day according to the recommendations of the SAFE complete care competence company). For self-ingestion, 4 doses of Safe® Geldiet Water (Safe SAS) corresponding to 400 mL were liquefied by heating in a microwave for 2 minutes and mixed in a beaker by magnetic stirring. Then, 12 µL of Pracetam® were added and after homogenization, the content of the beaker was transferred into the original jars, identified, weighed and solidified at 4°C for one hour. Treatment (30 mg/kg/day) was held over a period of 5 consecutive days. For gavage, mice were force-fed with 200 µL of water or water + APAP. For self-medication, one dose of Gel Water or Gel Water + APAP was placed on the floor of each cage of 4 mice and weighted at 24h to monitor consumption. In Gel Water, APAP concentration is identical. However, the dose ingested by the mice is different, because in Gel Water, the mice self-administer the drug to mimic natural mouse behavior. In all conditions, water was available ad libitum. Mice behavior using Marble burying test (anxiety test) The Marble Burying test/anxiety test 22 was performed regularly (D-2 = D0 before infection, D0 = infection, D5, D10, D21, D38) on infected mice. Each mouse was placed individually in a T3 type cage filled with 3 cm of litter for 5 minutes in order to get used to the new environment. The mouse was removed, then 15 beads (1.6 cm diameter) were placed in 5 rows and 3 columns. The mouse was returned to the cage and a score was assessed on each bead until 20 minutes (intact marble = 0, partially buried = 1, buried but visible = 2, completely buried = 3). Infection of mice by gavage with Toxoplasma gondii cysts The cysts were obtained from brains of CBA/J mice infected by gavage 2 months before with the T. gondii strain 76K. Mouse brains were homogenized in 5 mL of RPMI 1640 medium, and the number of tissue cysts per brain was determined microscopically by counting 3 samples (10 µL each) of each homogenate. The “lethal dose” groups received 120 cysts while the “chronic dose” groups received 15 cysts in the final volume of 200 µl by gavage 23 . The mice receiving 120 cysts were killed before day 12 of infection before they reached the end point previously defined. Blood tests for serum monitoring and biochemical markers analysis Blood samples (120 µl final serum) were taken from the mandibular vein with a sterile lancet on a sterile 2 mL dry tube. Blood samples were pooled as mouse pairs which stayed the same throughout the study. Cytotoxicity of APAP was analyzed by kinetic analysis of biochemical parameters of target organs: liver (alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase), kidneys (creatinine and urea) and glucose as a general parameter. Serum samples (diluted 1/10 and 1/61 in M-Scan II diluent) were placed in Select-6V crowns and analyzed using the M-ScanII Biochemical analyzer (Melet Schloesing Laboratories 24 ). Organ histology Spleen, brain, lung and stomach tissues fixed in 4% formalin (Carbo-Erba Reagents, Val de Breuil) for two weeks were embedded in paraffin wax (Paraplast plus, Leica) using an automatic device (TP1020, Leica). With a manual rotary microtome (RM2235, Leica), tissue sections 5 µm thick were deposited on Superfrost plus® slides (Thermo Fisher Scientific, Artenay) before being dried at 37 ° C overnight. The usual topographic staining by Haemalun-Eosin was used to observe the tissue structure of the different samples. The tissues were kept between slides and coverslips to be observed under a microscope (Eclipse 80i, Nikon). Cell staining Single-cell splenocyte suspensions were obtained from spleen first pressed and then filtered through a nylon mesh. Hypotonic shock (0.155 M NH 4 Cl, pH 7.4) was used to remove erythrocytes. The cells were then suspended in RPMI 1640 medium supplemented with 5% fetal calf serum (FCS), 25 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µM 2-β-mercaptoethanol, and 1 mM penicillin-streptomycin and counted. Splenocytes were seeded at 5.10 5 /200 µL into 96-well round-bottom culture plates. After centrifugation for 5 minutes at 700 g, the supernatants were discarded and 100 µL of each antibody diluted in PBS 5% FCS were added. Antibodies for the detection of CD4 (clone GK1.5), CD8 (clone eBioH35-17.2), CD19 (clone ebio1D3) and F4/80 (clone BM8) and their respective isotype were purchased from eBioscience and cells stained as described 25 . The cells were homogenized and the plates incubated for 30 minutes at 4°C. The plates were centrifuged, the supernatants removed and cells were washed with 100 µL of PBS containing 5% FCS. This step was repeated, then 100 µL of 2% paraformaldehyde (w/v) in PBS were added and the plates were placed at 4° C until analysis of 10000 events by flow cytometry (MACSquant, Miltenyi). Cytokine quantification For cytokine detection, splenocytes were recovered and purified as described above. Splenocytes were seeded at 5.10 5 /200 µL medium into 96-well flat-bottomed plates and stimulated for 72h with 10 µg/mL Toxoplasma extract (TE) or with concanavalin A at 5 µg/mL. Levels of mouse cytokines were quantified in the culture supernatants by using IL-6, IFN-γ, IL-12p40 specific sandwich enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s instructions (cytokine mouse uncoated ELISA kit, Invitrogen) as described 25 . Detection of anti-toxoplasmic immunoglobulins-G in serum by ELISA Titers of Toxoplasma -specific IgG antibodies were performed by ELISA on sera. Flat-bottomed 96-well plates (Nunc) were coated overnight with 10 µg/mL TE in 50 mM carbonate buffer (pH 9.6). The plates were washed with PBS-Tween 0.05% and blocked with PBS-4% bovine serum albumin (BSA, Sigma-Aldrich). Serial dilutions of serum were performed in PBS-BSA 4%, and the plates were incubated for 2h at 37°C. The plates were then washed again and incubated 1h at 37°C with goat anti-mouse IgG alkaline phosphatase (1:5000, Sigma-Aldrich). After washes, para-nitro-phenyl-phosphate (Sigma-Aldrich) diluted in DEA-HCl at 10 mg/mL was added. The absorbance of each sample was measured at 405 nm. Titers of IgG antibodies were determined as the highest serum dilution that exhibited an absorbance at least twice that of the mean absorbance of eight wells containing the negative control serum. DNA extraction and qPCR DNA was extracted from 25 mg tissue (brain, lungs or spleen) using Nucleospin tissue extraction kit (Macherey-NaGel). Quantitative PCR (qPCR) was performed on 200 ng of genomic DNA in a total volume of 20 µl containing LightCycler® Taqman® Master mix (Roche Diagnostics), 0.5 µM of 2 primers (TG III: 5’-CCT TGG CCG ATA GGT CTA GG-3’; TG IIb: 5’-GGC ATT CCT CGT TGA AGA TT-3’, and 180 nM of the probe FAM-5’-FAM-TGC AAT AAT CTA TCC CCA TCA CGA TGC ATA CTC AC-TAMRA-3’ (Eurofins Genomics, France). The qPCR program was 2 min at 50°C, 5 min at 95°C, 50 cycles of 20s at 95°C/60s at 65°C with the LightCycler® 2.0 Instrument (Roche Diagnostics, France). Standard curves were generated with DNA of known amounts of tachyzoites alone or extracted with the DNA of brain, lungs or spleen of non-infected mice as described 26 , 27 . Enumeration of brain cysts Brains were homogenized in 5 mL of RPMI 1640 medium in a glass potter, and the number of tissue cysts per brain was determined microscopically by counting 10 samples (10 µL each) of each homogenate. Statistical analyzes Statistical analyzes were performed using GraphPad Prism software, version 6.0 (GraphPad, San Diego). A two-way ANOVA analysis was performed to show the effects of treatments on the measured parameters. Nonparametric Kruskal Wallis tests were carried out to analyze the treatment effect between the groups. All statistical tests were two-sided and a value of P < 0.05 was considered statistically significant. Results Lack of toxicity of acetaminophen (APAP) treatment in non-infected CBA/J mice The temperature and weight of the mice were monitored from D-3 to D7, with APAP treatment from D0 to D5 (Table 2 ). Before treatment, mice had an average weight between 18.80 ± 0.92 g and 20.00 ± 1.20 g. The mice gained weight in accordance with the reference growth curve of the CBA/J strain, with no significative difference between groups, treated or not with APAP. Likewise, no significant variation has been observed for body temperature, which varied between 37.27 ± 0.69°C and 38.40 ± 0.68°C. Table 2 Zootechnical monitoring of CBA/J mice during the acetaminophen treatment phase (weight/temperature, D-3, D0, D5, D7, expressed as average standard deviation, n = 8/group) Day − 3 Day 0 Day 5 Day 7 Weight (g) Temperature (°C) Weight (g) Temperature (°C) Weight (g) Temperature (°C) Weight (g) Temperature (°C) Control 18.80 ± 0.92 37.56 ± 0.33 19.20 ± 0.92 37.81 ± 0.63 19.70 ± 1.49 38.40 ± 0.68 19.70 ± 1.49 38.40 ± 0.68 Water by gavage 20.13 ± 0.99 37.86±0.41 19.50±1.41 37.29±0.61 20.25±1.16 37.76±0.71 20.25±1.16 37.76±0.71 APAP by gavage 20.00 ± 1.20 37.80 ± 0.47 20.13 ± 0.99 37.90 ± 0.29 20.50 ± 1.41 37.34 ± 0.76 20.50 ± 1.41 37.34 ± 0.76 Gel water 19.13 ± 1.13 37.31 ± 0.50 18.88 ± 1.64 37.39 ± 0.45 19.25 ± 1.16 37.65 ± 0.47 19.25 ± 1.16 37.65 ± 0.47 APAP + Gel water 19.75 ± 1.28 37.40 ± 0.35 20.50 ± 1.31 37.44 ± 0.61 20.13 ± 0.99 37.27 ± 0.69 20.13 ± 0.99 37.27 ± 0.69 For each blood sample, biochemical quantification of alkaline phosphatase, aspartate transaminase, alanine aminotransferase, urea and glucose (Table 3 ) was carried out to assess the potential toxicity of APAP on the organs involved in its degradation and elimination (liver, kidneys). Except for elevated levels of alkaline phosphatase, all values were within the range of norms given by the supplier for the mouse species. For all parameters quantified, no significant difference between the groups and in comparison, to the day before the treatment was observed. Creatinine was measured but not detectable (< 1.9 mg/mL). Table 3 Monitoring of serum biochemical parameters, indicators of drug toxicity in CBA/J mice. (n = 4 serum samples per group, except 5 serum samples for the control, corresponding to n = 8 mice /group and n = 10 mice for control group). ALP = alkaline phosphatase, AST = aspartate transaminase, ALAT = alanine aminotransferase. Groups ALP (U/L) AST (GTP, U/L) ALAT (GOT, U/L) Urea (g/L) Glucose (g/L) Day 0 Control 332 ± 23 85 ± 18 23 ± 2 0.34 ± 0.05 1.22 ± 0.22 Water by gavage 236 ± 56 69 ± 24 9 ± 3 0.41 ± 0.04 1.72 ± 0.32 APAP by gavage 250 ± 52 94 ± 27 32 ± 11 0.40 ± 0.05 1.51 ± 0.45 Gel water 294 ± 29 102 ± 39 22 ± 6 0.41 ± 0.03 1.53 ± 0.03 APAP + Gel water 265 ± 50 66 ± 21 34 ± 3 0.42 ± 0.03 1.89 ± 0.19 Day 5 Control 234 ± 37 94 ± 21 41 ± 12 0.39 ± 0.05 1.60 ± 0.21 Water by gavage 192 ± 34 132 ± 12 41 ± 3 0.40 ± 0.09 1.60 ± 0.15 APAP by gavage 218 ± 40 88 ± 27 38 ± 6 0.37 ± 0.03 1.63 ± 0.28 Gel water 271 ± 37 105 ± 10 43 ± 2 0.33 ± 0.04 1.39 ± 0.15 APAP + Gel water 193 ± 6 106 ± 22 42 ± 2 0.41 ± 0.03 1.70 ± 0.11 Range of normal values (min-max ) 62–209 59–247 28–132 0.38–0.67 0.9–1.92 To further observed the effect of treatment, histological observations of target tissues (spleen, liver, kidneys, stomach) following treatment were performed. Figure 2 represents a sample of the tissues taken after the APAP treatment on the different groups. Figure 2 A to 2 E showed a sample of the spleen of each group. After treatment for 5 consecutive days and regardless of the groups, no change in tissue architecture nor cell infiltration could be observed. Figure 2 F through 2 J show the liver for each group. For all groups, no difference between hepatocytes was observed. Indeed, their clarity and the general organization of the tissue is different in the case of hepatotoxicity 28 . No difference in connective tissue containing centrilobular veins was noticeable, just as treatment with APAP did not impact the hepatic lobes surrounding them and the hepatic arteries. No change caused by APAP in the appearance of the stomach layers was observed (Fig. 2 K to 2 O). Figures 2 P to 2 T represent the sections of the right kidney of the mice in each group. No difference in structure and organization was observed on the two main areas of the kidney: the medulla (central area) and the cortical (peripheral area). For each animal, both kidneys were observed and were not different in their histology. To further assess the toxicity of APAP, in particular on the liver and its impact on the immune response, the IL-6 level in serum was measured on the first and last day of treatment. IL-6 is a good marker of liver toxicity. Its expression enables liver cells to regenerate. Thus, IL-6 could not be detected (below the detection threshold of 8 pg/mL) before treatment. No increase was observed after treatment regardless of the group (data not shown). Spleens were weighted and splenocyte number counted (Fig. 3 A, B). No significant difference in spleen weight and splenocytes numbers could be observed between the control groups without treatment and the groups treated with APAP by gavage or self-medication. However, higher splenocyte counts were observed in the groups treated with APAP Gel Water (P = 0.03). The average percentages of CD4 + , CD8 + , CD19 + and F4/80 + (macrophages) splenocytes were measured. No significant difference could be observed between the groups when compared to the control group (data not shown). At this dose, APAP treatment did not induce significant changes in zootechnical characteristics, mice behavior and tissue’s structure. Effect of acetaminophen (APAP) treatment on the pathophysiology of toxoplasmosis Zootechnical, clinical and behavioral monitoring of mice Early after infection (from D0 to D4) with 120 cysts (lethal dose), normal values of temperature (37.4 ± 0.7 ° C) were found regardless of the group (Fig. 4 A). An increase in temperature was observed from the D5 for all infected groups. This increase was followed by a significant hyperthermic peak up to 39.5°C (P < 0.01) in comparison to the control group on D6 and D7. Following this hyperthermic peak, the temperatures of the infected mice decreased to reach hypothermia (around 36°C) at D10 compared to the control group (P < 0.01). The APAP treatment from D7 to D11, either by gavage or self-medication, did not significantly modify the temperature curve of the non-treated infected group (Fig. 4 A). Mice infected with a lethal dose die between 12 and 15 days p.i. In the groups infected with 15 cysts (dose leading to chronic toxoplasmosis), temperature slightly increased, but also in the non-infected control group (Fig. 4 C). A temperature drop started at D10 and was significantly different from the control group at D1 (P < 0.01), although the temperatures remained above 36°C. The temperatures never returned to normal values or close to the control group (< 37.5°C) until the end of the experiment (between 36.5 and 37.2°C). APAP treatment by gavage from D7 to D11 significantly slowed the temperature drop from D10 and until the end of treatment (P = 0.04) (Fig. 4 C). The weight of the mice was followed throughout the experiment and expressed as percentage of the initial weight before infection with T. gondii . For the group infected with the 120 cysts without treatment, a weight loss up to more than 10% could be observed at D10 and D12 (Fig. 4 B). This weight loss was reduced in the two APAP-treated groups from D8 and until the end of treatment (P < 0.05, Fig. 4 B). For the groups infected with 15 cysts, the weight loss was less than 5% of the non-infected group. Only the group treated with APAP by gavage showed no weight loss at D8 and D10 (P = 0.05, Fig. 4 D). After the acute phase, all mice regained normal weight, equivalent to the control group (data not shown). To better assess animal pain and a possible effect of APAP during the acute phase of toxoplasmosis, a clinical score was set up with daily observation of the mice. Clinical signs (Table 1 ) appeared from D6 post-infection and were more important at D12. Logically, the clinical score was lower in groups infected with 15 cysts than in groups infected with 120 cysts (prostration, hypothermia, reduced movement). For both doses, a clear significant improvement in the score could be observed in the mice treated with APAP by gavage (Fig. 5 ), from D9 or D10 until D12, one day after the end of treatment. APAP therefore seems to have had a noticeable and significant effect on the well-being of mice, in particular a softening of the face (less tension, normal position of the whiskers), a conservation of social contact between individuals and an increase in mobility. Marble burying tests assess mice anxiety score. Animal distress is inversely proportional to the test score. Non-infected controls had an average score of 28 ± 2.7. Before infection and up to D6, no significant difference between groups could be measured, regardless of the infectious dose (data not shown). Between D6 and D10, a change in the overall behavior of the mice with the highest cysts dose was observed (prostration, reduced movement), which was less marked for the APAP-treated mice. For mice infected with 15 cysts, little change in behavior was observed regardless of the group. At D10, the score of the Marble burying test was significantly reduced for all infected groups versus the control groups, with lower scores for mice infected with 120 cysts (average score of 7, Fig. 6 A) than mice infected with 15 cysts (average score of 10, Fig. 6 B). However, an improvement in test scores was observed in infected groups treated with APAP by gavage (P < 0.05, Fig. 6 ), with score around 12 and 20, respectively. The score of chronic dose infected mice remained low compared with the control group at D21 (Control = 38 ± 4, versus 9 ± 7 for the other 3 groups). A progressive increase in score at D38 was observed to a greater extent in the APAP-treated groups (Control = 33 ± 3, versus infected only 15 ± 3 and infected + APAP 20 ± 3 and infected + APAP + Gel Water 16 ± 6) (data not shown). After infection with 120 cysts, significant changes in splenic tissue architecture can be observed, accompanied by massive infiltration of immune cells, such as macrophages, predominantly for the infected group alone. The white pulp is completely unorganized (Fig. 7 B, C, D) in the tissue of all infected mice, not allowing to see the germinal center (G) and its crown (Co) as seen in control mice (Fig. 7 A). Likewise, spleen of infected mice appeared to show a higher density of cells, especially at the ends and edges of the splenic capsule. Treatment with APAP does not appear to have altered the inflammatory induction associated with infection with the parasite. In lung section of mice from the control group (Fig. 7 E), the alveolar channels communicated with the alveoli (Al) connected to each other and appeared normal because they were scattered. Nuclei can also be seen in the figures, belonging either to endothelial cells or to alveolar cells. Finally, terminal bronchioles (Br) can be seen in the figures as well as a pulmonary arteriole (PA) containing visible red blood cells. On lung sections of infected mice, an overall thickening of the alveolar epithelium was very clearly observed (white circle, Fig. 7 F, G, H). The walls of the bronchioles were also thickened by the infection, with no difference observed between the groups that received treatment and the group without treatment. In brain hippocampus sections, astrocytes and neurons could be observed (Fig. I, black arrows), but no difference in tissue organization (e.g. lesions) was induced, either by infection with T. gondii or treatment with APAP (Fig. 7 J, K, L). In organ sections from mice infected with 15 cysts, only infiltration of immune cells into the spleen and thickening of the pulmonary epithelium could be observed in all infected groups, treated with APAP or not (Fig. 8 ). Cellular and humoral immune responses after infection and acetaminophen (APAP) treatment Main cytokines involved in the resolution of infection (IL-12 and IFN γ) were quantified in splenocyte cultures after activation with specific antigens of T. gondii . All samples from control non-infected group were under the detection threshold for the cytokines tested. No significant difference in IFN-γ and IL-6 concentrations in the groups treated with APAP compared to the infected group have been shown, regardless the number of cysts (Fig. 9 A, C, D, F). For lethal dose groups, IL-12 concentrations were higher in the group treated with APAP in Gel Water compared to the infected group without treatment (P < 0.05) but not found in the group treated by gavage (Fig. 9 B). There was no significant difference in IL-12 concentrations between groups infected with 15 cysts (Fig. 9 E). The average percentages of CD4+ (T lymphocytes), CD8+ (T lymphocytes), CD19+ (B lymphocytes) and F4/80+ (macrophages) populations within the splenocytes were analyzed. In the mice infected with 120 cysts, the percentage of F4/80 + cells increased (Fig. 10 A), whereas the percentage of CD4+ (Fig. 10 C) and CD19+ (Fig. 10 G) cells decreased. When infected mice were treated with APAP, the percentage of F4/80+, CD4 + and CD19 + cells did not change, whereas the percentage of CD8 + cells decreased, and significantly for the treatment by gavage (Fig. 10 E). In the mice infected with 15 cysts, the percentage of F4/80 + cells also increased (Fig. 10 B) and the percentage of CD4 + cells also decreased (Fig. 10 D). In contrast, the percentage of CD8 + cells decreased (Fig. 10 F) and the percentage of CD19 + cells was unchanged (Fig. 10 H). The treatment with APAP showed an impact only on the increase of F4/80 + cells, which was not so important as in the non-treated group, and only in the case of administration by gavage (Fig. 10 B). In summary, the treatment had a minor impact on population distribution after infection. Specific IgG were detected in the sera collected at different times during the experiment to observe seroconversion kinetics following infection (Fig. 11 A et 11B). A significant increase in optical density resulting from infection at D11. No difference was observed between the groups treated with APAP and the untreated group, regardless of the infectious dose administered, showing that APAP had a weak impact on the humoral immune response. Antibody titers have also been performed to get quantitative data and no significant difference between groups were observed (data not shown). Parasite distribution and dissemination were followed by qPCR in brain and lungs. No significant difference between infected groups and infected groups that received the APAP-treatment (Fig. 12 A, 12 B) during the acute phase of infection was observed. In the lungs of the mice in chronic phase, no difference was detected between groups by qPCR (Fig. 12 C). Brain cysts of the mice were counted in order to determine the parasite load at the end of the chronic phase (Fig. 12 D). The values illustrate high variability between individuals. However, no difference on encystment between the infected and treated groups was observed (Fig. 12 E). Discussion Pressure around animal experimentation is growing and the most invasive procedures are likely to be controversial and criticized. Animal models are essential in vaccine and therapeutic development against infectious organisms. However, the impact of infections on animals is sometimes severe and leads to problematic clinical signs from an ethical point of view (prostration, long-term hyperthermia, dehydration, even mortality 29 , 30 . The use of pharmacological tools to relieve pain remain underutilized in research rodents especially in infectious model despite the general acceptance of both the ethical imperative and regulatory requirements 7 , 31 . In way to optimize the conduct of experiments and increase their acceptability in compliance with regulations in animal experimentation focusing on one of the 3Rs, the refinement 32 , 33 , we used APAP as a new therapeutic refinement tool to relieve the mice in an infectious model of acute and chronic toxoplasmosis. In the present study, analyzes of the toxicity of APAP showed that its administration had no impact on weight, temperature and behavior of the mice during the 5 days of treatment. Two routes of oral administration were used by gavage and by consumption of Gel Water. APAP is administered orally, as its bioavailability and pharmacokinetics are documented by this route of administration and simpler for use in experimental conditions. This mode of administration has been widely validated by studies on the efficacy of APAP 13 . No dehydration of the mice was observed when APAP was given in Gel Water suggesting and confirms the previous data 34 . Biochemical serum analyzes (ALP, AST, ALAT, urea, glucose) showed that no toxicity was observable. In the case of APAP toxicity, transaminases levels as well as kidneys biomarkers, could increase because of hepatocytes lysis, which was not the case here 35 , 36 . These results confirm data published on different rodent models, showing that this dose of 30 mg/kg/day is not toxic during 5 consecutive days of treatment 21 , 37 . The weighing of the spleens and the number of splenocytes in non-infected mice were significantly different in the groups having been treated via Gel Water, only for the one with APAP. However, administration of APAP by gavage had no effect on splenocytes number or spleen weight. Gel Water is composed of 99% water. However, it can be assumed that other components of the Gel such as hydrocolloids (texturizing agent) or fibers (< 1.8%) could have an immunostimulatory effect 38 . No toxicity could be observed after histological analyzes on target organs (spleen, liver, kidneys, stomach). This lack of toxicity for this APAP dose is consistent with current published data 20 , 21 . IL-6 is known to have a dual role in fever regulation and hepatotoxicity after APAP overdose 39 . Serum concentrations of IL-6 were under the detection limit in all groups. The lack of IL-6, for this dose, confirmed that there is no hepatotoxicity and agrees with published data 40 , which shows the importance of IL-6 in the regeneration of hepatocytes in case of deregulation and abnormal functioning of the liver. After T. gondii infection, the clinical signs already known in toxoplasmosis were recorded (prostration, thermal peaks, face tightness, ears in back position 41 ). Although this had never been described in the literature for this infectious model of toxoplasmosis, hypothermia is the most commonly reported predictor of mice imminent death in several infectious model 42 . APAP treatment was not sufficient to suppress hyperthermia but it was significantly slowed down from the third day of treatment, mainly for mice treated by gavage. The first clinical signs observed from the fifth day post-infection were the onset of weight loss. The treatment made it possible to reduce weight loss, probably due to a general improvement of well-being for the mice infected with the lethal dose of cysts. This was not so obvious for the mice infected with the lowest dose because the weight loss was lower and delayed. Other symptoms appeared such as prostration, disheveled hair, tightness of the face and the shape of the eyes, lack of activity. Self-medication with APAP in Gel Water does not seem to have a noticeable effect on the behavior of mice which may be explained by a too low daily Gel consumption by the infected mice, especially with the lethal dose of cysts. However, mice infected with 15 cysts were able to ingest enough Gel Water with APAP to get an improvement in their well-being. The dose of APAP could be increased to 100 mg/kg/day as described in various studies 37 , 43 , but after 5 consecutive days, this dose has been shown to induce hepatic toxicity in rodents and pigs 21 . The detection of pain mild to moderate is difficult to record in mice. Burrowing performance have proved valuable tools to assess brain damage or malfunction 44 . This behavior is reduced by pain and stress, suggesting its use as behavioral parameter to assess general well-being in mice 45 . As behavior can be observed easily in a non-invasive manner it has been suggested as a relevant approach to assess both pain severity and the efficiency of pain management drugs. We used the Marble Burying Test to evaluate pain and pain relief after treatment. This test demonstrated a strong decrease in the activity of stressed or prostrated mice due to a general unease associated with the temperature variation during both acute and chronic phases of toxoplasmosis. These are completely consistent with the clinical signs observed. Other types of behavior tests could be used, for example the cross maze which could be used to study the discovery of a new environment like the Marble Burying test does, although the latter makes it easier to quantify anxiety 22 . On the other hand, in our study, a clear significant improvement was observed in the clinical signs and the general behavior of the mice on the 3rd day of treatment with APAP for both infectious doses. The hypothesis is that these two parameters can be related. Indeed, clinical score and behavior tests can be linked because a mouse with reduced mobility due to symptoms will naturally bury fewer marbles compared to a healthier mouse. A higher score of the Marble Burying test seems to illustrate an increase in the locomotion of the mice in relation to the general well-being effect induced by APAP, which also allowed the mice to be able to continue eating and hydrate (decrease of weight loss in mice treated by gavage) for both infectious doses. Similar results were found in the literature, on a non-parasitic mouse model 46 . This is a positive point for the use of APAP by gavage mainly to relieve the animal's discomfort. These first results are completely innovative in murine toxoplasmosis with these behavioral approaches in an infectious process and in agreement with the known effect of APAP on the general improvement of the individual 21 . To date, only one publication describes the use of buprenorphine to relieve pain and distress after acute T. gondii infection 47 . They only observed time to death and relief of pain distress. They don’t look at parasite dissemination nor immune response. Analysis of immune populations (CD4+, CD8 + and CD19 + lymphocytes, macrophages), did not reveal any significant differences with the exception of CD8 + T cells, which were lower in the groups treated with APAP by gavage compared to control infected group. Lymphocyte depletion in lymphoid organs due to APAP has already been observed, but at high dose and it was associated with hepatotoxicity 41 , 48 . Cytokines of interest were measured: IL-12 and IFN-γ as primary responses to T. gondii infection, IL-6 for its role in fever regulation and hepatotoxicity. Concentrations of these cytokines after stimulation of the splenocytes were slightly modified. A non-significant decrease in the IL-6 concentration in spleen could be observed in the APAP-gavage treated group. Fever is partly regulated by IL-6 20,21 . However, APAP is an antipyretic and even if it has no direct effect on the amounts of IL-6, it could induce a change in its concentration 49 . IL12 concentration was also significantly increased in the group treated by APAP in Gel Water in the group infected with the lowest dose of cysts. This may be consistent with the observation of a potential immunostimulatory effect due to the component of the gel 38 . Specific serum IgG were not modified for both infectious doses by treatment with APAP at the reference dose of 30 mg/kg/day. This shows that APAP had a weak impact on the cellular and humoral immune responses in our study conditions. Parasite load was assessed using qPCR for organs obtained at the end of acute phase (lungs, brain) and at the end of the chronic phase (lung) and also by counting the brain cysts. No significant difference in parasite load has been observed in both tissues regardless of the treatment. Related to brain and chronic phase behavior, no difference in cyst number was observed, indicating that APAP potentially improved behavior by reducing symptoms without affecting parasite load in the mice, showing a very limited impact on the physiopathology. Finally, the histological analysis showed no difference in the tissue organization of the target organs due to the treatment, in particular the hippocampus involved in the behavior and the exploration of a new environment. Since T. gondii is known to induce changes in behavior when installed in the brain, it was interesting to study the relation between APAP-treatment and behavior. Indeed, it was important that improvement in behavior during both phases of the infection was due to APAP reducing the severity of symptoms rather than by modifying the parasite distribution, using histological data obtained, especially in the brain. It is also important to note that APAP’s mode of action could be linked with serotonin levels 50 , one of the main mediators of anxiety in the central nervous system 51 . As for the distribution of cysts in the brain, we might have expected to detect fewer of these cystic structures impacting behavior following treatment with APAP. A homogeneous distribution of cysts also seems to be preserved under our experimental conditions and according to brain tissue analysis, consistent with the literature 52 . Conclusion Acetaminophen (APAP) administered by gavage appears to be a good pharmacological tool to relieve an animal infected with Toxoplasma gondii and therefore refine infectious process by improving well-being, as shown here on the model species. Our project therefore involved highlighting the combination of individual or collective markers specific to toxoplasmosis in mice (zootechnical, behavioral, immune response, parasite dissemination). The application of APAP during the acute phase did not modify all these parameters linked to the pathophysiology of murine toxoplasmosis. However, this treatment did significantly improve the general condition of the mice, thus contributing to the animal's well-being during experimentation. These initial results confirm our hypothesis and validate our proof of concept, i.e. that APAP at this reference dose and in our infectious model, is a new pharmacological tool for experimentation in infectiology to improve animal welfare. From a scientific and academic point of view, these research studies will also lead to a better understanding of the mode of action of APAP in veterinary medicine and in host-pathogen interactions, with no change in initial pathophysiology and improving the welfare of animals. This work will perhaps make fall a dogma on the use of APAP in veterinary health from animal experiments to breeding. Declarations Declaration of Competing Interests The author(s) declared no potential conflicts of interest with respect to research, authorship and/or publication of the article. The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author Ethics statement Experiments were carried out according to EU directives and French regulations (Directive 2010/63 / EU, 2010; Rural Code, 2018; Decree n ° 2013 − 118, 2013, https://www.legifrance.gouv.fr/loda/id/JORFTEXT000027038013/ . ). All experimental procedures have been evaluated and approved by the Ministry of Higher Education and Research (APAFIS # 2018021917268751.V3–13634). The procedures involving mice were evaluated by the Val de Loire ethics committee (CEEA VdL, committee number 19) and took place at the INRAE Platform for Experimental Infection PFIE (UE-1277 PFIE, INRAE Centre-Val-de Loire) Valley research, Nouzilly, France, https://doi.org/10.15454/1.5535888072272498e12 ). Funding The author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: the European Union’s Horizon 2020 Research and Innovation Programme (grant N° 731014, VetBioNet, Veterinary Biocontained facility Network to Sasha Trapp and Frédéric Lantier), the grants allocated by the Animal Health Department and from INRAE’s own funds. Author Contribution Conceptualization, MR, CR, NM and IDP; data curation, MR, NM; formal analysis, MR, AC, CR, NM; funding acquisition, MR and NM; investigation, MR, AC, CR, CoB, LaM, EL, NM and FDG; methodology, MR, AC, CR, CoB, LaM, EL, NM, FDG; project administration, MR, NM; resources, MR, AC, CR, CoB, LaM, EL, NM and IDP; supervision, MR, NM; validation, MR, NM and visualization, MR, NM and IDP; writing–original draft, AC, MR, CR, NM, FDG; writing–review & editing, MR, CR, NM and FDG. All authors have read and agreed to the published version of the manuscript. Acknowledgement We would like to thank the different partners that supported this project: the society Biolog-animal (Philippe Cordier, telemetric system), the SAFE group (Gel Water and use the acetaminophen in Gel Water), CEVA France for supplying Pracétam® (veterinary Acetaminophen) and the local ethical committee in Loire Valley (number 19). We are also grateful to the direction of PFIE (Stephane Abrioux) to accept this project and more particularly, for the experimental step, the staff (mice team) of the Experimental Infectiology Platform (UE-1277 PFIE, INRAE Centre de Recherche Val de Loire, Nouzilly, France, https://doi.org/10.15454/1.5535888072272498e12). We are also grateful to Dr. Caroline Prouillac for her participation in the pharmacologic aspects and preparation of APAP of these experiments and Sybille Brochard and Marion Guionnière for her technical assistance. We would also like to thank Pr. Alice de Boyer des Roches (UMR Herbivores, INRAE) and Dr. Dominique Autier-Derian (Animal-welfare-consulting company) for their expertise in this project. PFIE is part of EMERG’IN, the national infrastructure for the control of animal and zoonotic emerging infectious diseases through in vivo investigation. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5790171","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":403073632,"identity":"fd943367-1ccb-4737-8d97-850f675e6c2b","order_by":0,"name":"Nathalie Moiré","email":"","orcid":"","institution":"INRAE-Université de Tours, UMR-1282 Infectiologie et Santé publique (ISP), Centre Val de Loire","correspondingAuthor":false,"prefix":"","firstName":"Nathalie","middleName":"","lastName":"Moiré","suffix":""},{"id":403073633,"identity":"f4de2b03-6203-408c-b75d-a0a0297611fa","order_by":1,"name":"Axel Cauty","email":"","orcid":"","institution":"INRAE, UE-1277 Plateforme d'Infectiologie expérimentale (PFIE)","correspondingAuthor":false,"prefix":"","firstName":"Axel","middleName":"","lastName":"Cauty","suffix":""},{"id":403073634,"identity":"4478050c-f594-46d9-a895-f9f61eeda828","order_by":2,"name":"Christelle Rossignol","email":"","orcid":"","institution":"INRAE-Université de Tours, UMR-1282 Infectiologie et Santé publique (ISP), Centre Val de Loire","correspondingAuthor":false,"prefix":"","firstName":"Christelle","middleName":"","lastName":"Rossignol","suffix":""},{"id":403073635,"identity":"eef7fe53-1dbb-4429-8183-0b0327ed7847","order_by":3,"name":"Corinne Beaugé","email":"","orcid":"","institution":"INRAE, UE-1277 Plateforme d'Infectiologie expérimentale (PFIE)","correspondingAuthor":false,"prefix":"","firstName":"Corinne","middleName":"","lastName":"Beaugé","suffix":""},{"id":403073636,"identity":"3f81a0e1-8939-4507-a563-d725c88b4fd1","order_by":4,"name":"Laetitia Mérat","email":"","orcid":"","institution":"INRAE, UE-1277 Plateforme d'Infectiologie expérimentale (PFIE)","correspondingAuthor":false,"prefix":"","firstName":"Laetitia","middleName":"","lastName":"Mérat","suffix":""},{"id":403073637,"identity":"b358fee2-815e-438a-830e-6bd6ee481538","order_by":5,"name":"Emilie Lortscher","email":"","orcid":"","institution":"INRAE, UE-1277 Plateforme d'Infectiologie expérimentale (PFIE)","correspondingAuthor":false,"prefix":"","firstName":"Emilie","middleName":"","lastName":"Lortscher","suffix":""},{"id":403073638,"identity":"db6b50f6-4531-483b-b4ca-b12f0e6292f2","order_by":6,"name":"Françoise Debierre-Grockiego","email":"","orcid":"","institution":"INRAE-Université de Tours, UMR-1282 Infectiologie et Santé publique (ISP), Centre Val de Loire","correspondingAuthor":false,"prefix":"","firstName":"Françoise","middleName":"","lastName":"Debierre-Grockiego","suffix":""},{"id":403073639,"identity":"21b2650c-2af8-4d4b-974a-bcfa28f12f3a","order_by":7,"name":"Isabelle Dimier-Poisson","email":"","orcid":"","institution":"INRAE-Université de Tours, UMR-1282 Infectiologie et Santé publique (ISP), Centre Val de Loire","correspondingAuthor":false,"prefix":"","firstName":"Isabelle","middleName":"","lastName":"Dimier-Poisson","suffix":""},{"id":403073640,"identity":"f0942691-4f4f-47d7-8fb6-7cb6b7a648ad","order_by":8,"name":"Mickaël Riou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYDACZiBkYLBgAFMfDCCCH4jQIgHWwjgDooVxBkF7wFpALB4GIrTotjM/NvhRIcFg3s58TNqm4E4+f3sDY3MFHi1mh9mME3vOSDDIHGZLk84xeGY548wBxsYzeLXwMB/gbZNgkGDmMQNqOWzAcCOB/WEDAS0H//6DarEAapG//4CxkZCWZN4GqBYGoBaDGwyEtLAZG8sck+CRYGZLtuwBajE8k9iIX8v5w48l39TYyEnwHz5448efwwZyxw8fxKsFBniQ2IzEaBgFo2AUjIJRgA8AALH7QieCxMZmAAAAAElFTkSuQmCC","orcid":"","institution":"INRAE, UE-1277 Plateforme d'Infectiologie expérimentale (PFIE)","correspondingAuthor":true,"prefix":"","firstName":"Mickaël","middleName":"","lastName":"Riou","suffix":""}],"badges":[],"createdAt":"2025-01-08 15:08:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5790171/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5790171/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-06849-2","type":"published","date":"2025-07-01T15:58:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":74283855,"identity":"63ea2a53-c579-4ba4-8b27-0dc1edac908f","added_by":"auto","created_at":"2025-01-20 15:56:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":142768,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental timelines\u003c/strong\u003e. A) Evaluation of APAP cytotoxicity in naïve CBA/J mice. B) Effect of APAP on \u003cem\u003eToxoplasma gondii \u003c/em\u003epathophysiology (acute and chronic phases) and impact on the immune response and behavior in CBA/J mice. MB: Marble burying test. (n=8/group).\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5790171/v1/849f8f8d0c4aa4a79b97d631.jpg"},{"id":74283868,"identity":"41c6db53-1b1f-4251-8380-4b3998fcd1fe","added_by":"auto","created_at":"2025-01-20 15:56:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2589677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological analyzes of target tissues (spleen, liver, kidney and stomach) following 5 consecutive days of APAP treatment\u003c/strong\u003e. Observation after Haemalun-Eosin staining under the Nikon Eclipse 80i microscope, scale bar: 100 µm. Images A to E: section of spleen (white circle: macrophage; square: white pulp; asterisk: red pulp); F to J: section of liver (HA: hepatic artery; BD: Bile Duct); K to O: stomach section (GF: Fundic Gland; M: Muscle); P to T: section of right kidney (Gl: tubular Glomerulus; Tub: collecting tube). (Representative of 4 animals).\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5790171/v1/df76cba11d7884b7495b6490.jpg"},{"id":74284408,"identity":"4250c86d-e230-40e8-87b0-6722dfb10a68","added_by":"auto","created_at":"2025-01-20 16:04:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":250147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpleen inflammation after APAP treatment. \u003c/strong\u003eA) Spleen weight\u003cstrong\u003e (\u003c/strong\u003en=8, except control n=2) and B) number of splenocytes (n=4) after 5 days of consecutive treatments with APAP. Data were analyzed by Kruskal-Wallis test and expressed as median ± interquartile range, * indicates P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5790171/v1/42957f8ad24adc937154b479.jpg"},{"id":74283851,"identity":"fc1b7c64-66b1-48f6-87db-b1df2b1800a4","added_by":"auto","created_at":"2025-01-20 15:56:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":446303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZootechnical parameters during infection. \u003c/strong\u003eMonitoring of temperature (A and C) and weight (B and D) of CBA/J mice during the acute phase following infection with 120 cysts (A and B) or 15 cysts (C and D), with or without APAP treatment from D7 to D11 (data expressed as mean± SEM, * indicates P\u0026lt;0.05, ** P \u0026lt; 0.01, Two-way ANOVA, n=8).\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5790171/v1/707063c64a8f5bfe86231127.jpg"},{"id":74283860,"identity":"cb7cdca3-6d57-4364-a0a7-b4db28c5fafd","added_by":"auto","created_at":"2025-01-20 15:56:04","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":213299,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical follow-up. \u003c/strong\u003eClinical score during the acute phase following infection with 120 cysts (A) or 15 cysts (B) with or without APAP treatment from D7 to D11 (data expressed as mean ± SEM, * P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001, Two-way ANOVA, n=8).\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5790171/v1/386d259512f3db5d42b39919.jpg"},{"id":74284407,"identity":"8b246ef4-2a90-4d77-aacc-09926ac4f15e","added_by":"auto","created_at":"2025-01-20 16:04:04","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":185549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of behavior during infection. \u003c/strong\u003eResults of the Marble Burying test on D10 after infection with 120 cysts (A) or 15 cysts (B) with or without APAP-treatment from D7 to D11. Data were analyzed by Kruskal-Wallis test and expressed asmedian ± interquartile range, * indicates P\u0026lt;0.05, n=8).\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5790171/v1/b62cdc19634148f6744991ce.jpg"},{"id":74283871,"identity":"eb454d52-2752-41a1-8012-ab3d0dcbb2d5","added_by":"auto","created_at":"2025-01-20 15:56:05","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1785011,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological analyzes of target tissues during acute phase. \u003c/strong\u003eHaemalun-Eosin staining of organs from mice of different groups infected with a lethal dose (120 cysts) for 12 days and treated with APAP from D7 to D11; observation under a Nikon Eclipse 80i microscope, scale bar: 100 µm. Images A to D: spleen section, Cap: marginal Capsule; Co: Corona; G: Germinal center; Ma: macrophage; WP and RP: White Pulp and Red Pulp); E to H: lung section (Al: pulmonary Alveolus, Br: bronchiole; BV: Blood Vessel); I to L: brain section (AS: Astrocyte; DG: Dentate Gyrus; Ne: neuron). Pictures representative of several sections from 3 different animals.\u003c/p\u003e","description":"","filename":"Figure7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5790171/v1/78ced3f802f531226dace873.jpg"},{"id":74283869,"identity":"119b3a2d-254a-4712-a6de-3f9b6777b6c6","added_by":"auto","created_at":"2025-01-20 15:56:05","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1871029,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological analyzes of target tissues during chronic phase. \u003c/strong\u003eHaemalun-Eosin staining of organs from mice of different groups infected with a chronic dose (15 cysts) for 12 days and treated with APAP from D7 to D11; observation under a Nikon Eclipse 80i microscope, Scale: 100 µm. Images A to D: spleen section; E to H: lung section; I to L: brain section. Pictures representative of several sections from 3 different animals.\u003c/p\u003e","description":"","filename":"Figure8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5790171/v1/88509d7d0285edaed7b914a1.jpg"},{"id":74283852,"identity":"e7c40fe5-2263-47c7-a11c-842d50833a0b","added_by":"auto","created_at":"2025-01-20 15:56:04","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":267336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalyze of post-infection cytokine response. \u003c/strong\u003eProduction of cytokines in supernatants of splenocytes activated \u003cem\u003ein vitro\u003c/em\u003e by specific antigens of \u003cem\u003eT. gondii\u003c/em\u003e from mice at D12 of infection with or without APAP-treatment. A, B, C infection with 120 cysts and D, E, F infected with 15 cysts. A and D) IL-6; B and E) IL-12; and C and F) IFN-g. Data were analyzed by Kruskal-Wallis test and are expressed as median ±interquartile range, * P \u0026lt; 0.05, n=5.\u003c/p\u003e","description":"","filename":"Figure9.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5790171/v1/371895e208d9a31cd4e1fed8.jpg"},{"id":74283887,"identity":"b2af1c83-38a7-42b4-b602-c23bbc8b8cdc","added_by":"auto","created_at":"2025-01-20 15:56:06","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":709806,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePost-infection recruitment analyses of induced immune populations. \u003c/strong\u003ePercentage of splenocyte populations after 12 days of infection with 120 cysts (A, C, E and G) or 42 days of infection with 15 cysts (B, D, F, H) and 5 days of APAP-treatment. A and B: F4/80\u003csup\u003e+\u003c/sup\u003e (macrophages), C and D: CD4\u003csup\u003e+\u003c/sup\u003e T cells; E and F: CD8\u003csup\u003e+\u003c/sup\u003e T cells; G and H: CD19\u003csup\u003e+\u003c/sup\u003e B cells. Data were analyzed by Kruskal-Wallis test and are expressed as median ± interquartile range, * P \u0026lt; 0.05, ** P \u0026lt; 0.01, n=5.\u003c/p\u003e","description":"","filename":"Figure10.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5790171/v1/3519450328134c5b0b80758b.jpg"},{"id":74283857,"identity":"9a91388d-5cd0-496f-b6a2-4707f4c438f1","added_by":"auto","created_at":"2025-01-20 15:56:04","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":182342,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHumoral response after infection. \u003c/strong\u003eHumoral response in mice infected with 120 cysts (A) or 15 cysts (B) and APAP-treated (data are expressed as mean ± SD, n=4).\u003c/p\u003e","description":"","filename":"Figure11.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5790171/v1/468ca568f689948422eef9b4.jpg"},{"id":74284742,"identity":"0d5d43af-acf8-474c-abab-8d42f37e12d5","added_by":"auto","created_at":"2025-01-20 16:12:06","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":412097,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eParasite load during infection with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. gondii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eby qPCR\u003c/strong\u003e. A to C \u003cem\u003eT. gondii\u003c/em\u003e parasite number in 250 ng of total tissue DNA at day 12 after infection with 120 cysts in lung (A) and brain (C) and at day 40 after infection with 15 cysts in lung (B). D brain cyst counts. Data were analyzed by Kruskal-Wallis test and expressed as median ± interquartile rang, n=5. E) Illustration of cyst in brain, in particular into hippocampus region.\u003c/p\u003e","description":"","filename":"Figure12.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5790171/v1/e2b948cafd30d1cc868cf1cf.jpg"},{"id":86179172,"identity":"314fa1d7-20fe-44cb-9a7f-7d7aa3837461","added_by":"auto","created_at":"2025-07-07 16:16:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10533423,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5790171/v1/ca102ff4-d5ba-4127-a203-8864c3e77881.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Acetaminophen, a new tool to refine experimental infectious processes: the case of murine toxoplasmosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, animal welfare has become more important in society, especially in scientific research. Since 2013, a European decree has been established regarding the use of animals in experimental procedures, and the need for precise ethical evaluation of each project to be carried out. This involves strengthening ethics committees but also reminding and highlighting a rule that has existed since 1959: the 3Rs\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The objectives of this rule are multiple and therefore go through three main points: To \u003cb\u003eR\u003c/b\u003eeduce as much as possible the number of animals used during experiments and to \u003cb\u003eR\u003c/b\u003eeplace animal models with in vitro or bioinformatics models when possible. A key point of this rule, to \u003cb\u003eR\u003c/b\u003eefine, refers to methods that minimize the pain, suffering, distress that may be experienced by research animals, and which improve their welfare. Pain and suffering can alter animal\u0026rsquo;s behavior, physiology and immunology that can lead to variation in experimental results that may impair the reliability of studies\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRefinement can include an enrichment of the animals\u0026rsquo; environment, for example a game or a reward which can improve the general well-being\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and the introduction of endpoints that allow humans to estimate animal suffering. Finally, pain management using analgesic and/or anti-pyretic substances has also an impact on scientific results based on the choice on appropriate analgesic or the lack of pain treatment. The use of drugs is not so straightforward in experiments involving animals, especially in infectious disease models because of the influence that pharmacological substances could have on the results of the study. A dilemma then arises because symptoms related to an infection that can lead to the death of the animal could be limited with the appropriate medication. However, untreated pain can also affect, by example, the immune system. In all cases experimental bias may occur\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Before being able to generalize the use of drugs to relieve animals, it is necessary to know their mechanisms of action, the possible toxicity they could induce, but also their influence on the immune response and whether a cause-and-effect relationship to the infectious agent used can be observed.\u003c/p\u003e \u003cp\u003eOpioids and nonsteroidal anti-inflammatory drugs (NSAIDs) are the two main groups of pain relievers available\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Buprenorphine and meloxicam are the most commonly used analgesics in rodents and extensive literature review of pharmacokinetics of these drugs are available. Although it is one of the most largely used analgesic antipyretic drug in the world for humans\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, acetaminophen (N-acetyl-p-aminophenol or APAP) is rarely used to treat rodents. It is known to have no anti-inflammatory effect, unlike other nonsteroidal anti-inflammatory drugs\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The mechanism by which it produces its analgesic effect is largely unknown\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. It is supposed that acetaminophen would inhibit the synthesis of prostaglandins comprising a COX site (active site of the majority of NSAIDs\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e) in the central nervous system, prostaglandins having a pro-nociceptive role and potentially causing fever in the hypothalamus on which acetaminophen would act\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Similar to its analgesic action, the mechanism of antipyretic action of this drug remains poorly understood.\u003c/p\u003e \u003cp\u003eHowever, its administration is easily done orally and its absorption by oral route is complete and rapid, as the maximum plasma concentration is reached within one hour of ingestion\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Following oral administration, its systemic bioavailability is dose-dependent and ranges from 70\u0026ndash;90% with differences between animal species\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Acetaminophen is distributed rapidly in all tissues and is quickly eliminated by reaction with reduced glutathione and then excreted in the urine after conjugation with cysteine and mercapturic acid\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Nevertheless, literature studying acetaminophen use in experimental infectious rodent models is rare and most use concern pain relief after severe invasive procedures (in combination with opioids) or in cancer model.\u003c/p\u003e \u003cp\u003eHere, we were more interested in the antipyretic effect of acetaminophen and how this could reduce overall stress and unease during the acute phase of an infectious disease. For this study, we focused on a murine toxoplasmosis model. The agent responsible for toxoplasmosis is \u003cem\u003eToxoplasma gondii\u003c/em\u003e (\u003cem\u003eT. gondii\u003c/em\u003e), an obligate intracellular protist. It is present all over the world and in a wide diversity of hosts\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. It exists in 3 stages: tachyzoites rapidly multiplying in the acute phase of infection, bradyzoites within latent cysts in tissue; and sporozoites within oocysts, a form of resistance in the environment\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Initial contact with the parasite triggers a protective immune response in immunocompetent animals or humans with no, or only few, symptoms. However, the infection can lead to clinical symptoms (weight loss, temperature drop, prostration\u0026hellip;) and death in some mouse models, depending on the infectious dose. \u003cem\u003eT. gondii\u003c/em\u003e induces a pro-inflammatory (TH1 type) immune response, mediated by IFN-γ and IL-12 production\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e by T lymphocytes and antigen presenting cells, respectively. This immune response induced the control of the parasite in the acute phase, that allows its persistence in different tissues as dormant cysts resulting in chronic infection. This transient inflammation characteristic of the acute phase of toxoplasmosis, can be responsible for clinical symptoms.\u003c/p\u003e \u003cp\u003eThe objective of the study was to find out whether treating infected mice with APAP could relieve the pain or discomfort induced during the acute phase of toxoplasmosis, without altering the dissemination of the parasite and the immune response while improving the living conditions of the animals. For this, the harmlessness of APAP in CBA/J mice has been checked at the dose used (30 mg/kg/day), by gavage or by self-medication. This dose is recommended by the supplier of the veterinary medicinal product already used in pigs\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe same treatment has been transposed to CBA/J mice infected with \u003cem\u003eToxoplasma gondii\u003c/em\u003e, to study the effects of APAP on the pathophysiology (parasite multiplication and installation) of the infectious model and the specific humoral and cellular immune responses. Zootechnical analyzes (temperature, weight, behavior) as well as immunological, biochemical and histological analyzes were also performed. The main objective of the entire study was to validate the proof of concept for the use of APAP as a pharmacological tool to refine animal experiments using the murine model of toxoplasmosis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and Ethics statement\u003c/h2\u003e \u003cp\u003eExperiments were carried out according to EU directives and French regulations (Directive 2010/63 / EU, 2010; Rural Code, 2018; Decree n \u0026deg; 2013\u0026thinsp;\u0026minus;\u0026thinsp;118, 2013, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.legifrance.gouv.fr/loda/id/JORFTEXT000027038013/\u003c/span\u003e\u003cspan address=\"https://www.legifrance.gouv.fr/loda/id/JORFTEXT000027038013/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All experimental procedures have been evaluated and approved by the Ministry of Higher Education and Research (APAFIS # 2018021917268751.V3\u0026ndash;13634). The procedures involving mice were evaluated by the Val de Loire ethics committee (CEEA VdL, committee number 19) and took place at the INRAE Platform for Experimental Infection PFIE (UE-1277 PFIE, INRAE Centre-Val-de Loire) Valley research, Nouzilly, France, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15454/1.5535888072272498e12\u003c/span\u003e\u003cspan address=\"10.15454/1.5535888072272498e12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA total of 90 female mice of the CBA/J line (Janvier Labs, Le Genest-Saint-Isle, France) aged 5 weeks at the start of the experiments were used. Mice were randomly identified using telemetric sensors (Biolog-Animal\u0026reg;, Paris) implanted subcutaneously in the dorsal region, under general anesthesia with 4% isoflurane (Vetflurane\u0026reg;, Virbac, France). At the implantation area, a small amount of Tronothane\u0026reg; 1% in Gel form (DELPHARM, L\u0026rsquo;Aigle) was applied to relieve the animal. After injection, a small massage at the injection site was performed to maintain the chip. These telemetric sensors allow individual monitoring and temperature measurement added to general condition, weight, and behavior throughout the protocols. Mice were housed in groups of 4 to allow social interaction in T2 type cages on bedding and in an enriched environment on a Techniplast ventilated rack (Techniplast, Louviers). Humidity (between 45% and 65%) and temperature (between 20\u0026deg;C and 24\u0026deg;C) were controlled daily. Mice were followed by daily zootechnical visits with a checklist of previously clinical signs allowing quotation of welfare (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDescription of clinical signs and quotation following infection with \u003cem\u003eT. gondii\u003c/em\u003e during the acute phase for the assessment of well-being. Quotation 5 corresponds to the limit points reached, leading to the animal's death.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eQuotation\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClinical signs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo symptom\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncrease or drop in temperature or weight loss compared to D0 (\u0026lt;\u0026thinsp;36.5 \u0026deg; or \u0026gt;\u0026thinsp;38.2 \u0026deg; C)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWeight loss and/or temperature drop compared to D0 and/or tousled hair\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWeight loss\u0026thinsp;+\u0026thinsp;temperature drop\u0026thinsp;+\u0026thinsp;tousled hairs or almond-shaped eyes (beginning of facial tension) or low vibrissae or prostration\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWeight loss\u0026thinsp;+\u0026thinsp;temperature drop\u0026thinsp;+\u0026thinsp;tousled hairs and/or low vibrissae and/or almond-shaped eyes (facial tension and/or ears back) and/or prostration\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWeight loss\u0026thinsp;+\u0026thinsp;temperature drop\u0026thinsp;+\u0026thinsp;tousled hairs\u0026thinsp;+\u0026thinsp;low vibrissae\u0026thinsp;+\u0026thinsp;almond eyes (face tightness, ear back)\u0026thinsp;+\u0026thinsp;prostration\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental designs\u003c/h3\u003e\n\u003cp\u003eAcetaminophen (N-acetyl-p-aminophenol or APAP) was administered to non-infected mice (first experiment) and to mice infected with \u003cem\u003eT. gondii\u003c/em\u003e cysts (second experiment) for five consecutive days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). Blood samples were taken and zootechnical data collected throughout the experiments. Organs (spleen, liver, kidneys, stomach) were sampled at the end of the experiments for histological analyzes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eOral administration of acetaminophen (APAP)\u003c/h3\u003e\n\u003cp\u003eAPAP solutions were prepared every morning for 5 days of treatment before their administration. For gavage, 75 \u0026micro;L of 40% Pracetam\u0026reg; (400 mg/mL, CEVA Sant\u0026eacute; Animal, Libourne, France) were mixed in 9925 \u0026micro;L of water to obtain the correct dose (30 mg/kg/day according to the recommendations of the SAFE complete care competence company). For self-ingestion, 4 doses of Safe\u0026reg; Geldiet Water (Safe SAS) corresponding to 400 mL were liquefied by heating in a microwave for 2 minutes and mixed in a beaker by magnetic stirring. Then, 12 \u0026micro;L of Pracetam\u0026reg; were added and after homogenization, the content of the beaker was transferred into the original jars, identified, weighed and solidified at 4\u0026deg;C for one hour. Treatment (30 mg/kg/day) was held over a period of 5 consecutive days. For gavage, mice were force-fed with 200 \u0026micro;L of water or water\u0026thinsp;+\u0026thinsp;APAP. For self-medication, one dose of Gel Water or Gel Water\u0026thinsp;+\u0026thinsp;APAP was placed on the floor of each cage of 4 mice and weighted at 24h to monitor consumption. In Gel Water, APAP concentration is identical. However, the dose ingested by the mice is different, because in Gel Water, the mice self-administer the drug to mimic natural mouse behavior. In all conditions, water was available \u003cem\u003ead libitum.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eMice behavior using\u003c/b\u003e \u003cb\u003eMarble burying test\u003c/b\u003e \u003cb\u003e(anxiety test)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe Marble Burying test/anxiety test\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e was performed regularly (D-2\u0026thinsp;=\u0026thinsp;D0 before infection, D0\u0026thinsp;=\u0026thinsp;infection, D5, D10, D21, D38) on infected mice. Each mouse was placed individually in a T3 type cage filled with 3 cm of litter for 5 minutes in order to get used to the new environment. The mouse was removed, then 15 beads (1.6 cm diameter) were placed in 5 rows and 3 columns. The mouse was returned to the cage and a score was assessed on each bead until 20 minutes (intact marble\u0026thinsp;=\u0026thinsp;0, partially buried\u0026thinsp;=\u0026thinsp;1, buried but visible\u0026thinsp;=\u0026thinsp;2, completely buried\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cp\u003e \u003cb\u003eInfection of mice by gavage with\u003c/b\u003e \u003cb\u003eToxoplasma gondii\u003c/b\u003e \u003cb\u003ecysts\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe cysts were obtained from brains of CBA/J mice infected by gavage 2 months before with the \u003cem\u003eT. gondii\u003c/em\u003e strain 76K. Mouse brains were homogenized in 5 mL of RPMI 1640 medium, and the number of tissue cysts per brain was determined microscopically by counting 3 samples (10 \u0026micro;L each) of each homogenate. The \u0026ldquo;lethal dose\u0026rdquo; groups received 120 cysts while the \u0026ldquo;chronic dose\u0026rdquo; groups received 15 cysts in the final volume of 200 \u0026micro;l by gavage\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The mice receiving 120 cysts were killed before day 12 of infection before they reached the end point previously defined.\u003c/p\u003e\n\u003ch3\u003eBlood tests for serum monitoring and biochemical markers analysis\u003c/h3\u003e\n\u003cp\u003eBlood samples (120 \u0026micro;l final serum) were taken from the mandibular vein with a sterile lancet on a sterile 2 mL dry tube. Blood samples were pooled as mouse pairs which stayed the same throughout the study. Cytotoxicity of APAP was analyzed by kinetic analysis of biochemical parameters of target organs: liver (alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase), kidneys (creatinine and urea) and glucose as a general parameter. Serum samples (diluted 1/10 and 1/61 in M-Scan II diluent) were placed in Select-6V crowns and analyzed using the M-ScanII Biochemical analyzer (Melet Schloesing Laboratories\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e).\u003c/p\u003e\n\u003ch3\u003eOrgan histology\u003c/h3\u003e\n\u003cp\u003eSpleen, brain, lung and stomach tissues fixed in 4% formalin (Carbo-Erba Reagents, Val de Breuil) for two weeks were embedded in paraffin wax (Paraplast plus, Leica) using an automatic device (TP1020, Leica). With a manual rotary microtome (RM2235, Leica), tissue sections \u003cspan refid=\"Sec20\" class=\"InternalRef\"\u003e5\u003c/span\u003e \u0026micro;m thick were deposited on Superfrost plus\u0026reg; slides (Thermo Fisher Scientific, Artenay) before being dried at 37 \u0026deg; C overnight. The usual topographic staining by Haemalun-Eosin was used to observe the tissue structure of the different samples. The tissues were kept between slides and coverslips to be observed under a microscope (Eclipse 80i, Nikon).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell staining\u003c/h2\u003e \u003cp\u003eSingle-cell splenocyte suspensions were obtained from spleen first pressed and then filtered through a nylon mesh. Hypotonic shock (0.155 M NH\u003csub\u003e4\u003c/sub\u003eCl, pH 7.4) was used to remove erythrocytes. The cells were then suspended in RPMI 1640 medium supplemented with 5% fetal calf serum (FCS), 25 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 \u0026micro;M 2-β-mercaptoethanol, and 1 mM penicillin-streptomycin and counted. Splenocytes were seeded at 5.10\u003csup\u003e5\u003c/sup\u003e/200 \u0026micro;L into 96-well round-bottom culture plates. After centrifugation for 5 minutes at 700 g, the supernatants were discarded and 100 \u0026micro;L of each antibody diluted in PBS 5% FCS were added. Antibodies for the detection of CD4 (clone GK1.5), CD8 (clone eBioH35-17.2), CD19 (clone ebio1D3) and F4/80 (clone BM8) and their respective isotype were purchased from eBioscience and cells stained as described\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe cells were homogenized and the plates incubated for 30 minutes at 4\u0026deg;C. The plates were centrifuged, the supernatants removed and cells were washed with 100 \u0026micro;L of PBS containing 5% FCS. This step was repeated, then 100 \u0026micro;L of 2% paraformaldehyde (w/v) in PBS were added and the plates were placed at 4\u0026deg; C until analysis of 10000 events by flow cytometry (MACSquant, Miltenyi).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCytokine quantification\u003c/h3\u003e\n\u003cp\u003eFor cytokine detection, splenocytes were recovered and purified as described above. Splenocytes were seeded at 5.10\u003csup\u003e5\u003c/sup\u003e/200 \u0026micro;L medium into 96-well flat-bottomed plates and stimulated for 72h with 10 \u0026micro;g/mL \u003cem\u003eToxoplasma\u003c/em\u003e extract (TE) or with concanavalin A at 5 \u0026micro;g/mL. Levels of mouse cytokines were quantified in the culture supernatants by using IL-6, IFN-γ, IL-12p40 specific sandwich enzyme-linked immunosorbent assay (ELISA) following the manufacturer\u0026rsquo;s instructions (cytokine mouse uncoated ELISA kit, Invitrogen) as described\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eDetection of anti-toxoplasmic immunoglobulins-G in serum by ELISA\u003c/h3\u003e\n\u003cp\u003eTiters of \u003cem\u003eToxoplasma\u003c/em\u003e-specific IgG antibodies were performed by ELISA on sera. Flat-bottomed 96-well plates (Nunc) were coated overnight with 10 \u0026micro;g/mL TE in 50 mM carbonate buffer (pH 9.6). The plates were washed with PBS-Tween 0.05% and blocked with PBS-4% bovine serum albumin (BSA, Sigma-Aldrich). Serial dilutions of serum were performed in PBS-BSA 4%, and the plates were incubated for 2h at 37\u0026deg;C. The plates were then washed again and incubated 1h at 37\u0026deg;C with goat anti-mouse IgG alkaline phosphatase (1:5000, Sigma-Aldrich). After washes, para-nitro-phenyl-phosphate (Sigma-Aldrich) diluted in DEA-HCl at 10 mg/mL was added. The absorbance of each sample was measured at 405 nm. Titers of IgG antibodies were determined as the highest serum dilution that exhibited an absorbance at least twice that of the mean absorbance of eight wells containing the negative control serum.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and qPCR\u003c/h2\u003e \u003cp\u003eDNA was extracted from 25 mg tissue (brain, lungs or spleen) using Nucleospin tissue extraction kit (Macherey-NaGel). Quantitative PCR (qPCR) was performed on 200 ng of genomic DNA in a total volume of 20 \u0026micro;l containing LightCycler\u0026reg; Taqman\u0026reg; Master mix (Roche Diagnostics), 0.5 \u0026micro;M of 2 primers (TG III: 5\u0026rsquo;-CCT TGG CCG ATA GGT CTA GG-3\u0026rsquo;; TG IIb: 5\u0026rsquo;-GGC ATT CCT CGT TGA AGA TT-3\u0026rsquo;, and 180 nM of the probe FAM-5\u0026rsquo;-FAM-TGC AAT AAT CTA TCC CCA TCA CGA TGC ATA CTC AC-TAMRA-3\u0026rsquo; (Eurofins Genomics, France). The qPCR program was 2 min at 50\u0026deg;C, 5 min at 95\u0026deg;C, 50 cycles of 20s at 95\u0026deg;C/60s at 65\u0026deg;C with the LightCycler\u0026reg; 2.0 Instrument (Roche Diagnostics, France). Standard curves were generated with DNA of known amounts of tachyzoites alone or extracted with the DNA of brain, lungs or spleen of non-infected mice as described\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEnumeration of brain cysts\u003c/h2\u003e \u003cp\u003eBrains were homogenized in 5 mL of RPMI 1640 medium in a glass potter, and the number of tissue cysts per brain was determined microscopically by counting 10 samples (10 \u0026micro;L each) of each homogenate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyzes\u003c/h2\u003e \u003cp\u003eStatistical analyzes were performed using GraphPad Prism software, version 6.0 (GraphPad, San Diego). A two-way ANOVA analysis was performed to show the effects of treatments on the measured parameters. Nonparametric Kruskal Wallis tests were carried out to analyze the treatment effect between the groups. All statistical tests were two-sided and a value of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLack of toxicity of acetaminophen (APAP) treatment in non-infected CBA/J mice\u003c/h2\u003e \u003cp\u003eThe temperature and weight of the mice were monitored from D-3 to D7, with APAP treatment from D0 to D5 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Before treatment, mice had an average weight between 18.80 ± 0.92 g and 20.00 ± 1.20 g. The mice gained weight in accordance with the reference growth curve of the CBA/J strain, with no significative difference between groups, treated or not with APAP. Likewise, no significant variation has been observed for body temperature, which varied between 37.27 ± 0.69°C and 38.40 ± 0.68°C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eZootechnical monitoring of CBA/J mice during the acetaminophen treatment phase (weight/temperature, D-3, D0, D5, D7, expressed as average standard deviation, n = 8/group)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eDay − 3\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eDay 0\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eDay 7\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWeight (g)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTemperature (°C)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWeight (g)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTemperature (°C)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWeight (g)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTemperature (°C)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eWeight (g)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTemperature (°C)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e18.80 ± 0.92\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e37.56 ± 0.33\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e19.20 ± 0.92\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e37.81 ± 0.63\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c6\"\u003e \u003cp\u003e19.70 ± 1.49\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c7\"\u003e \u003cp\u003e38.40 ± 0.68\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c8\"\u003e \u003cp\u003e19.70 ± 1.49\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c9\"\u003e \u003cp\u003e38.40 ± 0.68\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater by gavage\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e20.13 ± 0.99\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e37.86±0.41\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e19.50±1.41\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e37.29±0.61\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c6\"\u003e \u003cp\u003e20.25±1.16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c7\"\u003e \u003cp\u003e37.76±0.71\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c8\"\u003e \u003cp\u003e20.25±1.16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c9\"\u003e \u003cp\u003e37.76±0.71\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAPAP by gavage\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e20.00 ± 1.20\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e37.80 ± 0.47\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e20.13 ± 0.99\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e37.90 ± 0.29\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c6\"\u003e \u003cp\u003e20.50 ± 1.41\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c7\"\u003e \u003cp\u003e37.34 ± 0.76\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c8\"\u003e \u003cp\u003e20.50 ± 1.41\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c9\"\u003e \u003cp\u003e37.34 ± 0.76\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGel water\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e19.13 ± 1.13\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e37.31 ± 0.50\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e18.88 ± 1.64\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e37.39 ± 0.45\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c6\"\u003e \u003cp\u003e19.25 ± 1.16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c7\"\u003e \u003cp\u003e37.65 ± 0.47\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c8\"\u003e \u003cp\u003e19.25 ± 1.16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c9\"\u003e \u003cp\u003e37.65 ± 0.47\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAPAP + Gel water\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e19.75 ± 1.28\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e37.40 ± 0.35\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e20.50 ± 1.31\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e37.44 ± 0.61\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c6\"\u003e \u003cp\u003e20.13 ± 0.99\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c7\"\u003e \u003cp\u003e37.27 ± 0.69\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c8\"\u003e \u003cp\u003e20.13 ± 0.99\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c9\"\u003e \u003cp\u003e37.27 ± 0.69\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eFor each blood sample, biochemical quantification of alkaline phosphatase, aspartate transaminase, alanine aminotransferase, urea and glucose (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) was carried out to assess the potential toxicity of APAP on the organs involved in its degradation and elimination (liver, kidneys). Except for elevated levels of alkaline phosphatase, all values were within the range of norms given by the supplier for the mouse species. For all parameters quantified, no significant difference between the groups and in comparison, to the day before the treatment was observed. Creatinine was measured but not detectable (\u0026lt; 1.9 mg/mL).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMonitoring of serum biochemical parameters, indicators of drug toxicity in CBA/J mice. (n = 4 serum samples per group, except 5 serum samples for the control, corresponding to n = 8 mice /group and n = 10 mice for control group). ALP = alkaline phosphatase, AST = aspartate transaminase, ALAT = alanine aminotransferase.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eALP (U/L)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAST (GTP, U/L)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eALAT (GOT, U/L)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUrea (g/L)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGlucose (g/L)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eDay 0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e332 ± 23\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85 ± 18\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23 ± 2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.34 ± 0.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.22 ± 0.22\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater by gavage\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e236 ± 56\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e69 ± 24\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9 ± 3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.41 ± 0.04\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.72 ± 0.32\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPAP by gavage\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e250 ± 52\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94 ± 27\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32 ± 11\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.40 ± 0.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.51 ± 0.45\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGel water\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e294 ± 29\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e102 ± 39\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22 ± 6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.41 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.53 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPAP + Gel water\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e265 ± 50\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66 ± 21\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34 ± 3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.42 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.89 ± 0.19\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eDay 5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e234 ± 37\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94 ± 21\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41 ± 12\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.39 ± 0.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.60 ± 0.21\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater by gavage\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e192 ± 34\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e132 ± 12\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41 ± 3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.40 ± 0.09\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.60 ± 0.15\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPAP by gavage\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e218 ± 40\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88 ± 27\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38 ± 6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.37 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.63 ± 0.28\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGel water\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e271 ± 37\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e105 ± 10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43 ± 2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.33 ± 0.04\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.39 ± 0.15\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPAP + Gel water\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e193 ± 6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e106 ± 22\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42 ± 2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.41 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.70 ± 0.11\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRange of normal values\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(min-max\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e62–209\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e59–247\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e28–132\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.38–0.67\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.9–1.92\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eTo further observed the effect of treatment, histological observations of target tissues (spleen, liver, kidneys, stomach) following treatment were performed. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e represents a sample of the tissues taken after the APAP treatment on the different groups. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA to \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE showed a sample of the spleen of each group. After treatment for 5 consecutive days and regardless of the groups, no change in tissue architecture nor cell infiltration could be observed. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF through \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ show the liver for each group. For all groups, no difference between hepatocytes was observed. Indeed, their clarity and the general organization of the tissue is different in the case of hepatotoxicity\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. No difference in connective tissue containing centrilobular veins was noticeable, just as treatment with APAP did not impact the hepatic lobes surrounding them and the hepatic arteries. No change caused by APAP in the appearance of the stomach layers was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK to \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO). Figures\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eP to \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eT represent the sections of the right kidney of the mice in each group. No difference in structure and organization was observed on the two main areas of the kidney: the medulla (central area) and the cortical (peripheral area). For each animal, both kidneys were observed and were not different in their histology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further assess the toxicity of APAP, in particular on the liver and its impact on the immune response, the IL-6 level in serum was measured on the first and last day of treatment. IL-6 is a good marker of liver toxicity. Its expression enables liver cells to regenerate. Thus, IL-6 could not be detected (below the detection threshold of 8 pg/mL) before treatment. No increase was observed after treatment regardless of the group (data not shown).\u003c/p\u003e \u003cp\u003eSpleens were weighted and splenocyte number counted (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). No significant difference in spleen weight and splenocytes numbers could be observed between the control groups without treatment and the groups treated with APAP by gavage or self-medication. However, higher splenocyte counts were observed in the groups treated with APAP Gel Water (P = 0.03). The average percentages of CD4\u003csup\u003e+\u003c/sup\u003e, CD8\u003csup\u003e+\u003c/sup\u003e, CD19\u003csup\u003e+\u003c/sup\u003e and F4/80\u003csup\u003e+\u003c/sup\u003e (macrophages) splenocytes were measured. No significant difference could be observed between the groups when compared to the control group (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt this dose, APAP treatment did not induce significant changes in zootechnical characteristics, mice behavior and tissue’s structure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of acetaminophen (APAP) treatment on the pathophysiology of toxoplasmosis\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eZootechnical, clinical and behavioral monitoring of mice\u003c/h2\u003e \u003cp\u003eEarly after infection (from D0 to D4) with 120 cysts (lethal dose), normal values of temperature (37.4 ± 0.7 ° C) were found regardless of the group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). An increase in temperature was observed from the D5 for all infected groups. This increase was followed by a significant hyperthermic peak up to 39.5°C (P \u0026lt; 0.01) in comparison to the control group on D6 and D7. Following this hyperthermic peak, the temperatures of the infected mice decreased to reach hypothermia (around 36°C) at D10 compared to the control group (P \u0026lt; 0.01). The APAP treatment from D7 to D11, either by gavage or self-medication, did not significantly modify the temperature curve of the non-treated infected group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Mice infected with a lethal dose die between 12 and 15 days p.i.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the groups infected with 15 cysts (dose leading to chronic toxoplasmosis), temperature slightly increased, but also in the non-infected control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). A temperature drop started at D10 and was significantly different from the control group at D1 (P \u0026lt; 0.01), although the temperatures remained above 36°C. The temperatures never returned to normal values or close to the control group (\u0026lt; 37.5°C) until the end of the experiment (between 36.5 and 37.2°C). APAP treatment by gavage from D7 to D11 significantly slowed the temperature drop from D10 and until the end of treatment (P = 0.04) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThe weight of the mice was followed throughout the experiment and expressed as percentage of the initial weight before infection with \u003cem\u003eT. gondii\u003c/em\u003e. For the group infected with the 120 cysts without treatment, a weight loss up to more than 10% could be observed at D10 and D12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This weight loss was reduced in the two APAP-treated groups from D8 and until the end of treatment (P \u0026lt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). For the groups infected with 15 cysts, the weight loss was less than 5% of the non-infected group. Only the group treated with APAP by gavage showed no weight loss at D8 and D10 (P = 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). After the acute phase, all mice regained normal weight, equivalent to the control group (data not shown).\u003c/p\u003e \u003cp\u003eTo better assess animal pain and a possible effect of APAP during the acute phase of toxoplasmosis, a clinical score was set up with daily observation of the mice. Clinical signs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) appeared from D6 post-infection and were more important at D12. Logically, the clinical score was lower in groups infected with 15 cysts than in groups infected with 120 cysts (prostration, hypothermia, reduced movement). For both doses, a clear significant improvement in the score could be observed in the mice treated with APAP by gavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), from D9 or D10 until D12, one day after the end of treatment. APAP therefore seems to have had a noticeable and significant effect on the well-being of mice, in particular a softening of the face (less tension, normal position of the whiskers), a conservation of social contact between individuals and an increase in mobility.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMarble burying tests assess mice anxiety score. Animal distress is inversely proportional to the test score. Non-infected controls had an average score of 28 ± 2.7. Before infection and up to D6, no significant difference between groups could be measured, regardless of the infectious dose (data not shown). Between D6 and D10, a change in the overall behavior of the mice with the highest cysts dose was observed (prostration, reduced movement), which was less marked for the APAP-treated mice. For mice infected with 15 cysts, little change in behavior was observed regardless of the group. At D10, the score of the Marble burying test was significantly reduced for all infected groups versus the control groups, with lower scores for mice infected with 120 cysts (average score of 7, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) than mice infected with 15 cysts (average score of 10, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). However, an improvement in test scores was observed in infected groups treated with APAP by gavage (P \u0026lt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), with score around 12 and 20, respectively. The score of chronic dose infected mice remained low compared with the control group at D21 (Control = 38 ± 4, versus 9 ± 7 for the other 3 groups). A progressive increase in score at D38 was observed to a greater extent in the APAP-treated groups (Control = 33 ± 3, versus infected only 15 ± 3 and infected + APAP 20 ± 3 and infected + APAP + Gel Water 16 ± 6) (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter infection with 120 cysts, significant changes in splenic tissue architecture can be observed, accompanied by massive infiltration of immune cells, such as macrophages, predominantly for the infected group alone. The white pulp is completely unorganized (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C, D) in the tissue of all infected mice, not allowing to see the germinal center (G) and its crown (Co) as seen in control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Likewise, spleen of infected mice appeared to show a higher density of cells, especially at the ends and edges of the splenic capsule. Treatment with APAP does not appear to have altered the inflammatory induction associated with infection with the parasite.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn lung section of mice from the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE), the alveolar channels communicated with the alveoli (Al) connected to each other and appeared normal because they were scattered. Nuclei can also be seen in the figures, belonging either to endothelial cells or to alveolar cells. Finally, terminal bronchioles (Br) can be seen in the figures as well as a pulmonary arteriole (PA) containing visible red blood cells. On lung sections of infected mice, an overall thickening of the alveolar epithelium was very clearly observed (white circle, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, G, H). The walls of the bronchioles were also thickened by the infection, with no difference observed between the groups that received treatment and the group without treatment. In brain hippocampus sections, astrocytes and neurons could be observed (Fig. I, black arrows), but no difference in tissue organization (e.g. lesions) was induced, either by infection with \u003cem\u003eT. gondii\u003c/em\u003e or treatment with APAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ, K, L).\u003c/p\u003e \u003cp\u003eIn organ sections from mice infected with 15 cysts, only infiltration of immune cells into the spleen and thickening of the pulmonary epithelium could be observed in all infected groups, treated with APAP or not (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCellular and humoral immune responses after infection and acetaminophen (APAP) treatment\u003c/h2\u003e \u003cp\u003eMain cytokines involved in the resolution of infection (IL-12 and IFN γ) were quantified in splenocyte cultures after activation with specific antigens of \u003cem\u003eT. gondii\u003c/em\u003e. All samples from control non-infected group were under the detection threshold for the cytokines tested.\u003c/p\u003e \u003cp\u003eNo significant difference in IFN-γ and IL-6 concentrations in the groups treated with APAP compared to the infected group have been shown, regardless the number of cysts (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA, C, D, F). For lethal dose groups, IL-12 concentrations were higher in the group treated with APAP in Gel Water compared to the infected group without treatment (P \u0026lt; 0.05) but not found in the group treated by gavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). There was no significant difference in IL-12 concentrations between groups infected with 15 cysts (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe average percentages of CD4+ (T lymphocytes), CD8+ (T lymphocytes), CD19+ (B lymphocytes) and F4/80+ (macrophages) populations within the splenocytes were analyzed. In the mice infected with 120 cysts, the percentage of F4/80 + cells increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA), whereas the percentage of CD4+ (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC) and CD19+ (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eG) cells decreased. When infected mice were treated with APAP, the percentage of F4/80+, CD4 + and CD19 + cells did not change, whereas the percentage of CD8 + cells decreased, and significantly for the treatment by gavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eE). In the mice infected with 15 cysts, the percentage of F4/80 + cells also increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB) and the percentage of CD4 + cells also decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eD). In contrast, the percentage of CD8 + cells decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eF) and the percentage of CD19 + cells was unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eH). The treatment with APAP showed an impact only on the increase of F4/80 + cells, which was not so important as in the non-treated group, and only in the case of administration by gavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, the treatment had a minor impact on population distribution after infection.\u003c/p\u003e \u003cp\u003eSpecific IgG were detected in the sera collected at different times during the experiment to observe seroconversion kinetics following infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA et 11B). A significant increase in optical density resulting from infection at D11. No difference was observed between the groups treated with APAP and the untreated group, regardless of the infectious dose administered, showing that APAP had a weak impact on the humoral immune response. Antibody titers have also been performed to get quantitative data and no significant difference between groups were observed (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eParasite distribution and dissemination were followed by qPCR in brain and lungs. No significant difference between infected groups and infected groups that received the APAP-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eA, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eB) during the acute phase of infection was observed. In the lungs of the mice in chronic phase, no difference was detected between groups by qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eC). Brain cysts of the mice were counted in order to determine the parasite load at the end of the chronic phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eD). The values illustrate high variability between individuals. However, no difference on encystment between the infected and treated groups was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":"\u003cp\u003ePressure around animal experimentation is growing and the most invasive procedures are likely to be controversial and criticized. Animal models are essential in vaccine and therapeutic development against infectious organisms. However, the impact of infections on animals is sometimes severe and leads to problematic clinical signs from an ethical point of view (prostration, long-term hyperthermia, dehydration, even mortality\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The use of pharmacological tools to relieve pain remain underutilized in research rodents especially in infectious model despite the general acceptance of both the ethical imperative and regulatory requirements\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In way to optimize the conduct of experiments and increase their acceptability in compliance with regulations in animal experimentation focusing on one of the 3Rs, the refinement\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, we used APAP as a new therapeutic refinement tool to relieve the mice in an infectious model of acute and chronic toxoplasmosis.\u003c/p\u003e\u003cp\u003eIn the present study, analyzes of the toxicity of APAP showed that its administration had no impact on weight, temperature and behavior of the mice during the 5 days of treatment. Two routes of oral administration were used by gavage and by consumption of Gel Water. APAP is administered orally, as its bioavailability and pharmacokinetics are documented by this route of administration and simpler for use in experimental conditions. This mode of administration has been widely validated by studies on the efficacy of APAP\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. No dehydration of the mice was observed when APAP was given in Gel Water suggesting and confirms the previous data\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Biochemical serum analyzes (ALP, AST, ALAT, urea, glucose) showed that no toxicity was observable. In the case of APAP toxicity, transaminases levels as well as kidneys biomarkers, could increase because of hepatocytes lysis, which was not the case here\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. These results confirm data published on different rodent models, showing that this dose of 30 mg/kg/day is not toxic during 5 consecutive days of treatment\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The weighing of the spleens and the number of splenocytes in non-infected mice were significantly different in the groups having been treated \u003cem\u003evia\u003c/em\u003e Gel Water, only for the one with APAP. However, administration of APAP by gavage had no effect on splenocytes number or spleen weight. Gel Water is composed of 99% water. However, it can be assumed that other components of the Gel such as hydrocolloids (texturizing agent) or fibers (\u0026lt; 1.8%) could have an immunostimulatory effect\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. No toxicity could be observed after histological analyzes on target organs (spleen, liver, kidneys, stomach). This lack of toxicity for this APAP dose is consistent with current published data\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. IL-6 is known to have a dual role in fever regulation and hepatotoxicity after APAP overdose\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Serum concentrations of IL-6 were under the detection limit in all groups. The lack of IL-6, for this dose, confirmed that there is no hepatotoxicity and agrees with published data\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, which shows the importance of IL-6 in the regeneration of hepatocytes in case of deregulation and abnormal functioning of the liver.\u003c/p\u003e\u003cp\u003eAfter \u003cem\u003eT. gondii\u003c/em\u003e infection, the clinical signs already known in toxoplasmosis were recorded (prostration, thermal peaks, face tightness, ears in back position\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e). Although this had never been described in the literature for this infectious model of toxoplasmosis, hypothermia is the most commonly reported predictor of mice imminent death in several infectious model\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. APAP treatment was not sufficient to suppress hyperthermia but it was significantly slowed down from the third day of treatment, mainly for mice treated by gavage.\u003c/p\u003e\u003cp\u003eThe first clinical signs observed from the fifth day post-infection were the onset of weight loss. The treatment made it possible to reduce weight loss, probably due to a general improvement of well-being for the mice infected with the lethal dose of cysts. This was not so obvious for the mice infected with the lowest dose because the weight loss was lower and delayed.\u003c/p\u003e\u003cp\u003eOther symptoms appeared such as prostration, disheveled hair, tightness of the face and the shape of the eyes, lack of activity. Self-medication with APAP in Gel Water does not seem to have a noticeable effect on the behavior of mice which may be explained by a too low daily Gel consumption by the infected mice, especially with the lethal dose of cysts. However, mice infected with 15 cysts were able to ingest enough Gel Water with APAP to get an improvement in their well-being. The dose of APAP could be increased to 100 mg/kg/day as described in various studies\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, but after 5 consecutive days, this dose has been shown to induce hepatic toxicity in rodents and pigs\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe detection of pain mild to moderate is difficult to record in mice. Burrowing performance have proved valuable tools to assess brain damage or malfunction\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. This behavior is reduced by pain and stress, suggesting its use as behavioral parameter to assess general well-being in mice\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. As behavior can be observed easily in a non-invasive manner it has been suggested as a relevant approach to assess both pain severity and the efficiency of pain management drugs. We used the Marble Burying Test to evaluate pain and pain relief after treatment.\u003c/p\u003e\u003cp\u003eThis test demonstrated a strong decrease in the activity of stressed or prostrated mice due to a general unease associated with the temperature variation during both acute and chronic phases of toxoplasmosis. These are completely consistent with the clinical signs observed. Other types of behavior tests could be used, for example the cross maze which could be used to study the discovery of a new environment like the Marble Burying test does, although the latter makes it easier to quantify anxiety\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. On the other hand, in our study, a clear significant improvement was observed in the clinical signs and the general behavior of the mice on the 3rd day of treatment with APAP for both infectious doses. The hypothesis is that these two parameters can be related. Indeed, clinical score and behavior tests can be linked because a mouse with reduced mobility due to symptoms will naturally bury fewer marbles compared to a healthier mouse. A higher score of the Marble Burying test seems to illustrate an increase in the locomotion of the mice in relation to the general well-being effect induced by APAP, which also allowed the mice to be able to continue eating and hydrate (decrease of weight loss in mice treated by gavage) for both infectious doses. Similar results were found in the literature, on a non-parasitic mouse model\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. This is a positive point for the use of APAP by gavage mainly to relieve the animal's discomfort. These first results are completely innovative in murine toxoplasmosis with these behavioral approaches in an infectious process and in agreement with the known effect of APAP on the general improvement of the individual\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. To date, only one publication describes the use of buprenorphine to relieve pain and distress after acute \u003cem\u003eT. gondii\u003c/em\u003e infection\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. They only observed time to death and relief of pain distress. They don’t look at parasite dissemination nor immune response.\u003c/p\u003e\u003cp\u003eAnalysis of immune populations (CD4+, CD8 + and CD19 + lymphocytes, macrophages), did not reveal any significant differences with the exception of CD8 + T cells, which were lower in the groups treated with APAP by gavage compared to control infected group. Lymphocyte depletion in lymphoid organs due to APAP has already been observed, but at high dose and it was associated with hepatotoxicity\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Cytokines of interest were measured: IL-12 and IFN-γ as primary responses to \u003cem\u003eT. gondii\u003c/em\u003e infection, IL-6 for its role in fever regulation and hepatotoxicity. Concentrations of these cytokines after stimulation of the splenocytes were slightly modified. A non-significant decrease in the IL-6 concentration in spleen could be observed in the APAP-gavage treated group. Fever is partly regulated by IL-6\u003csup\u003e20,21\u003c/sup\u003e. However, APAP is an antipyretic and even if it has no direct effect on the amounts of IL-6, it could induce a change in its concentration\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. IL12 concentration was also significantly increased in the group treated by APAP in Gel Water in the group infected with the lowest dose of cysts. This may be consistent with the observation of a potential immunostimulatory effect due to the component of the gel\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Specific serum IgG were not modified for both infectious doses by treatment with APAP at the reference dose of 30 mg/kg/day. This shows that APAP had a weak impact on the cellular and humoral immune responses in our study conditions.\u003c/p\u003e\u003cp\u003eParasite load was assessed using qPCR for organs obtained at the end of acute phase (lungs, brain) and at the end of the chronic phase (lung) and also by counting the brain cysts. No significant difference in parasite load has been observed in both tissues regardless of the treatment. Related to brain and chronic phase behavior, no difference in cyst number was observed, indicating that APAP potentially improved behavior by reducing symptoms without affecting parasite load in the mice, showing a very limited impact on the physiopathology. Finally, the histological analysis showed no difference in the tissue organization of the target organs due to the treatment, in particular the hippocampus involved in the behavior and the exploration of a new environment. Since \u003cem\u003eT. gondii\u003c/em\u003e is known to induce changes in behavior when installed in the brain, it was interesting to study the relation between APAP-treatment and behavior. Indeed, it was important that improvement in behavior during both phases of the infection was due to APAP reducing the severity of symptoms rather than by modifying the parasite distribution, using histological data obtained, especially in the brain. It is also important to note that APAP’s mode of action could be linked with serotonin levels\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, one of the main mediators of anxiety in the central nervous system\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. As for the distribution of cysts in the brain, we might have expected to detect fewer of these cystic structures impacting behavior following treatment with APAP. A homogeneous distribution of cysts also seems to be preserved under our experimental conditions and according to brain tissue analysis, consistent with the literature\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAcetaminophen (APAP) administered by gavage appears to be a good pharmacological tool to relieve an animal infected with \u003cem\u003eToxoplasma gondii\u003c/em\u003e and therefore refine infectious process by improving well-being, as shown here on the model species. Our project therefore involved highlighting the combination of individual or collective markers specific to toxoplasmosis in mice (zootechnical, behavioral, immune response, parasite dissemination). The application of APAP during the acute phase did not modify all these parameters linked to the pathophysiology of murine toxoplasmosis. However, this treatment did significantly improve the general condition of the mice, thus contributing to the animal's well-being during experimentation. These initial results confirm our hypothesis and validate our proof of concept, i.e. that APAP at this reference dose and in our infectious model, is a new pharmacological tool for experimentation in infectiology to improve animal welfare. From a scientific and academic point of view, these research studies will also lead to a better understanding of the mode of action of APAP in veterinary medicine and in host-pathogen interactions, with no change in initial pathophysiology and improving the welfare of animals. This work will perhaps make fall a dogma on the use of APAP in veterinary health from animal experiments to breeding.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declared no potential conflicts of interest with respect to research, authorship and/or publication of the article. The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author\u003c/p\u003e\u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eExperiments were carried out according to EU directives and French regulations (Directive 2010/63 / EU, 2010; Rural Code, 2018; Decree n \u0026deg; 2013\u0026thinsp;\u0026minus;\u0026thinsp;118, 2013, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.legifrance.gouv.fr/loda/id/JORFTEXT000027038013/\u003c/span\u003e\u003cspan address=\"https://www.legifrance.gouv.fr/loda/id/JORFTEXT000027038013/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. ).\u003c/p\u003e \u003cp\u003eAll experimental procedures have been evaluated and approved by the Ministry of Higher Education and Research (APAFIS # 2018021917268751.V3\u0026ndash;13634). The procedures involving mice were evaluated by the Val de Loire ethics committee (CEEA VdL, committee number 19) and took place at the INRAE Platform for Experimental Infection PFIE (UE-1277 PFIE, INRAE Centre-Val-de Loire) Valley research, Nouzilly, France, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15454/1.5535888072272498e12\u003c/span\u003e\u003cspan address=\"10.15454/1.5535888072272498e12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: the European Union\u0026rsquo;s Horizon 2020 Research and Innovation Programme (grant N\u0026deg; 731014, VetBioNet, Veterinary Biocontained facility Network to Sasha Trapp and Fr\u0026eacute;d\u0026eacute;ric Lantier), the grants allocated by the Animal Health Department and from INRAE\u0026rsquo;s own funds.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, MR, CR, NM and IDP; data curation, MR, NM; formal analysis, MR, AC, CR, NM; funding acquisition, MR and NM; investigation, MR, AC, CR, CoB, LaM, EL, NM and FDG; methodology, MR, AC, CR, CoB, LaM, EL, NM, FDG; project administration, MR, NM; resources, MR, AC, CR, CoB, LaM, EL, NM and IDP; supervision, MR, NM; validation, MR, NM and visualization, MR, NM and IDP; writing\u0026ndash;original draft, AC, MR, CR, NM, FDG; writing\u0026ndash;review \u0026amp; editing, MR, CR, NM and FDG. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003e We would like to thank the different partners that supported this project: the society Biolog-animal (Philippe Cordier, telemetric system), the SAFE group (Gel Water and use the acetaminophen in Gel Water), CEVA France for supplying Prac\u0026eacute;tam\u0026reg; (veterinary Acetaminophen) and the local ethical committee in Loire Valley (number 19). We are also grateful to the direction of PFIE (Stephane Abrioux) to accept this project and more particularly, for the experimental step, the staff (mice team) of the Experimental Infectiology Platform (UE-1277 PFIE, INRAE Centre de Recherche Val de Loire, Nouzilly, France, https://doi.org/10.15454/1.5535888072272498e12). We are also grateful to Dr. Caroline Prouillac for her participation in the pharmacologic aspects and preparation of APAP of these experiments and Sybille Brochard and Marion Guionni\u0026egrave;re for her technical assistance. We would also like to thank Pr. Alice de Boyer des Roches (UMR Herbivores, INRAE) and Dr. Dominique Autier-Derian (Animal-welfare-consulting company) for their expertise in this project. PFIE is part of EMERG\u0026rsquo;IN, the national infrastructure for the control of animal and zoonotic emerging infectious diseases through in vivo investigation.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.All data generated or analysed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRussell, W. M. S. \u0026amp; Burch, R. L. 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Immun.\u003c/em\u003e \u003cb\u003e80\u003c/b\u003e, 88\u0026ndash;108 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"welfare, acetaminophen, toxoplasmosis, mice, behavior, immune response","lastPublishedDoi":"10.21203/rs.3.rs-5790171/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5790171/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOver the past few years, animal welfare has taken more place in society and especially in scientific research. It has become necessary to refine the experimental procedures as much as possible in infectiology as in our reference model, toxoplasmosis, in agreement with the 3Rs rule and different ethical concerns. Thus, the establishment of a treatment using analgesics would relieve animals acutely infected with \u003cem\u003eToxoplasma gondii\u003c/em\u003e. However, the use of this analgesic should in no way alter the pathophysiology of the disease and the immune response of the host, so as not to interfere with the initial scientific study. Currently, little is known about the use of acetaminophen in an infectious model. In the present work, we studied the impact of acetaminophen at a reference dose of 30 mg/kg/day in a murine model of acute toxoplasmosis. To do this, zoonotic, telemetric, behavioral, histological and immune parameters were analyzed to better characterize the consequences of a treatment with acetaminophen either \u003cem\u003evia\u003c/em\u003e gavage or \u003cem\u003evia\u003c/em\u003e self-medication in Gel Water. Acetaminophen administered by gavage did not induce cellular or tissue toxicity and did not alter the physiological development of mice either. Moreover, the very nature of Gel Water, independently of APAP, has had an impact on the immune response. The acetaminophen improved the general well-being and slowed down the appearance of clinical signs without modifying the physiopathology or the immune responses induced by \u003cem\u003eT. gondii\u003c/em\u003e. These first results in mice validated our initial hypothesis that acetaminophen appears to be a pharmacological tool to refine and improve animal welfare during the acute phase of toxoplasmosis. Therefore, our project has highlighted the combination of specific markers to contribute to animal welfare in mice. In the long term, the use of acetaminophen could be extended to other infectious models with other target animal species.\u003c/p\u003e","manuscriptTitle":"Acetaminophen, a new tool to refine experimental infectious processes: the case of murine toxoplasmosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-20 15:55:58","doi":"10.21203/rs.3.rs-5790171/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-13T03:33:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-10T19:42:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-06T18:04:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"155520345591301204884469867958089224984","date":"2025-01-27T16:10:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59460445684922169765151314229714541352","date":"2025-01-25T12:36:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-25T07:28:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-25T07:26:17+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-01-16T14:43:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-15T12:48:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-01-08T14:52:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"07927869-993e-4384-a4d5-db522666590c","owner":[],"postedDate":"January 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":42973349,"name":"Health sciences/Pathogenesis/Immunopathogenesis/Adaptive immunity"},{"id":42973350,"name":"Health sciences/Pathogenesis/Immunopathogenesis/Innate immunity"},{"id":42973351,"name":"Health sciences/Pathogenesis/Inflammation/Acute inflammation"},{"id":42973352,"name":"Health sciences/Pathogenesis/Inflammation/Chronic inflammation"},{"id":42973353,"name":"Health sciences/Health care/Medical ethics"}],"tags":[],"updatedAt":"2025-07-07T16:05:38+00:00","versionOfRecord":{"articleIdentity":"rs-5790171","link":"https://doi.org/10.1038/s41598-025-06849-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-01 15:58:03","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-01-20 15:55:58","video":"","vorDoi":"10.1038/s41598-025-06849-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-06849-2","workflowStages":[]},"version":"v1","identity":"rs-5790171","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5790171","identity":"rs-5790171","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}