Development of chlorine-induced lung injury in the anesthetized, spontaneously breathing pig.

Chlorine is a toxic industrial chemical produced in vast quantities globally, being used in a range of applications such as water purification, sanitation and industrial processes. Its use and transport cannot be restricted; exposure may occur following accidental or deliberate releases. The OPCW recently verified the use of chlorine gas against civilians in both Syria and Iraq. Chlorine inhalation produces damage to the lungs, which may result in the development of an acute lung injury, respiratory failure and death. Treatment remains an intractable problem. Our objective was to develop a clinically relevant pre-clinical model of a moderate to severe lung injury in the pig. This would enable future assessment of therapeutic drugs or interventions to be implemented in the pre-hospital phase after exposure. Due to the irritant nature of chlorine, a number of strategies for exposing terminally anesthetized pigs needed to be investigated. A number of challenges (inconsistent acute changes in respiratory parameters; early deaths), resulted in a moderate to severe lung injury not being achieved. However, most pigs developed a mild lung injury by 12 h. Further investigation is required to optimize the model and enable the assessment of therapeutic candidates. In this paper we describe the exposure strategies used and discuss the challenges encountered in establishing a model of chlorine-induced lung injury. A key aim is to assist researchers navigating the challenges of producing a clinically relevant model of higher dose chlorine exposure where animal welfare is protected by use of terminal anesthesia.

Sampling optimization for blend monitoring of a low dose formulation in a tablet press feed frame using spatially resolved near-infrared spectroscopy.

In-line measurements of low dose blends in the feed frame of a tablet press were performed for API concentration levels as low as 0.10% w/w. The proposed methodology utilizes the advanced sampling capabilities of a Spatially Resolved Near-Infrared (SR-NIR) probe to develop Partial Least-Squares calibration models. The fast acquisition speed of multipoint spectra allowed the evaluation of different numbers of co-adds and feed frame paddle speeds to establish the optimum conditions of data collection to predict low potency blends. The interaction of the feed frame paddles with the SR-NIR probe was captured with high resolution and allowed the implementation of a spectral data selection criterion to remove the effect of the paddles from the calibration and testing process. The method demonstrated accuracy and robustness when predicting drug concentrations across different feed frame paddle speeds.

Bioavailability of subcutaneous and intramuscular administrated buprenorphine in New Zealand White rabbits.

BACKGROUND: Buprenorphine is one of the most used analgesics for postoperative pain in rabbits. The recommended dose in rabbits (0.01-0.05 mg/kg) is the same for intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration, despite lack of pharmacokinetic data. Five male and five female New Zealand White rabbits (mean ± SD body weight 3.1 ± 0.3 kg) were administered 0.05 mg/kg buprenorphine by the IV, IM and SC routes and 0.1 mg/kg by the SC route, in a cross-over design with two-week wash-out periods between treatments. Blood was collected before, and up to 8 h post buprenorphine injection, for determination of serum levels by UPHLC-MS/MS. RESULTS: The area under the time concentration curve (AUC0-t) was lower after SC (398 ± 155 ng/mL/min) than IM (696 ± 168 ng/mL/min, p < 0.001) and IV (789 ± 189 ng/mL/min, p < 0.001) administration. The maximum serum concentration was lower after SC (2.2 ± 1.4 ng/mL) than after IM (11 ± 3.2 ng/mL) administration (p < 0.001). The bioavailability was lower after SC (50 ± 19%) than after IM (95 ± 21%) administration (p = 0.006). The elimination half-life was longer after SC (260 ± 120 min) than after IM (148 ± 26 min, p = 0.002) as well as IV (139 ± 33 min) injection (p < 0.001). An increase in the SC dose from 0.05 to 0.1 mg/kg resulted in an increase in the area under the time concentration curve of 50% in female (p = 0.022) and 165% in male rabbits (p < 0.001). The bioavailability did not change in the females (36 ± 14%, p = 0.6), whereas it increased in the males (71 ± 23%, p = 0.008). CONCLUSIONS: The lower bioavailability of 0.05 mg/kg buprenorphine after SC administration could explain the lack of efficacy seen in clinical pain studies in rabbits, using this route. For immediate pain relief, IV or IM administration is therefore be recommended, whereas SC administration may be useful to sustain analgesic serum levels, once efficient pain relief has been achieved. The current data do not support an increase in dose to compensate for the lower SC bioavailability.

Assessing the safety and suitability of nesting material for singly housed mice with surgically fitted head plates.

Nesting material, for example shredded paper, is a common form of enrichment for laboratory mice. However, there has been limited research performed regarding its apparent safety when given to mice fitted with exteriorised devices such as head plates. Anecdotally, shredded paper has been deemed unsafe for use with such animals due to frequently observed entanglement. This study assessed the safety of four nesting materials (Pure Comfort White, Rodent Roll, Short Paper Shavings and Facial Tissue) to identify a suitable alternative to shredded paper. The four nesting materials were each tested on 5 head plate mice over a 14-day period. The quality of the nests produced was scored throughout the trial period and incidences of tangling monitored. Tangling was only observed in the Facial Tissue group, and the highest quality nest scores were recorded in the Pure Comfort White group. As shredded paper has been anecdotally alleged to cause entanglement, Pure Comfort White, Rodent Roll and Short Paper Shavings may present more suitable options, given that their use did not result in any incidences of tangling throughout this trial. Pure Comfort White was concluded to be the safest and most suitable nesting material for head plate mice as it produced nests of high quality while reducing the risk of entanglement.

Differences in monocyte: lymphocyte ratio and Tuberculosis disease progression in genetically distinct populations of macaques.

Monocyte:lymphocyte ratio (M:L) has been identified as a risk factor in development of TB disease in children and those undergoing treatment for HIV in co-infected individuals. Retrospective analysis was performed using M:L data collected from TB modelling studies performed in Rhesus macaques of Indian genotype (RM), cynomolgus macaque of Chinese genotype (CCM) and cynomolgus macaque of Mauritian genotype (MCM), which found that the more susceptible populations (RM and MCM) had higher M:L ratios than the least susceptible population (CCM). Following Mycobacterium tuberculosis exposure, significant increases in M:L ratio were observed in susceptible RM and MCM within 12 weeks of TB infection, whereas M:L in CCM remained stable, suggesting that changes in M:L ratio may also act as a biomarker of TB disease progression. The frequency of PPD-specific interferon gamma (IFNγ) secreting cells (SFU) were compared, with the more susceptible macaque populations showing an association between M:L and IFNγ SFU frequency. Investigation of the genes associated with monocyte-derived antigen presenting cells revealed differences between RM and CCM, highlighting differences in their monocyte populations, as well as overall M:L ratio. Differences in M:L ratio between macaque populations could be used to explore immunological mechanisms in susceptible populations that would complement human population studies.

Increased expression of CRF and CRF-receptors in dorsal striatum, hippocampus, and prefrontal cortex after the development of nicotine sensitization in rats.

BACKGROUND: Nicotine addiction supports tobacco smoking, a main preventable cause of disease and death in Western countries. It develops through long-term neuroadaptations in the brain reward circuit by modulating intracellular pathways and regulating gene expression. This study assesses the regional expression of the transcripts of the CRF transmission in a nicotine sensitization model, since it is hypothesised that the molecular neuroadaptations that mediate the development of sensitization contribute to the development of addiction. METHODS: Rats received intraperitoneal nicotine administrations (0.4 mg/kg) once daily for either 1 day or over 5 days. Locomotor activity was assessed to evaluate the development of sensitization. The mRNA expression of CRF and CRF1 and CRF2 receptors was measured by qPCR in the ventral mesencephalon, ventral striatum, dorsal striatum (DS), prefrontal cortex (PFCx), and hippocampus (Hip). RESULTS: Acute nicotine administration increased locomotor activity in rats. In the sub-chronic group, locomotor activity progressively increased and reached a clear sensitization. Significant effects of sensitization on CRF mRNA levels were detected in the DS (increasing effect). Significantly higher CRF1 and CRF2 receptor levels after sensitization were detected in the Hip. Additionally, CRF2 receptor levels were augmented by sensitization in the PFCx, and treatment and time-induced increases were detected in the DS. Nicotine treatment effects were observed on CRF1R levels in the DS. CONCLUSIONS: This study suggests that the CRF transmission, in addition to its role in increasing withdrawal-related anxiety, may be involved in the development of nicotine-habituated behaviours through reduced control of impulses and the aberrant memory plasticity characterising addiction.

When neuroscience met clinical pathology: partitioning experimental variation to aid data interpretation in neuroscience.

In animal experiments, neuroscientists typically assess the effectiveness of interventions by comparing the average response of groups of treated and untreated animals. While providing useful insights, focusing only on group effects risks overemphasis of small, statistically significant but physiologically unimportant, differences. Such differences can be created by analytical variability or physiological within-individual variation, especially if the number of animals in each group is small enough that one or two outlier values can have considerable impact on the summary measures for the group. Physicians face a similar dilemma when comparing two results from the same patient. To determine whether the change between two values reflects disease progression or known analytical and physiological variation, the magnitude of the difference between two results is compared to the reference change value. These values are generated by quantifying analytical and within-individual variation, and differences between two results from the same patient are considered clinically meaningful only if they exceed the combined effect of these two sources of 'noise'. In this article, we describe how the reference change interval can be applied within neuroscience. This form of analysis provides a measure of outcome at an individual level that complements traditional group-level comparisons, and therefore, introduction of this technique into neuroscience can enrich interpretation of experimental data. It can also safeguard against some of the possible misinterpretations that may occur during analysis of the small experimental groups that are common in neuroscience and, by illuminating analytical error, may aid in design of more efficient experimental methods.

Influence of intertrial interval on basal and drug-induced impulsive action in the 5-choice serial reaction time task: Effects of d-amphetamine and (±)-2,5-dimethoxy-4-iodoamphetamine (DOI).

Impulsivity is a characteristic of a number of neuropsychiatric disorders such as attention-deficit/hyperactivity disorder. The 5-choice serial reaction time task (5-CSRTT) is a rodent paradigm extensively used to assess attention and impulsivity. Notably, 5-CSRTT studies do not typically account for the reduction in premature responding, the measure of impulsive action, occurring upon repeated exposure to test sessions with long or variable intertrial intervals (ITIs). This present 5-CSRTT study investigated the use of variable ITIs (5, 10 or 15s) across 15 test days (4 training days followed by 1 drug test day per week for three weeks) as previous experience had shown that 4 training days would be sufficient to induce consistent premature response levels in male C57BL/6J mice. Once a steady state was achieved, the effects of dextroamphetamine (AMPH) and (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) were then assessed using a Latin-square design to determine whether pharmacological-induced impulsive actions depended on ITI length. Mice habituated to the variable ITI schedule after only 3days and showed consistently lower premature response levels until the end of the study. AMPH (p<0.05) and DOI (p<0.05) increased the percentage of premature responses at 15s ITI trials, while only DOI (p<0.05) increased impulsive action at 10s ITI trials. Additionally, DOI increased omission rates (p<0.001), mean correct latency (p<0.01), reward collection latency (p<0.001), and reduced the total attempted trials (p<0.001). In summary, we demonstrated that mice habituate to the variable ITI schedule, suggesting that using the variable ITI schedule during training allowed premature response rates to stabilize before commencing pharmacological testing. Moreover, in these habituated mice AMPH and DOI significantly enhanced impulsive action at the long ITI trials only. We propose that experimental design considerations can improve the sensitivity of the 5-CSRTT to detect pharmacologicallyinduced impulsive action.

The Experimental Design Assistant.

Addressing the common problems that researchers encounter when designing and analysing animal experiments will improve the reliability of in vivo research. In this article, the Experimental Design Assistant (EDA) is introduced. The EDA is a web-based tool that guides the in vivo researcher through the experimental design and analysis process, providing automated feedback on the proposed design and generating a graphical summary that aids communication with colleagues, funders, regulatory authorities, and the wider scientific community. It will have an important role in addressing causes of irreproducibility.

Using InVivoStat to perform the statistical analysis of experiments.

The need to improve reproducibility and reliability of animal experiments has led some journals to increase the stringency of the criteria that must be satisfied before manuscripts can be considered suitable for publication. In this article we give advice on experimental design, including minimum group sizes, calculating statistical power and avoiding pseudo-replication, which can improve reproducibility. We also give advice on normalisation, transformations, the gateway analysis of variance strategy and the use of p-values and confidence intervals. Applying all these statistical procedures correctly will strengthen the validity of the conclusions. We discuss how InVivoStat, a free-to-use statistical software package, which was designed for life scientists, especially animal researchers, can be used to help with these principles.

ADX71943 and ADX71441, novel positive allosteric modulators of the GABAB receptor with distinct central/peripheral profiles, show efficacy in the monosodium iodoacetate model of chronic osteoarthritis pain in the rat.

We tested novel positive allosteric modulators (PAMs) of the γ-aminobutyric acid receptor B (GABAB), ADX71943 and ADX71441in the monosodium iodoacetate model of chronic osteoarthritis pain in rats with the objective to delineate the role of peripheral versus central GABAB receptor populations in modulation of chronic pain. Anesthetized Sprague-Dawley rats received an injection of monosodium iodoacetate into the knee and were tested for hyperalgesia starting post-MIA day 14. Effects of compounds on ipsilateral joint compression threshold were evaluated on post-MIA day 14 (after acute treatment), as well as after repeated, daily treatment on days 21 and 28 (ADX71943 only) and were compared to those of celecoxib (30mg/kg, p.o.). The PAMs were also tested in the rat rotarod test for potential muscle-relaxant effects. Acutely, ADX71943 (1-30mg/kg, p.o.), the peripherally restricted PAM, resulted in similar increases in pain threshold across the doses on day 14, while showing reduced efficacy on day 21 and no efficacy on day 28. A clear reduction in the efficacy of celecoxib across testing was also noted in this experiment. Acutely ADX71441 (0.3-15mg/kg, p.o.), the central-peripheral PAM, resulted in over 2-fold increases in pain threshold at 15mg/kg (but not at lower doses) on day 14, while causing more modest effects on day 21. Celecoxib increased pain threshold after both acute and daily treatment, showing overall similar efficacy. Thus, early, presumably more inflammatory phase of osteoarthritis pain in more sensitive to GABAB PAMs with peripherally restricted profile, while later, presumably more neuropathic phase is more sensitive to PAMs with central-peripheral profile.

Influenza A Virus Challenge Models in Cynomolgus Macaques Using the Authentic Inhaled Aerosol and Intra-Nasal Routes of Infection.

Non-human primates are the animals closest to humans for use in influenza A virus challenge studies, in terms of their phylogenetic relatedness, physiology and immune systems. Previous studies have shown that cynomolgus macaques (Macaca fascicularis) are permissive for infection with H1N1pdm influenza virus. These studies have typically used combined challenge routes, with the majority being intra-tracheal delivery, and high doses of virus (> 107 infectious units). This paper describes the outcome of novel challenge routes (inhaled aerosol, intra-nasal instillation) and low to moderate doses (103 to 106 plaque forming units) of H1N1pdm virus in cynomolgus macaques. Evidence of virus replication and sero-conversion were detected in all four challenge groups, although the disease was sub-clinical. Intra-nasal challenge led to an infection confined to the nasal cavity. A low dose (103 plaque forming units) did not lead to detectable infectious virus shedding, but a 1000-fold higher dose led to virus shedding in all intra-nasal challenged animals. In contrast, aerosol and intra-tracheal challenge routes led to infections throughout the respiratory tract, although shedding from the nasal cavity was less reproducible between animals compared to the high-dose intra-nasal challenge group. Intra-tracheal and aerosol challenges induced a transient lymphopaenia, similar to that observed in influenza-infected humans, and greater virus-specific cellular immune responses in the blood were observed in these groups in comparison to the intra-nasal challenge groups. Activation of lung macrophages and innate immune response genes was detected at days 5 to 7 post-challenge. The kinetics of infection, both virological and immunological, were broadly in line with human influenza A virus infections. These more authentic infection models will be valuable in the determination of anti-influenza efficacy of novel entities against less severe (and thus more common) influenza infections.

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa.

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.

A common control group - optimising the experiment design to maximise sensitivity.

Methods for choosing an appropriate sample size in animal experiments have received much attention in the statistical and biological literature. Due to ethical constraints the number of animals used is always reduced where possible. However, as the number of animals decreases so the risk of obtaining inconclusive results increases. By using a more efficient experimental design we can, for a given number of animals, reduce this risk. In this paper two popular cases are considered, where planned comparisons are made to compare treatments back to control and when researchers plan to make all pairwise comparisons. By using theoretical and empirical techniques we show that for studies where all pairwise comparisons are made the traditional balanced design, as suggested in the literature, maximises sensitivity. For studies that involve planned comparisons of the treatment groups back to the control group, which are inherently more sensitive due to the reduced multiple testing burden, the sensitivity is maximised by increasing the number of animals in the control group while decreasing the number in the treated groups.

Characterization of the novel positive allosteric modulator of the metabotropic glutamate receptor 4 ADX88178 in rodent models of neuropsychiatric disorders.

There is growing evidence that activation of metabotropic glutamate receptor 4 (mGlu4) leads to anxiolytic- and antipsychotic-like efficacy in rodent models, yet its relevance to depression-like reactivity remains unclear. Here, we present the pharmacological evaluation of ADX88178 [5-methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine], a novel potent, selective, and brain-penetrant positive allosteric modulator of the mGlu4 receptor in rodent models of anxiety, obsessive compulsive disorder (OCD), fear, depression, and psychosis. ADX88178 dose-dependently reduced the number of buried marbles in the marble burying test and increased open-arm exploration in the elevated plus maze (EPM) test, indicative of anxiolytic-like efficacy. Target specificity of the effect in the EPM test was confirmed using male and female mGlu4 receptor knockout mice. In mice, ADX88178 reduced the likelihood of conditioned freezing in the acquisition phase of the fear conditioning test, yet had no carryover effect in the expression phase. Also, ADX88178 dose-dependently reduced duration of immobility in the forced swim test, indicative of antidepressant-like efficacy. ADX88178 reduced DOI (2,5-dimethoxy-4-iodoamphetamine)-mediated head twitches (albeit with no dose-dependency), and MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]-induced locomotor hyperactivity in mice, but was inactive in the conditioned avoidance response test in rats. The compound showed good specificity as it had no effect on locomotor activity in mice and rats at efficacious doses. Thus, allosteric activation of mGlu4 receptors can be a promising new therapeutic approach for treatment of anxiety, OCD, fear-related disorders, and psychosis.

Angiopoietin-1 regulates microvascular reactivity and protects the microcirculation during acute endothelial dysfunction: role of eNOS and VE-cadherin.

The growth factor angiopoietin-1 (Ang-1) plays an essential role in angiogenesis and vascular homeostasis. Nevertheless, the role of Ang-1 in regulating vascular tone and blood flow is largely unexplored. Endothelial nitric oxide synthase (eNOS) and the junctional protein VE-cadherin are part of the complex signalling cascade initiated by Ang-1 in endothelial cells. In this study, we aimed to investigate the mechanisms underlying acute effects of Ang-1 on microvascular reactivity, permeability and blood flow, and hypothesise that eNOS and VE-cadherin underpin Ang-1 mediated vascular effects that are independent of angiogenesis and proliferation. Myography of isolated microarterioles from male C3H/HeN mice (7-10 weeks) was employed to measure vascular reactivity in vitro. Microcirculatory function in vivo was evaluated by intravital microscopy and Doppler fluximetry in dorsal window chambers. Ang-1 and its stable variant MAT.Ang-1 induced a concentration-dependent vasodilation of arterioles in vitro, which was blocked with nitric oxide (NO) synthesis inhibitor l-NAME. In vivo, MAT.Ang-1 restored to control levels l-NAME induced peripheral vasoconstriction, decreased blood flow and microvascular hyperpermeability. Tissue protein expression of VE-cadherin was reduced by NOS inhibition and restored to control levels by MAT.Ang-1, whilst VE-cadherin phosphorylation was increased by l-NAME and subsequently reduced by MAT.Ang-1 administration. Moreover, MAT.Ang-1 alone did not modulate systemic levels of angiogenetic factors. Our novel findings report that Ang-1 induces arteriolar vasodilation via release of NO, suggesting that Ang-1 is an important regulator of microvascular tone. As MAT.Ang-1 ameliorates detrimental effects on the microcirculation induced by inhibition of NO synthesis and stabilizes the endothelial barrier function through VE-cadherin, we propose that this Ang-1 variant may serve as a novel therapeutic agent to protect the microcirculation against endothelial dysfunction.

Assessment of the protective effect of Imvamune and Acam2000 vaccines against aerosolized monkeypox virus in cynomolgus macaques.

To support the licensure of a new and safer vaccine to protect people against smallpox, a monkeypox model of infection in cynomolgus macaques, which simulates smallpox in humans, was used to evaluate two vaccines, Acam2000 and Imvamune, for protection against disease. Animals vaccinated with a single immunization of Imvamune were not protected completely from severe and/or lethal infection, whereas those receiving either a prime and boost of Imvamune or a single immunization with Acam2000 were protected completely. Additional parameters, including clinical observations, radiographs, viral load in blood, throat swabs, and selected tissues, vaccinia virus-specific antibody responses, immunophenotyping, extracellular cytokine levels, and histopathology were assessed. There was no significant difference (P > 0.05) between the levels of neutralizing antibody in animals vaccinated with a single immunization of Acam2000 (132 U/ml) and the prime-boost Imvamune regime (69 U/ml) prior to challenge with monkeypox virus. After challenge, there was evidence of viral excretion from the throats of 2 of 6 animals in the prime-boost Imvamune group, whereas there was no confirmation of excreted live virus in the Acam2000 group. This evaluation of different human smallpox vaccines in cynomolgus macaques helps to provide information about optimal vaccine strategies in the absence of human challenge studies.

A comparison of InVivoStat with other statistical software packages for analysis of data generated from animal experiments.

InVivoStat is a free-to-use statistical software package for analysis of data generated from animal experiments. The package is designed specifically for researchers in the behavioural sciences, where exploiting the experimental design is crucial for reliable statistical analyses. This paper compares the analysis of three experiments conducted using InVivoStat with other widely used statistical packages: SPSS (V19), PRISM (V5), UniStat (V5.6) and Statistica (V9). We show that InVivoStat provides results that are similar to those from the other packages and, in some cases, are more advanced. This investigation provides evidence of further validation of InVivoStat and should strengthen users' confidence in this new software package.