When exposed AC220 to repeated levels of disturbance throughout the day, the net effect is an altered activity budget, in which killer whales spend less time feeding in the presence of boats than during no-boat, control conditions

(Lusseau et al., 2009 and Williams et al., 2006). A number of studies have demonstrated effects of noise from large ships on a variety of cetacean species, including Cuvier’s beaked whale (Aguilar Soto et al., 2006), North Atlantic right whale (Nowacek et al., 2004 and Rolland et al., 2012), beluga (Erbe and Farmer, 1998 and Erbe and Farmer, 2000) and fin whales (Castellote et al., 2012). These studies provide a hint that ship noise can reduce a whale’s foraging efficiency (Aguilar Soto et al., 2006); elevate the risk of ship strikes (Nowacek et al., 2004); and cause physiological stress that is detectable in hormone levels (Rolland et al., 2012). A combination of captive experiments and computer models (Erbe and Farmer, 2000) enabled researchers to estimate that icebreaker noise is audible to belugas and capable of eliciting behavioral responses and causing http://www.selleckchem.com/products/PTC124.html communication masking at ranges to 62 km. A temporary hearing shift was modeled to occur if a beluga stayed within 1–4 km of the icebreaker

for at least 20 min. Whales have evolved in an ocean environment that becomes naturally noisy during storms and surf zones, and they have evolved some mechanisms to compensate for noise. Fin whales change their song characteristics to try to maintain communication in high levels

of shipping noise (Castellote et al., 2012). There is some evidence to suggest that killer whales can compensate for increases in ambient noise by lengthening their calls (Foote et al., 2004) or increasing the source level of social calls (Holt et al., 2008). There is no evidence that killer whales can adjust their echolocation patterns to compensate for masked signals used in foraging, and no information on the upper limit to Selleckchem Fludarabine the whales’ compensatory mechanisms. In many behavioral response studies, the received levels that trigger responses are rarely known (but see (Williams et al., 2002a)). A recurring theme in the literature describing marine mammals and noise is that the most rigorous behavioral studies rarely report information on the acoustic stimulus, and the best acoustic studies often have very small sample size for inferring behavioral responses (Nowacek et al., 2007). No studies have yet examined the responses of killer whales to presence and activities of large ships. Such studies are needed (Wright, 2008). Global shipping represents a large and growing contributor to ocean ambient soundscapes (Hildebrand, 2009), and creative solutions are needed to quantify and mitigate impacts of chronic ocean noise on sensitive marine mammals (Wright et al., 2011).

Thus, the amaranth flour film should meet some requirements, regarding mechanical strength, flexibility, and permeability to water vapor and gases, in order to ensure food preservation during storage. Therefore, the aim of this work was to examine the effect of the drying conditions find more on the mechanical, solubility, barrier properties, and drying time of amaranth flour films plasticized with glycerol or sorbitol and optimize the drying process by using a response surface methodology and multi-response analyses, targeting the production

of films with low solubility and good mechanical properties. The Amaranthus cruentus BRS Alegria seeds were grown in the state of Santa Catarina (Brazil) at 18.8–22 °C, soil pH of 5.5. The seeds were harvested in early October, transported to Campinas (Brazil), cleaned, and stored at 10 °C. The amaranth flour was obtained by using a modification to the alkaline wet milling method of Perez, Bahnassey, and Breene (1993), as proposed by Tapia-Blácido et al. (2005a). The composition of amaranth flour is: moisture content 8.3 ± 0.4 g/100 g, ashes 2.1 ± 0.0 g/100 g, lipids 7.9 ± 0.2 g/100 g,

protein 14.1 ± 0.3 g/100 g, and starch 75.7 ± 0.3 g/100 g (11.9 ± 0.3 g amylose/100 g flour) (db). All the reagents were analytical PI3K Inhibitor Library chemical structure grade. Sorbitol and NaOH were purchased from Synth (São Paulo, Brazil). All the solutions were prepared with deionized water. The films were produced by the casting method. Amaranth flour films were prepared by using the methodology proposed by Tapia-Blácido et al. (2005a). A suspension ifoxetine of flour in water (4 g/100 g) was homogenized in a mixer for 25 min, and the pH was regulated to 10.7 with 0.1 mol equi/L NaOH, to dissolve the protein. This suspension was then heated at 75 °C for 15 min, followed by addition of the plasticizer (29.6 g sorbitol/100 g flour or 20.02 g glycerol/100 g flour). For each film, 85 ± 3 g of the film-forming solution was poured onto acrylic plates (18 × 21 cm), in order to obtain a constant thickness

of 80 ± 5 μm. The films were dried under different drying conditions by using an oven with air circulation and controlled temperature (model MA 415UR, Marconi, Piracicaba, Brazil). The studied drying conditions were 30 °C, 40% RH; 30 °C, 70% RH; 50 °C, 40% RH; 50 °C, 70% RH; 25.9 °C, 55% RH; 54.1 °C, 55% RH; 40 °C, 33.8% RH; 40 °C, 76.2% RH; and 40 °C, 55% RH, defined according to the experimental design that was being used (Tables 1 and 2). The drying kinetics curves of the amaranth flour films were determined for all the studied conditions. Prior to characterization, all the films were preconditioned for at least 48 h in desiccators containing a saturated NaBr solution (25 ± 3 °C, 58 ± 2% RH). The thickness of the films was measured with a digital micrometer Fowler (average of 8 measurements).

To have a representative set of the

liver contigs of B. microlepidotus, reads of each individual were mapped back to the assembled transcriptome using the alignment program TMAP (http://github.com/iontorrent/TMAP/tarball/tmap.0.3.7) (for more details see Supplementary methods) and contigs showing expression in the three individuals were chosen. In total 13,724 contigs (Supplementary information 1) with an average length of 836.8 bp were retained for the functional annotation ( Table 1; Fig. 1A). The raw sequence data is accessioned in the NCBI Sequence Read Archive (SRA accession SRP046041). The Blastx function was performed with a minimum E-value score of 1.0E− 06 and the gene ontology (GO) terms of molecular function, cellular component, and biological process were GKT137831 price assigned to the 13,724 retained contigs using the Blast2GO software (Conesa et al., 2005). A total of 2803 sequences presented Blast results and 7938 (57.8%) sequences were successfully annotated (Fig. S1, Supplementary information 2). As expected, the species distribution of the Blast hits showed that most hits correspond to fish species (Fig. S2, Supplementary information 2). A total of 40,814 annotations (Supplementary information 3) for the 13,724 contigs were obtained; the biological processes class was the most highly represented (44.2%), followed

by molecular function (35%) and cellular component (20.8%) (Fig. 1B). These proportions were similar to these described for Oncorchynchus mykiss ( Fox et al.,

2014). For B. microlepidotus, the biological see more processes involved mainly the diversity of gene expression, with Mannose-binding protein-associated serine protease predominance of cellular, metabolic and single-organism processes ( Fig. 2A), while the GO annotations for molecular functions were mostly represented by binding and catalytic activity ( Fig. 2B). The cellular component class was mainly composed of cell, organelle, membrane and macromolecular complex components ( Fig. 2C). See Supplementary methods for details regarding functional annotation. The following are the supplementary data related to this article. Supplementary methods We thank C Quezada-Romegialli, JP Oyanedel and P Muñoz-Rojas for support during field work and Dr. Arne Nolte for support during the analyses. The authors thank R Espejo and Omics-Solutions Chile for sequencing. DV thanks Basal Grant PFB 023, ICM P05-002 and Nucleo Milenio NC120030; CVR thanks Conicyt Doctoral Fellowship 21090188 and doctoral thesis fellowship 24121005. All analyses were conducted in Chile and complied with its existing laws (Resolución Exenta No. 3329 Subsecretaria de Pesca). “
“Brine shrimp (Artemia franciscana) are small crustaceans found worldwide, mainly in hypersaline environments. This zooplanktonic organism has been extensively used in fish aquaculture as larval feed for over 85% of cultured species ( Kayim et al., 2010). Besides this role in aquaculture, Artemias spp.

One of the advantages of our study was the large number of participants in the study compared to previous researches, 84 patients with MS and 115 healthy controls. Most of the participants in our study were RRMS and SPMS, with a small percentage of PPMS. We recommend future studies to include other types of MS in the evaluation to check for differences between all types of the disease. As there is controversy between different studies assessing CCSVI criteria in MS patients and above-mentioned reports about IJV resection consequences, reconsidering the criteria may be an option. Another reason for these controversies might be differences

in techniques, instruments, anatomical site and patient’s position when performing sonography, which can be decreased by using the same method and mode of sonography. The person who performed sonographic evaluations was not blind to patient’s group in our http://www.selleckchem.com/products/DAPT-GSI-IX.html study. Blinding the assessors also can decrease the bias in the future studies. The authors would like to thank Dr. Jalil Kouhpayezadeh for his confidential

supports in statistical procedures and sample size calculation. Also we would like to appreciate the staff of Firoozgar Clinical Research Development Center (FCRDC) for their technical supports and helps. “
“Optic Neuritis (ONe) is a common feature of Multiple Sclerosis (MS) both in the early phase and during the disease course [1]. Pembrolizumab order MS and ONe are due to demyelination [2], but it has been postulated Urease that vascular mechanisms may have a role in MS and

ONe pathogenesis [3], [4], [5] and [6]. According to a recent hypothesis, cerebrospinal venous system alterations may contribute to the development of the disease and may drive its clinical course [7] and [8]. As a matter of fact, a correlation between the hemodynamic pattern of Chronic Cerebrospinal Venous Insufficiency (CCSVI) and the clinical features in patients with MS has been described [9]. In particular, ONe at onset seems to be associated with Internal Jugular Veins (IJV) and/or of proximal Azygous Vein (AV) high grade stenosis, with consequent reflux in the deep cerebral veins. The blood then flows to the pterygoid plexus, and from there to the facial veins via the deep facial vein, to the cavernous sinus and to the ophthalmic veins. While changes in the hemodynamics of the eye’s arterial system, detected by Doppler ultrasound sonography, have been previously described in MS patients with both acute and chronic ONe [10], [11], [12] and [13], the venous flow has not been studied yet, as far as we know. Taking into account the peculiar environment of the arterial-venous system supplying and draining the Optic Nerve, we have considered it as a representative site for studying the relationship between veins and nervous parenchyma.

Samples were obtained with informed consent. A detailed protocol learn more for gastric culture is provided in the Supplementary materials. Briefly, glands were extracted from 1 cm2 of human tissue using EDTA in cold chelation buffer,17 seeded in Matrigel (BD Biosciences), and overlaid with medium containing advanced Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with penicillin/streptomycin, 10 mmol/L HEPES, GlutaMAX, 1 × B27 (all

from Invitrogen), and 1 mmol/L N-acetylcysteine (Sigma-Aldrich). Growth factors were added to the basal medium as indicated in the Figures. The final human stomach culture medium contained the following essential components: 50 ng/mL epidermal growth factor (EGF) (Invitrogen), 10% noggin-conditioned medium, 10% R-spondin1–conditioned medium, 50% Wnt-conditioned medium, 200 ng/mL fibroblast growth factor (FGF)10

(Peprotech), 1 nmol/L gastrin (Tocris), and 2 μmol/L transforming growth factor (TGF)βi (A-83-01; Tocris). The facultative component was 10 mmol/L nicotinamide (Sigma-Aldrich). After seeding, 10 μmol/L RHOKi (Y-27632; Sigma-Aldrich) was added. Additional tested components were as follows: 100 ng/mL insulin-like growth factor (IGF) (Peprotech), 10 μmol/L p38 inhibitor (SB202190; Sigma-Aldrich), 3 μmol/L GSK3β inhibitor (CHIR99021; Axon Medchem), and 500 nmol/L prostaglandin E (PGE)2 (Tocris). Approximately 1 cm2 of cancer tissue was cut into small fragments and washed in cold chelation buffer until the supernatant was clear. Fragments were subjected to enzymatic Ergoloid digestion by 1.5 mg/mL collagenase (Gibco) and VX-809 nmr 20 μg/mL hyaluronidase (Sigma) in 10 mL advanced

DMEM/F12 (Gibco), supplemented with antibiotics (Primocin; Invivogen), for 1 hour at 37°C with shaking. Cells were washed twice in advanced DMEM/F12, seeded into Matrigel, and overlayed with medium containing HEPES, GlutaMAX, penicillin, streptomycin, B27, n-acetylcysteine, EGF, R-spondin1, noggin, Wnt, FGF10, gastrin, TGFβ inhibitor, and RHOK inhibitor as described earlier. Bacterial strains and culture conditions are specified in the Supplementary materials. For infection studies, organoids were seeded in 50 μL Matrigel in 4-well multidishes (Thermo Scientific). Antibiotic-free medium was refreshed every 2–3 days, with a minimum of 3 medium changes before infection to allow removal of antibiotics from the culture. Organoids were microinjected on day 10 after seeding with an approximate multiplicity of infection (MOI) of 50 unless otherwise stated. For calculation of MOI, organoids were disrupted into single cells by EDTA and cells were counted (approximately 4000 cells/organoid). To achieve a final MOI of 50, bacteria were suspended in advanced DMEM/F12 at a density of 1 × 109/mL and organoids were injected with approximately 0.2 μL bacterial suspension using a micromanipulator and microinjector (M-152 and IM-5B; Narishige) under a stereomicroscope (MZ75; Leica) inside a sterile bench (CleanAir).

Cellular

monolayer Selleck GDC-941 is comprised of midgut epithelial cells and is surrounded on its basal side by a well-established extracellular space. Muscle cells and tracheoles were found adjacent to the extracellular space ( Fig. 1C). Columnar and goblet cells are the most abundant cell types and no specific distribution pattern was observed ( Fig. 1C–E). Both cells define the monolayer height and present luminal-oriented microvilli. They differ by the presence of the goblet cell cavity (GV), a specific luminal space (besides EcS and EnS), rich in microvilli. Vesicles could be detected in the columnar cell, suggesting a trafficking route, perhaps involving multivesicular bodies ( Fig. 1D). Regenerative cells were a less often observed cell type limited to the basal side of the cellular monolayer. As vesicles could be observed inside the epithelial cells cytoplasm, we proceeded towards detecting PolyP stores

using both the modified exopolyphosphatase GSK458 research buy PolyP-binding domain (PPBD) (Saito et al., 2005) and DAPI staining on OCT embedded sections. DAPI has been used as PolyP reporter as its interaction with PolyP yields fluorescence in a different wavelength than the blue emission from DAPI–DNA (Allan and Miller, 1980 and Aschar-Sobbi et al., 2008). Although PolyP stores were present along both columnar and goblet cells, goblet cell cavities and its surroundings were the major regions of accumulation of HAS1 PolyP stores (Fig. 2A and B). To confirm storage of PolyP inside epithelial cells, tissue homogenates were analyzed using a recombinant yeast exopolyphosphatase-based assay (Ruiz et al., 2001b). PolyP strongly concentrated in the posterior midgut of A. gemmatalis but was also detected in the anterior midgut ( Fig. 3A). In that regard, both regions were used in the following experiments. After mechanical lysis and decantation, we could obtain

a fraction rich in PolyP granules as detected by DAPI staining ( Fig. 3B). Under the transmission electron microscope, midgut PolyP granules presented an electron dense morphology ( Fig. 3C, inset) similar to PolyP granules from other models. X-ray microanalysis showed an elemental composition identical to previously found spherite profiles ( Fig. 3C). In that regard, detectable levels of metallic atoms like calcium, magnesium, potassium, sodium, and zinc were present. Phosphorous and chloride were also detected. Manganese, iron and sulfur were less often detected ( Fig. 3D). In our samples, calcium peaks were only observed inside spherites and allowed us to use calcium as a spherite reporter in subsequent experiments. A specific group of polyP-containing organelles from protozoans have been shown to contain bafilomycin A1-sensitive V-ATPases (Docampo et al., 2005 and Scott et al., 1995a) and vanadate-sensitive Ca+2-ATPases (Docampo et al., 1995b) important for metal uptake.

Fig. 3a–c shows the elution profile of three CRPcys-XL preparations,

following cross-linking by SPDP. This procedure may vary, occasionally yielding GDC-0449 price CRPcys-XL of low potency. The preparations selected for this study were of high (3a), acceptable (3b) and low (3c) platelet-reactivity, the latter being unsuitable for use. The contribution of each molecular species and its corresponding Stokes Radius were calculated. Up to 55% of the total peptide formed soluble helix aggregates (Fig. 3a). The amount of soluble helical aggregate increased with potency in CRPcys-XL-induced platelet aggregation (Suppl. Fig. 1). As would be expected from a peptide that can bind, and therefore cluster, at most three GpVI molecules [30], monomeric CRPcys acts as a weak partial agonist [1] and can therefore antagonize the effect of CRPcys-XL, a potent agonist at platelet GpVI. We therefore investigated the triple-helical and polymeric states of our peptides over time to see what degree of polymerization of CRPcys in solution may be caused by oxidative disulfide formation. When CRPcys was freshly dissolved in cold buffer from freeze-dried stock, gel filtration showed that 78% of find more the peptide was triple-helical, with 14% being monomeric and ∼8% being in polymers of 2–4 triple helices (Fig. 4a). Over 14 d at 4 °C, re-folding reduced the monomer content to 9%, while oxidation increased polymers of triple-helices to 14% (Fig. 4b). Freezing

the solution immediately after dissolving the peptide kept it mostly reduced: peptide monomer

was 11% and helical polymers 16% after 80 d (data not shown). However, when the peptide was stored for long periods at 4 °C Idoxuridine or was repeatedly freeze–thawed, much more extensive oxidation occurred, shown in Fig. 4c–e. These polymers, however, still have smaller Stokes Radius compared to CRPcys-XL due to the different cross-linking mechanism. Repeating this experiment with peptide GPPcys in N2-saturated solution resulted in negligible oxidation over 14 d. Likewise, investigating short (1–14 d) and long-term (18 month) storage of GPPcys in air-saturated buffer gave very similar results to those obtained for CRPcys (data not shown). We used a TCEP-reduced sample of III-24 to establish the elution time of its triple-helix (Fig. 5a). The non-reduced control (Fig. 5b) showed an additional feature, a minor peak corresponding to a disulfide-linked peptide dimer (labeled d). Non-reduced solutions equilibrated at 4 °C for 12 h or longer (Fig. 5c) contained significantly less monomeric peptide (labeled m) than the TCEP-reduced sample. As peptide was flash-frozen from a room temperature solution before freeze-drying for storage, the re-dissolved peptide in Fig. 5b reflects the initial room-temperature equilibrium composition, with more monomer. Monomer content falls over 14 d at 4 °C (Fig. 5c), partly due to peptide oxidation and increased dimer content, but mainly due to folding as triple helices (as in Fig.

g. silver in quantum dots); and metals in other technologies (e.g. scandium in solid oxide fuel cells and neodymium in high performance magnets) (Du C59 wnt price and Graedel, 2011). It is necessary to establish baseline background levels so that changes over time can be tracked and to allow an exposure assessment of workers in these industries and those workers involved in the ‘end of product life’ recycling industries. Reference values for many of these elements in the UK population are limited. In 1998 White and Sabbioni, reported reference ranges for thirteen

elements in 200 non-exposed persons in the UK (White and Sabbioni, 1998) and in 2012 reference ranges for seventeen elements analysed in 24 h collections from 111 patients from a renal stones clinic in Southampton (Sieniawska et al., 2012) were reported. In addition,

a CEFIC (European chemical industries association) funded study was reported in 2012 where 436 UK individuals provided urine samples for a range of background analytes to be measured including two metals, mercury and cadmium (Bevan et al., 2012). Several European countries have established human biomonitoring programmes and networks, such as those in Belgium (Schoeters et al., 2012), France (Fréry et al., 2011), Czech Republic (Cerna et al., 2007) and Germany (Schulz et al., 2011 and Schulz et al., 2007). In the U.S., the ‘The National Report on Human Exposure to Environmental Chemicals’ (NHANES, 2011) provides an on-going assessment of the exposure of the U.S. population to environmental chemicals using biological PD-1 antibody monitoring. Although this is an extensive and informative study the utility of the data is restricted because geographic, industrial and dietary differences exist between the US and the UK and because the NHANES programme only reports levels for thirteen

elements. There have also been several European studies that have looked at reference ranges including a recent Belgian RVX-208 study, where Hoet et al. published a comprehensive list of the reference values for 26 trace elements in urine samples from 1022 adults (Hoet et al., 2013). However, as reference values are known to be influenced by environment, lifestyle factors and may differ from countries/regions and if possible they should be established at a national/regional level (Hoet et al., 2013). The data reported in this paper contribute to valuable information on background levels for a wide range of elements in urine samples from non-occupationally exposed adults. The sample cohort is not representative of the whole UK population but this dataset offers information on current levels for the largest number of elements undertaken in any UK study. This study measured repeat samples from the cohort of non-occupationally exposed people to provide an idea of variation of elemental concentrations both between and within individuals. The samples were analysed using modern analytical techniques and instrumentation with good limits of detection.

The network’s gamma oscillations were generated in the model on a local spatial scale within each hypercolumn due to strong lateral feedback inhibition (Whittington et al., 2000 and Brunel and Wang, 2003). A hypercolumn was in fact defined by the spatial extent of this recurrent

inhibition. This localized aspect of feedback inhibition was motivated by histology (Yoshimura et al., 2005 and Yuan et al., 2011). As a result, local coherence was high but on a global scale it considerably dropped, in line with experimental findings (Gray and Singer, 1989, Jacobs et al., ROCK inhibitor 2007 and Sirota et al., 2008). The gamma cycle dynamics allowed small shifts in the excitability of individual neurons to have considerable impact on the spiking output (Fries et al., 2007 and Lundqvist et al., 2010). Therefore, small top-down attentional excitation or external stimulation modulating spike timing

can have a strong effect on networks operating in the gamma regime with fast switching between competing assemblies (Buehlmann http://www.selleckchem.com/products/AZD0530.html and Deco, 2008 and Lundqvist et al., 2010). As a result, this type of gamma oscillations has several interesting features in functional networks. It underlies a winner-take-all mechanism (Fries et al., 2007 and Lundqvist et al., 2010), provides low firing rates in the synchronous irregular regime (Brunel and Wang, 2003), and yet allows for fast stimulus/attention driven switching between competitors (Borgers et al., 2005, Fries et al., 2007 and Lundqvist et al., 2010). The strong dependence of coherence on spatial distance evident for gamma oscillations (Sirota et al., 2008) reflected the local nature of the computations they mediated in the model. The global coherence was still however significantly above zero and it was even higher for short-lived rather than stationary attractors. This effect was due to the fact that the gamma oscillations were nested on the highly coherent theta rhythm providing the synchronization

framework within Dichloromethane dehalogenase a short period of time following the attractor onset. An effect of increased gamma synchrony, reported in experiments during memory tasks (Miltner et al., 1999 and Lutzenberger et al., 2002) could thus potentially reflect burstiness or nesting on the slower rhythms. Theta oscillations exhibited considerably higher global coherence than the gamma rhythm. They reflected the activation of a distributed memory pattern in the network. The finite dwell time of attractors resulting in theta oscillations was governed by neural fatigue, but could equally well have been implemented with a second type of interneurons (Krishnamurthy et al., 2012).

In 1959, Russell and Burch performed a study based upon the philosophical concept of humanity, in which they observed that some biological experiments could be classed as “inhumane” based upon the levels of pain, distress and lasting harm experienced by the test CX-5461 ic50 animals (Russell et al., 1959). Their research provided the systematic basis of the 3R’s: Replace, Reduce and Refine the use

of sentient beings in experimental biology. This led to a general expansion of funding sources for ex vivo and in vitro alternative methods, to reduce the dependency on live animal testing, whilst also creating a political climate whereby alternative procedures were incorporated into federal and government legislation ( Stephens and Mak, 2013). In this review, we will provide an overview of established and newly developed ocular toxicity tests and discuss their advantages and potential limitations. Live animals have selleck chemicals llc been used to assess and evaluate potentially harmful products to the eyes since the 18th century (Wilhelmus, 2001). The international standard assay for acute ocular toxicity is the rabbit in vivo Draize eye test ( Draize et al., 1944) which was developed in the 1940s by the Food and Drugs Administration (FDA) in response to new laws implemented following permanent eye injuries occurring due to cosmetics use in

the 1930s ( Calabrese, 1987). Draize testing is a government endorsed protocol accepted by the Organization for Economic Co-operation DOK2 and Countries Development (OECD, test guidance [TG] 405) ( Huhtala et al., 2008 and OECD, 2012b). New Zealand white (NZW) rabbits are most commonly used as they have large eyes with a well described anatomy and physiology, are easy to handle, readily available and are relatively inexpensive

( Wilhelmus, 2001). The procedure involves the application of 0.1 ml (or 0.1 g solid) test substance onto the cornea and conjunctival sac of one eye of a conscious rabbit for up to 72 h while the other eye serves as an untreated control ( Draize et al., 1944). The original Draize protocol used at least six rabbits per test, but this was reduced to three animals or a single animal when serious ocular damage is expected, with those with severe lesions being humanely euthanized. The latest Draize test guidelines include the application and delivery of analgesics and anesthetics ( OECD, 2012b) to reduce animal pain and suffering. Rabbits are observed at selected intervals for up to 21 days for signs of irritation including redness, swelling, cloudiness, edema, hemorrhage, discharge and blindness ( Huhtala et al., 2008). In cases where severe eye irritation or pain is observed, it is recommended that the animals are euthanized or removed from the study prior to the 21 day time point ( OECD, 2012b).