Male CD-1 Mice Research Articles

The Role of Nrf2 and Inflammation on the Dissimilar Cardiotoxicity of Doxorubicin in Two-Time Points: a Cardio-Oncology In Vivo Study Through Time

Doxorubicin (DOX) is a topoisomerase II inhibitor used in cancer therapy. Despite its efficacy, DOX causes serious adverse effects, such as short- and long-term cardiotoxicity. This work aimed to assess the short- and long-term cardiotoxicity of DOX and the role of inflammation and antioxidant defenses on that cardiotoxicity in a mice model. Adult CD-1 male mice received a cumulative dose of 9.0 mg/kg of DOX (2 biweekly intraperitoneal injections (ip), for 3 weeks). One week (1W) or 5 months (5M) after the last DOX administration, the heart was collected. One week after DOX, a significant increase in p62, tumor necrosis factor receptor (TNFR) 2, glutathione peroxidase 1, catalase, inducible nitric oxide synthase (iNOS) cardiac expression, and a trend towards an increase in interleukin (IL)-6, TNFR1, and B-cell lymphoma 2 associated X (Bax) expression was observed. Moreover, DOX induced a decrease on nuclear factor erythroid-2 related factor 2 (Nrf2) cardiac expression. In both 1W and 5M, DOX led to a high density of infiltrating M1 macrophages, but only the 1W-DOX group had a significantly higher number of nuclear factor κB (NF-κB) p65 immunopositive cells. As late effects (5M), an increase in Nrf2, myeloperoxidase, IL-33, tumor necrosis factor-α (TNF-α), superoxide dismutase 2 (SOD2) expression, and a trend towards increased catalase expression were observed. Moreover, B-cell lymphoma 2 (Bcl-2), cyclooxygenase-2 (COX-2), and carbonylated proteins expression decreased, and a trend towards decreased p38 mitogen-activated protein kinase (MAPK) expression were seen. Our study demonstrated that DOX induces adverse outcome pathways related to inflammation and oxidative stress, although activating different time-dependent response mechanisms.Graphical

SAT047 Regulation Of The Insulin Receptor And The Insulin-like Growth Factor 1 Receptor By miR-322-5p, A miR-16 Family Member, In Hypothalamic Neuronal Models

Abstract Disclosure: W. He: None. N. Loganathan: None. K.W. Mak: None. E. McIlwraith: None. D.D. Belsham: None. Insulin signals through the insulin receptor (INSR) and the insulin-like growth factor 1 receptor (IGF1R) in hypothalamic neurons to control food intake and peripheral metabolism. A contributor to obesity, and subsequent comorbidities such type 2 diabetes and heart disease, is the development of cellular insulin resistance in hypothalamic neurons. However, the molecular changes to neuronal insulin signaling remain to be fully elucidated. MicroRNAs (miRNAs) inhibit translation of specific mRNAs; thus, this study aimed to understand the involvement of miRNAs in the regulation of insulin signaling and resistance in hypothalamic neurons. To profile miRNAs expressed in hypothalamic neurons, RNA from the whole hypothalami of 14-week-old CD1 male mice (n = 4), as well as the immortalized hypothalamic neuronal cell lines mHypoE-46 (n = 3) and mHypoA-59 (n = 3), each co-expressing neuropeptide Y (NPY) and agouti-related peptide (AgRP), were assessed using the Affymetrix GeneChip miRNA 4.0 Array. Notably, miR-16 family members, including miR-16-5p, miR-15b-5p, and miR-322-5p, were among the most highly expressed miRNAs in each case. In the mHypoA-59 neurons, overexpression of miR-322-5p for 24 hours decreased the mRNA levels of Igf1r (-26.1%; p = 0.0007; n = 4) and the protein level of INSR-beta (-31.8%; p = 0.0023). These results suggest that the miR-16 family plays an inhibitory role in insulin signaling in hypothalamic neurons. To study the involvement of miRNAs in hyperinsulinemia-induced neuronal insulin resistance, the mHypoE-46 neurons were treated with 100 nM insulin for 24 hours to induce cellular insulin resistance, as characterized by decreased INSR-beta protein (-96.3%; p < 0.0001; n = 4) and a resulting decrease in insulin-induced phosphorylation of protein kinase B (AKT) (-67.6%; p = 0.01; n = 4). GeneChip miRNA array analysis showed that insulin overexposure disrupted the expression of 48 miRNAs (p < 0.05, n = 3), including the upregulation of miR-18a-3p, mir-322, miR-494-3p, and miR-671-3p. Upon RT-qPCR validation (n = 3), we established that prolonged (24 hours), but not acute (1-6 hours), insulin exposure upregulated miR-18a-3p (+62%; p = 0.0104), miR-671-3p (+111%; p = 0.0079), and miR-1983 (+97%; p = 0.0181). miR-671-3p, miR-494-3p, and miR-18a-3p have been shown to decrease phosphatase and tensin homolog (PTEN) levels, which can promote neuronal insulin resistance based on bioinformatic analysis. Overall, these results suggest hyperinsulinemia disrupts the expression of specific miRNAs in hypothalamic neurons to promote cellular insulin resistance. Knowledge derived from these studies will provide insight into hypothalamus-derived miRNAs that could be targeted for miRNA-based diagnostics and therapeutics for early central insulin resistance in humans.(Supported by the CRC, CIHR, NSERC, EndoSoc, and BBDC) Presentation: Saturday, June 17, 2023

FRI443 Optimizing Conditions Of Tubule-like Structures In Testicular Organoids That Will Support Spermatogenesis

Abstract Disclosure: O. Printy: None. Testicular organoids are increasingly being investigated as a potential means of fertility restoration. These organoids have been shown to self-organize to recapitulate the inner architecture of the native testis, forming tubule-like structures that resemble the seminiferous tubules. However, these tubule-like structures lack dimensionality and do not contain a large, meiotically active germ cell population. We hypothesized that if testicular organoids are cultured under appropriate microenvironmental conditions, we will observe the development of tubule-like structures with diameters that resemble what is seen in vivo, in addition to appropriate somatic cell localization and Sertoli-germ cell interactions. To test this hypothesis, we generated organoids from 5dpp male CD1 mice at three cellular seeding densities: low density (170,000 cells per well), medium density (280,000 cells per well), and high density (310,000 cells per well). The heterogenous testicular cell suspension formed compact organoids at all three seeding densities and organoid diameter increased with increasing cell number. To assess the ability of the organoids to mimic the inner architecture of a testis, we performed immunohistochemistry on 14-day-old organoids: for the major somatic cell types of the testis Leydig cells (HSD3B2), peritubular myoid cells (ACTA1), and Sertoli cells (SOX9). Thus far, only medium density organoids appear to recapitulate the compartmentalization seen in native testes. These organoids tend to contain semi-continuous ring-like structures composed exclusively of ACTA1positive cells; these structures encapsulate a population of SOX9-positive cells, while HSD3B2-expressing cells are located on the periphery of the organoids. To assess the organoids’ ability to fuse to form a larger aggregate, organoids were transferred to 3D printed troughs. However, organoids did not merge and appeared to die. However, when organoids are cultured together in microcentrifuge tubes and alginate structures, they fused to form a larger aggregate. In the future, immunohistochemistry will be used to evaluate whether any extended tubule-like structures formed within the aggregates. Additionally, spermatogonial stem cells will be injected into the aggregates and ELISA assays will be performed to assess the spermatogenic and hormonal functionality of the organoids. Presentation: Friday, June 16, 2023

Ultrastructural alterations due to sodium metavanadate treatment in the blood stages of Plasmodium yoelii yoelii

Malaria is a potentially mortal disease caused by parasites of the genus Plasmodium spp. It has a wide distribution in the world and unfortunately there are several factors that make its control difficult; among which the development of pharmacological resistance to the different drugs used to treat this disease stands out, which makes it necessary to design new compounds that have an antimalarial effect. Previous studies have shown that vanadium has a broad antiparasitic spectrum and is also safe for the host, so the objective of this research was to evaluate the antimalarial potential of sodium metavanadate (SM) and to analyze the ultrastructural changes in parasites exposed. The method consisted of inoculating CD-1 male mice with Plasmodium yoelii yoelii and administering a 10 mg/kg/day dose of SM orally for 4 days. On the fifth day, whole blood samples were obtained, processed for ultrastructural analysis, and the changes in the different parasite stages were compared against the control. Our results showed that SM decreased parasitemia compared to the group that did not receive treatment and modified the ultrastructure in all parasitic stages because it damaged the membranes, causing alterations mainly in the nucleus and in the mitochondria as well as the loss of cellular organization, which could affect the integrity of these parasites and decrease its viability.

Neurofilament light chain in plasma as a sensitive diagnostic biomarker of peripheral neurotoxicity: In Vivo mouse studies with oxaliplatin and paclitaxel - NeuroDeRisk project

Identifying compounds that are neurotoxic either toward the central or the peripheral nervous systems (CNS or PNS) would greatly benefit early stages of drug development by derisking liabilities and selecting safe compounds. Unfortunately, so far assays mostly rely on histopathology findings often identified after repeated-dose toxicity studies in animals. The European NeuroDeRisk project aimed to provide comprehensive tools to identify compounds likely inducing neurotoxicity. As part of this project, the present work aimed to identify diagnostic non-invasive biomarkers of PNS toxicity in mice. We used two neurotoxic drugs in vivo to correlate functional, histopathological and biological findings. CD1 male mice received repeated injections of oxaliplatin or paclitaxel followed by an assessment of drug exposure in CNS/PNS tissues. Functional signs of PNS toxicity were assessed using electronic von Frey and cold paw immersion tests (oxaliplatin), and functional observational battery, rotarod and cold plate tests (paclitaxel). Plasma concentrations of neurofilament light chain (NF-L) and vascular endothelial growth factor A (VEGF-A) were measured, and histopathological evaluations were performed on a comprehensive list of CNS and PNS tissues. Functional PNS toxicity was observed only in oxaliplatin-treated mice. Histopathological findings were observed dose-dependently only in paclitaxel groups. While no changes of VEGF-A concentrations was recorded, NF-L concentrations were increased only in paclitaxel-treated animals as early as 7 days after the onset of drug administration. These results show that plasma NF-L changes correlated with microscopic changes in the PNS, thus strongly suggesting that NF-L could be a sensitive and specific biomarker of PNS toxicity in mice.

Comprehensive evaluation of the pharmacological and toxicological effects of γ-valerolactone as compared to γ-hydroxybutyric acid: Insights from in vivo and in silico models

Γ-valerolactone (GVL), marketed online as “Tranquilli-G” and “excellent Valium”, is used as a legal substitute for γ-hydroxybutyric acid (GHB); however, until now, GVL has only been connected to one Drug-Facilitated Sexual Assault (DFSA) case. Moreover, the pharmaco-toxicological effects of GVL are poorly studied. The aim of this study was to investigate the 1) in vivo effects of gavage administration of GVL (100–3000 mg/kg) on neurological (myoclonia, convulsions), sensorimotor (visual, acoustic, and overall tactile) responses, righting reflex, thermoregulation, motor activity (bar, drag, and accelerod test) and cardiorespiratory changes (heart rate, breath rate, oxygen saturation, and pulse distension) in CD-1 male mice and the 2) in silico ADMET profile of GVL in comparison to GHB and the open active form γ-hydroxyvaleric acid (GHV). The present study demonstrates that GVL inhibits, in a dose-dependent manner, sensorimotor and motor responses and induces cardiorespiratory depression (at a dose of 3000 mg/kg) in mice. The determination of the ED50 in sensorimotor and motor responses revealed that GVL is about 4–5 times less potent than GHB. In silico prediction of ADMET profiles revealed toxicokinetic similarities between GHB and GHV, and differences with GVL. These results suggest that GVL could be used as a substitute for GHB and should be added to forensic toxicology screenings.

The role of inflammation and antioxidant defenses in the cardiotoxicity of doxorubicin in elderly CD-1 male mice

Doxorubicin (DOX) is a potent chemotherapeutic agent used against several cancer types. However, due to its cardiotoxic adverse effects, the use of this drug may be also life-threatening. Although most cancer patients are elderly, they are poorly represented and evaluated in pre-clinical and clinical studies. Considering this, the present work aims to evaluate inflammation and oxidative stress as the main mechanisms of DOX-induced cardiotoxicity, in an innovative approach using an experimental model constituted of elderly animals treated with a clinically relevant human cumulative dose of DOX. Elderly (18–20 months) CD-1 male mice received biweekly DOX administrations, for 3 weeks, to reach a cumulative dose of 9.0 mg/kg. One week (1W) or two months (2 M) after the last DOX administration, the heart was collected to determine both drug’s short and longer cardiac adverse effects. The obtained results showed that DOX causes cardiac histological damage and fibrosis at both time points. In the 1W-DOX group, the number of nuclear factor kappa B (NF-κB) p65 immunopositive cells increased and a trend toward increased NF-κB p65 expression was seen. An increase of inducible nitric oxide synthase (iNOS) and interleukin (IL)-33 and a trend toward increased IL-6 and B-cell lymphoma-2-associated X (Bax) expression were seen after DOX. In the same group, a decrease in IL-1β, p62, and microtubule-associated protein 1A/1B-light chain 3 (LC3)-I, p38 mitogen-activated protein kinase (MAPK) expression was observed. Contrariwise, the animals sacrificed 2 M after DOX showed a significant increase in glutathione peroxidase 1 and Bax expression with persistent cardiac damage and fibrosis, while carbonylated proteins, erythroid-2-related factor 2 (Nrf2), NF-κB p65, myeloperoxidase, LC3-I, and LC3-II expression decreased. In conclusion, our study demonstrated that in an elderly mouse population, DOX induces cardiac inflammation, autophagy, and apoptosis in the heart in the short term. When kept for a longer period, oxidative-stress-linked pathways remained altered, as well as autophagy markers and tissue damage after DOX treatment, emphasizing the need for continuous post-treatment cardiac monitoring.

Comparison of Systemic Extended-release Buprenorphine and Local Extended-release Bupivacaine-Meloxicam as Analgesics for Laparotomy in Mice.

Extended-release (ER) local anesthetics can be used in multi-modal analgesia or in situations in which systemic analgesics may alter animal physiology and thus introduce interpretational confounds. In this study, we compared the analgesic efficacy of an ER buprenorphine formulation with that of a synergistic combination of ER bupivacaine and meloxicam. Female and male CD1 mice were randomly assigned to receive subcutaneous buprenorphine (3.25mg/kg) preemptively, subcutaneous infiltration of bupivacaine???meloxicam (0.03mL at incision closure (bupivacaine, 35mg/kg; meloxicam, 1mg/kg), or saline (10mL/kg SC) after induction of anesthesia. After laparotomy, mice were assessed for changes in daily body weight, rearing frequency, nest consolidation scores, time-to-integrate-nest test (TINT), and response to von Frey testing at 4, 8, 24, 48, and 72h after surgery. Daily weight, nest consolidation scores and rearing frequency were not significantly different among the 3 groups. TINT had fallen significantly response at 24 and 48h after injection in the ER buprenorphine group as compared with the saline and ER bupivacaine-meloxicam groups. Nociceptive thresholds, as assessed with von Frey testing, differed between saline controls and both analgesic groups at 4, 8, 24, 48, and 72 h after surgery. None of the mice in the bupivacaine???meloxicam group developed signs of neurotoxicity, a potential side effect of high-dose local anesthetics. This study demonstrates that local ER bupivacaine???meloxicam may be a useful alternative to systemic, ER buprenorphine for the relief of pain after laparotomy in mice.

Hydroxytyrosol produced by engineered Escherichia coli strains activates Nrf2/HO-1 pathway: An in vitro and in vivo study.

This study explores the biological effects of hydroxytyrosol (HT), produced by the metabolic engineering of Escherichia coli, in a series of in vitro and in vivo experiments. In particular, a metabolically engineered Escherichia coli strain capable of producing HT was constructed and utilized. HEK293 and HeLa cells were exposed to purified HT to determine non-toxic doses that can offer protection against oxidative stress (activation of Nrf2/HO-1 signaling pathway). Male CD-1 mice were orally supplemented with HT to evaluate (1) renal and hepatic toxicity, (2) endogenous system antioxidant response, and (3) activation of Nrf2/HO-1 system in the liver. HT protected cells from oxidative stress through the activation of Nrf2 regulatory network. Activation of Nrf2 signaling pathway was also observed in the hepatic tissue of the mice. HT supplementation was safe and produced differential effects on mice's endogenous antioxidant defense system. HT biosynthesized from genetically modified Escherichia coli strains is an alternative method to produce high-quality HT that exerts favorable effects in the regulation of the organism's response to oxidative stress. Nonetheless, further investigation of the multifactorial action of HT on the antioxidant network regulation is needed.

A Clinically Relevant Dosage of Mitoxantrone Disrupts the Glutathione and Lipid Metabolic Pathways of the CD-1 Mice Brain: A Metabolomics Study.

Long-term cognitive dysfunction, or "chemobrain", has been observed in cancer patients treated with chemotherapy. Mitoxantrone (MTX) is a topoisomerase II inhibitor that binds and intercalates with DNA, being used in the treatment of several cancers and multiple sclerosis. Although MTX can induce chemobrain, its neurotoxic mechanisms are poorly studied. This work aimed to identify the adverse outcome pathways (AOPs) activated in the brain upon the use of a clinically relevant cumulative dose of MTX. Three-month-old male CD-1 mice were given a biweekly intraperitoneal administration of MTX over the course of three weeks until reaching a total cumulative dose of 6 mg/kg. Controls were given sterile saline in the same schedule. Two weeks after the last administration, the mice were euthanized and their brains removed. The left brain hemisphere was used for targeted profiling of the metabolism of glutathione and the right hemisphere for an untargeted metabolomics approach. The obtained results revealed that MTX treatment reduced the availability of cysteine (Cys), cysteinylglycine (CysGly), and reduced glutathione (GSH) suggesting that MTX disrupts glutathione metabolism. The untargeted approach revealed metabolic circuits of phosphatidylethanolamine, catecholamines, unsaturated fatty acids biosynthesis, and glycerolipids as relevant players in AOPs of MTX in our in vivo model. As far as we know, our study was the first to perform such a broad profiling study on pathways that could put patients given MTX at risk of cognitive deficits.

Sex and age differences in mice models of effort-based decision-making and anergia in depression: the role of dopamine, and cerebral-dopamine-neurotrophic-factor.

Mesolimbic dopamine (DA) regulates vigor in motivated behavior. While previous results have mainly been performed in male rodents, the present studies compared CD1 male and female mice in effort-based decision-making tests of motivation. These tests offered choices between several reinforcers that require different levels of effort (progressive ratio/choice task and 3-choice-T-maze task). Sweet reinforcers were used in both tasks. In the operant tasks, females worked harder as the task required more effort to access a 10% sucrose solution. Although males and females did not differ in preference for 10% vs 3% solutions under free concurrent presentation, females consumed more of the 10% solution when tested alone. The operant task requires a long period of training and changes in the DA system due to age can be mediating long-term changes in effort. Thus, age and sex factors were evaluated in the T-maze task, which requires only a short training period. Both sexes and ages were equally active when habituated to the running wheel (RW), but females consumed more sweet pellets than males, especially at an older age. Both sexes had a strong preference for the RW compared to more sedentary reinforcers in the 3-choice-T-maze test, but older animals spent less time running and ate more than the young ones. The DA-depleting agent tetrabenazine reduced time running in older mice but not in adolescents. Cerebral-dopamine-neurotrophic-factor was reduced in older mice of both sexes compared to adolescent mice. These results emphasize the importance of taking into account differences in sex and age when evaluating willingness to exert effort for specific reinforcers.

STRUCTURAL REACTIONS OF CD-1 MICE HEART AND LIVER TO A SINGLE INJECTION OF BOROCAPTATE SODIUM

Boron neutron capture therapy is considered a promising method for the treatment of malignant tumors of the head and neck. It is believed that to increase the effectiveness of this type of therapy, the use of large doses of boron is required, which may entail damaging effects on healthy tissue. One of the substances used in the clinical practice of boron neutron capture therapy is sodium boroncaptate Na2B12H11SH (BSH), enriched with the 10B boron isotope. The purpose of the study was to study the structural reactions of the myocardium and liver of CD-1 mice after administration of BSH. A light-optical and polarization-microscopic study of the myocardium and liver of male CD-1 mice (n=56) was carried out after injection of a boron-containing substance in doses of 100 and 1000 mg/kg, once, intraperitoneally. Assessment of structural changes in the myocardium and liver was carried out 1, 3 and 7 days after BSH administration. A single injection of BSH at a dose of 100 mg/kg did not lead to the death of animals, whereas 3 hours after the injection of BSH at a dose of 1000 mg/kg, 1 animal died. The body weight of the animals changed slightly during the experiment. Analysis of heart weight showed a decrease in this indicator on days 3 and 7 compared with indicators in the same groups on day 1 of the experiment. When analyzing the dynamics of changes in liver mass, no significant changes were revealed during the experiment. The main structural changes in the myocardium included lytic and contractural damage to cardiomyocytes, hemodynamic disturbances in the form of pronounced venous and capillary congestion. Liver damage was manifested in dystrophic changes in hepatocytes, the appearance after 3 days of monocellular necrosis of hepatocytes and pericentral mononuclear infiltrates. The data obtained indicate that the used doses of BSH 100 and 1000 mg/kg with a single injection cause structural changes in the myocardium and liver of varying severity, which persist for 7 days of observation.

Lactobacillus Reuteri 6475 Prevents Bone Loss in a Clinically Relevant Oral Model of Glucocorticoid-Induced Osteoporosis in Male CD-1 Mice.

Glucocorticoids (GCs) are commonly used anti-inflammatory medications with significant side effects, including glucocorticoid-induced osteoporosis (GIO). We have previously demonstrated that chronic subcutaneous GC treatment in mice leads to gut barrier dysfunction and trabecular bone loss. We further showed that treating with probiotics or barrier enhancers improves gut barrier function and prevents GIO. The overall goal of this study was to test if probiotics could prevent GC-induced gut barrier dysfunction and bone loss in a clinically relevant oral-GC model of GIO. Eight-week-old male CD-1 mice were treated with vehicle or corticosterone in the drinking water for 4 weeks and administered probiotics Lactobacillus reuteri ATCC 6475 (LR 6475) or VSL#3 thrice weekly via oral gavage. As expected, GC treatment led to significant gut barrier dysfunction (assessed by measuring serum endotoxin levels) and bone loss after 4 weeks. Serum endotoxin levels significantly and negatively correlated with bone volume. Importantly, LR 6475 treatment effectively prevented both GC-induced increase in serum endotoxin and trabecular bone loss. VSL#3 had intermediate results, not differing from either control or GC-treated animals. GC-induced reductions in femur length, cortical thickness, and cortical area were not affected by probiotic treatment. Taken together, these results are the first to demonstrate that LR 6475 effectively prevents the detrimental effects of GC treatment on gut barrier, which correlates with enhanced trabecular bone health in an oral mouse model of GIO. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Efficacy of oltipraz in preventing acetaminophen-induced liver injury in mice.

Oltipraz (OPZ) is a synthetic dithiolethione with potential as a cancer chemopreventive agent, which can work by inducing detoxification enzymes. OPZ is an activator of nuclear factor erythroid 2-related factor 2 (Nrf2), suggesting its involvement in enzyme induction and possible protection against drug-induced liver injury. In this study, we present OPZ-mediated protection of mice against acetaminophen (APAP)-induced liver injury and discuss its possible contributing factors. Overnight-fasted male CD-1 mice were administered APAP intraperitoneally, and some mice were administered OPZ 16h before APAP. Hepatotoxicity was assessed by measuring serum alanine aminotransferase leakage and histopathological evaluation. The hepatic mRNA expressions of CYP2E1, glutamate cysteine ligase (GCL), and NAD(P)H:quinone oxidoreductase (NQO1) were measured by real-time reverse-transcription polymerase chain reaction. OPZ protected mice from APAP-induced liver injury in a dose-dependent manner, but did not alter hepatic glutathione (GSH) content or GCL expression in control mice, indicating that its hepatoprotective effect is not due to changes in basal GSH levels. OPZ did not affect CYP2E1 expression or APAP-induced early GSH depletion, suggesting it does not inhibit the metabolic activation of APAP to produce N-acetyl-p-benzoquinone imine. In contrast, after GSH depletion, OPZ accelerated hepatic GSH recovery. APAP significantly increased GCL expression during liver injury, but OPZ treatment only led to additional NQO1 expression. This suggests that NQO1 is responsible for the enhanced GSH recovery and protection against APAP-induced liver injury seen in OPZ-treated mice. In summary, OPZ protects against APAP-induced liver injury by inducing NQO1 expression and resulting in improved GSH recovery.

ECoG spiking activity and signal dimension are early predictive measures of epileptogenesis in a translational mouse model of traumatic brain injury

The latency between traumatic brain injury (TBI) and the onset of epilepsy (PTE) represents an opportunity for counteracting epileptogenesis. Antiepileptogenesis trials are hampered by the lack of sensitive biomarkers that allow to enrich patient's population at-risk for PTE. We aimed to assess whether specific ECoG signals predict PTE in a clinically relevant mouse model with ∼60% epilepsy incidence. TBI was provoked in adult CD1 male mice by controlled cortical impact on the left parieto-temporal cortex, then mice were implanted with two perilesional cortical screw electrodes and two similar electrodes in the hemisphere contralateral to the lesion site. Acute seizures and spikes/sharp waves were ECoG-recorded during 1 week post-TBI. These early ECoG events were analyzed according to PTE incidence as assessed by measuring spontaneous recurrent seizures (SRS) at 5 months post-TBI. We found that incidence, number and duration of acute seizures during 3 days post-TBI were similar in PTE mice and mice not developing epilepsy (No SRS mice). Control mice with cortical electrodes (naïve, n = 5) or with electrodes and craniotomy (sham, n = 5) exhibited acute seizures but did not develop epilepsy. The daily number of spikes/sharp waves at the perilesional electrodes was increased similarly in PTE (n = 15) and No SRS (n = 8) mice vs controls (p < 0.05, n = 10) from day 2 post-injury. Differently, the daily number of spikes/sharp waves at both contralateral electrodes showed a progressive increase in PTE mice vs No SRS and control mice. In particular, spikes number was higher in PTE vs No SRS mice (p < 0.05) at 6 and 7 days post-TBI, and this measure predicted epilepsy development with high accuracy (AUC = 0.77, p = 0.03; CI 0.5830–0.9670). The cut-off value was validated in an independent cohort of TBI mice (n = 12). The daily spike number at the contralateral electrodes showed a circadian distribution in PTE mice which was not observed in No SRS mice. Analysis of non-linear dynamics at each electrode site showed changes in dimensionality during 4 days post-TBI. This measure yielded the best discrimination between PTE and No SRS mice (p < 0.01) at the cortical electrodes contralateral to injury. Data show that epileptiform activity contralateral to the lesion site has the the highest predictive value for PTE in this model reinforcing the hypothesis that the hemisphere contralateral to the lesion core may drive epileptogenic networks after TBI.

Cognitive dysfunction and impaired neuroplasticity following repeated exposure to the synthetic cannabinoid JWH-018 in male mice.

Psychotic disorders have been reported in long-term users of synthetic cannabinoids. This study aims at investigating the long-lasting effects of repeated JWH-018 exposure. Male CD-1 mice were injected with vehicle, JWH-018 (6mg·kg-1 ), the CB1 -antagonist NESS-0327 (1mg·kg-1 ) or co-administration of NESS-0327 and JWH-018, every day for 7days. After 15 or 16 days washout, we investigated the effects of JWH-018 on motor function, memory, social dominance and prepulse inhibition (PPI). We also evaluated glutamate levels in dialysates from dorsal striatum, striatal dopamine content and striatal/hippocampal neuroplasticity focusing on the NMDA receptor complex and the neurotrophin BDNF. These measurements were accompanied by in vitro electrophysiological evaluations in hippocampal preparations. Finally, we investigated the density of CB1 receptors and levels of the endocannabinoid anandamide (AEA) and 2-arachidonoylglycerol (2-AG) and their main synthetic and degrading enzymes in the striatum and hippocampus. The repeated treatment with JWH-018 induced psychomotor agitation while reducing social dominance, recognition memory and PPI in mice. JWH-018 disrupted hippocampal LTP and decreased BDNF expression, reduced the synaptic levels of NMDA receptor subunits and decreased the expression of PSD95. Repeated exposure to JWH-018, reduced hippocampal CB1 receptor density and induced a long-term alteration in AEA and 2-AG levels and their degrading enzymes, FAAH and MAGL, in the striatum. Our findings suggest that repeated administration of a high dose of JWH-018 leads to the manifestation of psychotic-like symptoms accompanied by alterations in neuroplasticity and change in the endocannabinoid system.

Familiar observers attenuate surgery-induced neuroinflammation and cognitive dysfunction in mice.

Postoperative cognitive dysfunction (POCD) is a common complication associated with poor outcome. Our previous study has shown that living with familiar observers in the same cage reduces anxiety of mice with surgery. Anxiety can impair learning and memory. Thus, this study was designed to determine whether living with familiar observers attenuated the dysfunction of learning and memory of mice with surgery. Six- to eight-week-old CD-1 male mice or 18-month-old C57BL/6 male mice had left carotid artery exposure under isoflurane anesthesia. They lived with non-surgery male mice at 2 (number of surgery mice) to 3 (number of non-surgery mice) ratio or with other surgery mice. Mice were subjected to light and dark box test 3 days after surgery to measure their anxiety levels and novel object recognition and fear conditioning tests from 5 days after surgery to measure their learning and memory. Blood and brain were harvested for biochemical analysis. Living with familiar observers that lived with surgery mice for at least 2 weeks before the surgery and then after surgery reduced the anxiety and dysfunction of learning and memory in young adult male mice. Living with unfamiliar observers that lived with surgery mice after the surgery but not before the surgery did not have those effects on the mice with surgery. Living with familiar observers attenuated learning and memory dysfunction after surgery also in old male mice. Living with familiar observers attenuated inflammatory response in the blood and brain and the activation of the lateral habenula (LHb)-ventral tegmental area (VTA) neural circuitry, which has been shown to be important for POCD. Wound infiltration with bupivacaine attenuated the activation of LHb-VTA. These results suggest that living with familiar observers attenuates POCD and neuroinflammation, possibly via inhibiting the activation of the LHb-VTA neural circuitry.

Sex differences in vanadium inhalation effects in non-ciliated bronchiolar cells.

The non-ciliated bronchiolar cell (NCBC) is responsible for the defense of the lung and responds to negative stimuli such as exposure to toxic pro-oxidant substances, which triggers the hyperproduction and hypersecretion of mucins and CC16 protein. The literature demonstrates that physiological and pathological responses in the lung can be influenced by the organism's sex. The objective of this report was to evaluate response differences to vanadium (V) inhalation in male and female CD-1 mice. Mice were exposed to V for four weeks. Hyperplasia of bronchiolar epithelium, small inflammatory foci and sloughing of the NCBC were observed, without changes between sexes and throughout the exposure time. Mucosecretory metaplasia was found in both males and females, however it was more drastic in males. The expression of CC16 increased in both sexes. This study demonstrated a different susceptibility between male and female mice exposed to V inhalation regarding mucosecretory metaplasia.

P-084 Oral antioxidant supplementation ameliorates the deleterious effects of plasticizers Bisphenol AF (BPAF) and Dibutyl Phthalate (DBP) on mouse sperm DNA integrity

Abstract Study question Do common chemicals in plastic, BPAF and DBP, negatively impact sperm parameters and DNA integrity, and can oral antioxidant supplementation mitigate their impact? Summary answer Short-term, low-dose exposure to BPAF or DBP did not impact testicular/epididymal histology but caudal epididymal sperm parameters, particularly DNA integrity was significantly compromised. What is known already Plasticizers are among the most commonly produced chemicals worldwide with 90% of people showing detectable levels in their urine. As endocrine disruptors (EDCs), they act as estrogen mimetics but can also induce oxidative stress and form DNA adducts. Numerous studies have linked exposure to EDCs to impaired spermatogenesis, sperm DNA damage and male infertility. Limited studies suggest antioxidants may alleviate the reproductive toxicity of EDCs. This study measured the effects of short-term exposures to two common environmental EDCs (BPAF &amp; DBP) on the post-testicular compartment epididymis and investigate the efficacy of an oral antioxidant supplement to safeguard sperm DNA integrity. Study design, size, duration Adult male CD1 mice were exposed to low doses (50mg/kg) of BPAF or DBP with or without co-administration of antioxidant supplementation (Fertilix®) for 14 days and evaluated after euthanasia. Participants/materials, setting, methods All experiments were performed at the University Clermont Auvergne, Clermont-Ferrand, France. Mice were supplemented with BPAF, DBP or/and Fertilix® in their drinking water. The results of exposure of males were evaluated against controls and included: testicular and epididymal histology, epididymal ROS status via 4-HNE levels in caput protein extracts, general semen parameters: (count, motility, viability, acrosome integrity), sperm nuclear/DNA parameters [decondensation (Toluidine blue stain), fragmentation (TUNEL test), oxidation (8-OHdG residue level)]. Main results and the role of chance The histology of the testis and epididymis was unaffected by the exposure conditions used. An oxidative stress situation was evident by the EDC exposures, demonstrated by increased level of 4-HNE adducts in the epididymal caput protein extracts. Viability, total and progressive motility, and acrosome integrity of caudal epididymal spermatozoa remained unaltered in exposed animals compared with controls. Exposure to BPAF but not DBP significantly decreased sperm count. Sperm nuclear/DNA integrity assessed by the percentages of sperm with decondensed and oxidized nuclei was significantly increased in EDC-exposed animals. Sperm DNA fragmentation did not significantly increase in any group of exposed animals. Co-administration with Fertilix® significantly corrected the defective sperm parameters (nuclear decondensation and DNA base oxidation) with the exception of sperm count which did not return to control levels in BPAF+ Fertilix® treated animals. These data confirm that even at low doses and short-term exposures, epididymal spermatozoa are susceptible to DNA damage induced by environmental ROS-generating EDCs, confirming its association with male infertility. Limitations, reasons for caution The translation of these findings to humans needs to be confirmed, although the nuclear compartment of human sperm is more susceptible to oxidative alterations than that of the mouse. Wider implications of the findings These data demonstrate the susceptibility of post-testicular sperm cells to common environmental chemicals. They also support the use of appropriate antioxidant supplementation as an effective therapeutic choice for individuals with multiple exposures to environmental stressors such as ROS-generating EDCs. Trial registration number not applicable

574-P: A Ketogenic Diet Improves Aerobic Capacity in Hyperglycemic Mice by Sex-Dependent Mechanisms

Hyperglycemia is associated with lower aerobic exercise capacity (VO2peak) in humans and rodent models. We tested whether reducing blood glucose levels with a dietary intervention can restore VO2peak in hyperglycemic mice. Male and female CD-1 mice were injected with low dose streptozotocin (STZ) to induce moderate hyperglycemia (&amp;gt;200 mg/dL). Hyperglycemic mice were randomized to receive a standard diet (STZ-CHOW) or a ketogenic diet (STZ-KETO). KETO significantly reduced blood glucose in STZ-treated mice. Next, both groups underwent similar volumes of exercise training by voluntary wheel running, or remained sedentary for 8 wks. In males, VO2peak during maximal exercise testing was similar in sedentary mice, regardless of diet. However, with training, VO2peak was significantly improved in STZ-KETO, but not STZ-CHOW males (see Figure). In females, KETO significantly increased VO2peak in sedentary mice, with no additional increase in response to training. Improved VO2peak with KETO in females was associated with a 50% increase in skeletal muscle mitochondrial ATP synthase protein. In contrast, ATP synthase was not regulated by diet in males. Our data demonstrate that a ketogenic diet can improve VO2peak in mice with hyperglycemia. In males, improved VO2peak with KETO was exercise-dependent, while in females, the beneficial effects of KETO on VO2peak and mitochondrial content were exercise-independent. Disclosure P.Pattamaprapanont: None. M.Formato: None. R.Nogueira soares: None. S.J.Lessard: None.