PT1 peptide isolated from the venom of spider Geolycosa sp. is a modulator of P2X3 receptors that play a role in the development of inflammation and the transmission of pain impulses. The anti-inflammatory and analgesic efficacy of the PT1 peptide was studied in a model of complete Freund\'s adjuvant-induced paw inflammation in CD-1 mice. The analgesic activity of PT1 peptide was maximum after intramuscular injection at a dose of 0.01 mg/kg, which surpassed the analgesic effect of diclofenac at a dose of 1 mg/kg. The anti-inflammatory activity was maximum after intramuscular injection at a dose of 0.0001 mg/kg; a decrease in paw thickness was observed as soon as 2 h after the administration of the PT1 peptide against the background of inflammation development. All tested doses of PT1 peptide showed high anti-inflammatory activity 4 and 24 h after administration. PT1 peptide at a dose of 0.01 mg/kg when injected intramuscularly simultaneously produced high anti-inflammatory and analgesic effects compared to other doses of the peptide. Increasing the dose of PT1 peptide led to a gradual decrease in its analgesic and anti-inflammatory activity; increasing the dose of intramuscular injection to 0.1 and 1 mg/kg is inappropriate.

Development of lethal models of Ebola virus disease has been achieved by the serial passage of virus isolates from human cases in mice and guinea pigs. Use of mice infected with non-adapted virus has been limited due to the absence of overt clinical disease. In recent years, newly recognized sequelae identified in human cases has highlighted the importance of continued investigations of non-lethal infection both in humans and animal models. Here, we revisit the use of rodent-adapted and non-adapted Ebola virus (EBOV) in mice to investigate infection tolerance and future utility of these models in pathogenesis and therapeutic intervention studies. We found that like non-adapted wild-type EBOV, guinea pig-adapted EBOV resulted in widespread tissue infection, variably associated with tissue pathology, and alterations in clinical and immunological analytes in the absence of overt disease. Notably, infection with either non-lethal variant did not greatly differ from lethal mouse-adapted EBOV until near the time end-point criteria are reached in these mice. These data support future investigations of pathogenesis, convalescence, and sequelae in mouse models of virus tolerance.

Rift Valley fever virus (RVFV) is an important mosquito-borne pathogen that causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. The development of an effective veterinary and human vaccine to protect against Rift Valley fever (RVF) disease remains a high priority. The live attenuated RVFV MP-12 is a promising vaccine candidate for the prevention of RVF in both human and domestic ruminants. The aim of this study was to determine the onset of protective immunity elicted in mice by a single dose of this vaccine. Groups of CD-1 mice were vaccinated intraperitoneally with RVFV MP-12 vaccine and challenged on days 2, 5, 6 and 7 post-vaccination (PV) with a lethal dose of virulent RVFV. The mice were observed once daily for terminal morbidity and blood samples were obtained from the retro-orbital sinus complex on days 23 and 28 PV of surviving mice to determine RVFV neutralizing antibody titers. In one test, 2 of 3 mice challenged on day 2 PV survived and all 3 mice challenged at days 5 and 7 PV also survived. A second test of 10 mice per group was performed, and half (5) of those challenged at day 2 PV survived while all (10) survived challenge at day 4 and 6 PV. All surviving animals develop antibody that ranged from 1:80 to 1:1,280 PV. In a separate experiment, RVFV MP-12 vaccinated CD-1 mice, but not challenged developed a low viremia for the first 3 days PV and neutralzing antibody was detected on days 5 through day 28 PV. These findings demonstrated that the RVFV MP-12 vaccine elicited a rapid protective immune response in mice as early as 2 days PV, thus further supporting the effectiveness of this vaccine candidate for preventing RVF among humans and domestic ruminants.

CO2 inhalation is currently the most common method of euthanasia for laboratory rats and mice, and it is often used for further terminal blood sampling for clinical biochemical assays. Lately, this method has been criticized due to animal welfare issues associated with some processes that develop after CO2 inhalation. The stress reaction and the value of the clinical laboratory parameters significantly depend on the used anesthetics, method, and the site of blood sampling. Especially in small rodents, an acute terminal state followed by a cascade of metabolic reactions that can affect the studied biochemical profile may develop and cause unnecessary suffering of animals. The aim of this study was to compare the stability of biochemical parameters of outbred Sprague Dawley rats and CD-1 mice serum collected after CO2 inhalation or the intramuscular injection of tiletamine-zolazepam-xylazine (TZX). The serum content of total protein and albumin, cholesterol, triglycerides, aspartate aminotransferase (AST), alanine aminotr ansferase (ALT), alkaline phosphatase (ALP), total bilirubin, and creatinine was decreased by the injection of TZX in comparison with CO2 inhalation. In addition, the levels of calcium, phosphates, chlorides and potassium were lowered by TZX vs. CO2 administration, while the level of sodium increased. Finally, the level of the majority of serum clinical biochemical parameters in rats and mice tend to be overestimated after CO2 inhalation, which may lead to masking the possible effect of anti-inflammatory drugs in animal tests. Injection anesthesia for small rodents with TZX is a more feasible method for terminal blood sampling, which also reduces the suffering of animals.

Achieving durable protective immunity following vaccination is dependent on many factors, including vaccine composition and antigen dose, and it has been investigated for various types of vaccines. Aim of the present study was to investigate the overall immune response elicited by two different booster doses in CD-1 mice, by exploiting the largely used 13-valent pneumococcal conjugate vaccine Prevnar 13® (PCV13). Immunization was performed by two primary doses of PCV13 two weeks apart, and a full or fractional (1/5) booster dose on week 10. Serotype-specific antibody titer, avidity, and opsonophagocytic activity were evaluated one week later, and compared to cell-mediated immunity (CMI) responses determined as the frequency of cytokines producing splenocytes by in vitro recall with the antigens (carrier protein and polysaccharides). Data showed that regardless of the booster dose, a comparable humoral response was produced, characterized by similar amounts of serotype-specific antibodies, with analog avidity and opsonophagocytic properties. On the other hand, when CMI was evaluated, the presence of CRM197-specific IL-5 and IL-2 producing cells was evident in splenocytes from mice immunized with the full dose, while in those immunized with the fractional booster dose, IFN-γ producing cells responsive to both protein and polysaccharide antigens were significantly increased, whereas the number of IL-5 and IL-2 positive cells remained unaffected. Overall the present findings show that PCV13 humoral response in mice is associated to a Th2 predominant response at the full booster dose, while the fractional one favors a mixed Th1/Th2 response, suggesting an important role of CMI besides measurement of functional protective antibodies, as an additional and important key information in vaccine development.

Fluorene-9-bisphenol (BHPF), a substitute for bisphenol A (BPA), has been widely used in the synthesis of polyester polymers. Studies have reported multiple BHPF toxicities but its effect on the liver remains unknown. In this study, we performed short-term and subchronic toxicity tests, as well as primary hepatocyte experiments, to investigate the hepatic toxicity of BHPF using CD-1 mice. And microarray was used to analyze the changes of global gene expression in the liver of mice treated with BHPF. The results showed that the liver coefficient and the activities of serum aminotransferases were obviously elevated by BHPF at doses of 27.8 mg/kg body weight (bw)/day or higher in mice treated for 10 days. Histological analysis showed obvious changes, including narrowed hepatic sinuses, dilated central vein, leucocyte infiltration, and cytoplasmic vacuolation, in the livers of mice treated with BHPF at dosages of 2 mg/kg bw/3-day and higher for 36 days. Microarray analyses revealed 2623 differentially expressed genes (DEGs) in the livers of mice treated with 50 mg/kg bw/day of BHPF for 3 days, which could be enriched in GO terms of T cell activation, leukocyte migration, and leukocyte chemotaxis and KEGG pathways of natural killer cell-mediated cytotoxicity and autoimmune thyroid disease. The top 10 hub DEGs, including LTF and MMP8, were observed in the protein-protein interaction network obtained via STRING database analysis, and are proposed as potential biomarkers for liver injury studies. Primary hepatocyte experiments demonstrated the hepatotoxicity of BHPF at concentrations of 10-6 M and higher. This study indicates that BHPF could cause liver injury at relatively low levels, suggesting that the risk of human BHPF exposure should be of concern.

Posidonia oceanica (L.) Delile is traditionally used for its beneficial properties. Recently, promising antioxidant and anti-inflammatory biological properties emerged through studying the in vitro activity of the ethanolic leaves extract (POE). The present study aims to investigate the anti-inflammatory and analgesic role of POE in mice. Inflammatory pain was modeled in CD-1 mice by the intraplantar injection of carrageenan, interleukin IL-1β and formalin. Pain threshold was measured by von Frey and paw pressure tests. Nociceptive pain was studied by the hot-plate test. POE (10-100 mg kg-1) was administered per os. The paw soft tissue of carrageenan-treated animals was analyzed to measure anti-inflammatory and antioxidant effects. POE exerted a dose-dependent, acute anti-inflammatory effect able to counteract carrageenan-induced pain and paw oedema. Similar anti-hyperalgesic and anti-allodynic results were obtained when inflammation was induced by IL-1β. In the formalin test, the pre-treatment with POE significantly reduced the nocifensive behavior. Moreover, POE was able to evoke an analgesic effect in naïve animals. Ex vivo, POE reduced the myeloperoxidase activity as well as TNF-α and IL-1β levels; further antioxidant properties were highlighted as a reduction in NO concentration. POE is the candidate for a new valid strategy against inflammation and pain.

Parkinson´s disease is the most important neuromotor pathology due to the prominent loss of dopaminergic neurons in the substantia nigra pars compacta. There is an inherent deficiency of dopamine in Parkinson´s disease, which is aggravated when neuroinflammatory processes are present. Several biomolecules are interesting candidates for the regulation of inflammation and possible neuroprotection, such as valerenic acid, one of the main components of Valeriana officinalis. A 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP)-induced mouse model of Parkinson\'s disease was developed to evaluate the motor effects of valerenic acid. The evaluation was carried out with four tests (an invert screen test for muscle strength, cross beam test, open field mobility test and lifting on hind legs test). Subsequently, the neuroinflammatory process was evaluated through ELISA of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and IFN-γ). The decreases in the inflammatory and neurodegenerative processes were evaluated by Western blot and immunohistochemistry analyses of the tissues, which included an evaluation of the tyrosine hydroxylase and GFAP proteins. Finally, the predicted mechanism of action of valerenic acid was supported by molecular docking calculations with the 5-HT5A receptor. The results indicate that the use of valerenic acid as a co-treatment decreases the neuroinflammation in Parkinson\'s disease induced by MPTP and provides evidence of a decrease in the evaluated pro-inflammatory cytokines and in the amount of GFAP in the mesencephalic area. Valerenic acid prevents neuroinflammation in a Parkinson\'s disease mouse model, which might reflect the neuroprotection of dopaminergic neurons with the recovery of motor ability.

We independently and retrospectively reviewed three studies that evaluated the toxicity of BIA 10-2474 (3-(1-(cyclohexyl(methyl)carbamoyl)-lH-imidazol-4-yl)pyridine 1-oxide), a novel fatty acid amide hydrolase (FAAH) inhibitor in male and female CD-1 mice based upon raw data obtained from Bial Portela & Companhia S.A. (São Mamede do Coronado, Portugal). These studies were carried out prior to the clinical trial with BIA 10-2474 and formed part of the regulatory submission. An initial oral dose range-finding study with BIA 10-2474 showed that doses from 600 mg/kg/day were poorly tolerated with a high mortality rate and signs of weakness, prostration, labored breathing, clear lacrimation, tachypnea/bradypnea and decreased activity. At lower doses (100 and 300 mg/kg/day) there were few signs but post-mortem analysis showed increased liver weight. In a 28-day study a third of the animals receiving 500 mg/kg/day died or required euthanasia, with similar signs to those seen in the dose-range finding study. At lower doses (i.e. 100 and 300 mg/kg/day) there were few clinical signs although there were dose-related decreases in erythrocyte count and hemoglobin. Histopathology was seen in the 300 and 500 mg/kg/day groups and included hepatocellular hypertrophy (with increased liver weight), nephropathy and enterocyte vacuolation. Finally, in the 13-week oral gavage study, BIA 10-2474 was administered to CD-1 mice of both sexes at dose levels of 25, 75 and 150 mg/kg/day. Under these conditions, there were almost no clinical signs apart from a tendency to increase body-weight. Cholesterol was increased at 75 and 150 mg/kg and remained high after recovery. Liver and spleen weights increased at 75 and 150 mg/kg/day. Histopathologically, there was a dose-dependent increase in sciatic nerve and myofiber degeneration, hepatocellular hypertrophy, nephropathy and inflammatory loci in the bladder. The nerve damage and nephropathy seen at 150 mg/kg/day persisted after a 4-week recovery period. Toxicokinetic analysis in the 4- and 13-week studies showed that exposure was broadly dose-proportional with no evidence of accumulation. On the basis of the changes seen during the 13-week study, the NOAEL was established at 75 mg/kg/day.

Primary mouse hepatocyte cultures are widely used in toxicological and pharmacological studies. However, the strain differences in alterations of metabolic enzymes and the regulation of gene expression in response to different stimuli remains unclear. To address this issue, we examined the expression of metabolic enzymes and the regulatory role of DNA methylation in the primary hepatocytes of two mouse strains, CD-1 and C57BL/6. Primary culture of mouse hepatocytes was established using collagen sandwich configuration. Analysis of gene expression of 24 phase I, 18 phase II, and 6 phase III metabolic enzymes on 4 consecutive days after cell seeding revealed that the basal levels of most enzymes in primary cultured hepatocytes differed greatly between the two mouse strains. However, the dynamic changes in most genes were identical between the two strains. In addition, treatment with 3-methylcholanthrene, phenobarbital, and rifampin led to the induction of cytochrome P-450 (cyp) 1a1 and cyp1a2, cyp2b10, cyp3a11. However, induction varied in degree between the two types of primary hepatocytes. The dynamic changes in global DNA methylation and the expression of DNA methylation regulatory factors of the two mouse strains were similar. Of the genes down-regulated over the culture period, hypermethylation of cyp2e1 gene appeared in both mouse strains and led to a suppression of gene expression. Taken together, these results demonstrate that the expression of metabolic enzymes and the response to agonists in primary hepatocytes differ between CD-1 and C57BL/6 mouse strains. Epigenetic regulation might be involved in the suppression of cyp 450s\' expression.