Contaminants and Where to Find Them: Microbiological Quality Control in Axenic Animal Facilities.

The use of axenic animal models in experimental research has exponentially grown in the past few years and the most reliable way for confirming their axenic status remains unclear. It is especially the case when using individual ventilated positive-pressure cages such as the Isocage. This type of cage are at a greater risk of contamination and expose animals to a longer handling process leading to more potential stress when opened compared to isolators. The aim of this study was to propose simple ways to detect microbial contaminants with Isocages type isolator resulting by developing, validating and optimizing three different methods (culture, microscopy, and molecular). These three approaches were also tested in situ by spiking 21 axenic mice with different microorganisms. Our results suggest that the culture method can be used for feces and surface station (IBS) swabs exclusively (in Brain Heart Infusion for 7 days at 25°C and 37°C in aerobic conditions, and at 30°C in anaerobic conditions), while microscopy (wet mounts) and molecular method (quantitative PCR) were only suitable for fecal matter analyses. In situ results suggests that the culture and molecular methods can detect up to 100% of bacterial contamination events while the microscopy approach generates many erroneous results when not performed by a skilled microscopist. In situ results also suggest that when an axenic mouse is contaminated by a microbial agent, the microorganism will colonize the mouse to such an extent that detection is obvious in 4 days, in average. This report validates simple but complimentary tests that can be used for optimal detection of contaminants in axenic animal facilities using Isocage type isolators.

Managing the bacterial contamination risk in an axenic mice animal facility.

A gap exists between good laboratory practices with axenic animals and the procedures applied. This work examined the efficacy of sodium dichloroisocyanurate (MB-10) and potassium peroxymonosulfate (Virkon™) disinfectants, as well as the appropriate soaking time for materials used with the ISOcage Biosafety Station™. We also compared the microbial load in cage systems hosting mice over 2 weeks in axenic rooms (ARs) and in typical specific-pathogen-free (SPF) non-axenic rooms (NARs) to identify resistant microorganisms, targeted for longer soaking disinfection, and evaluated the necessary procedures for reducing the microbial load in AR. Staphylococcus was the most frequently isolated genus (in both ARs and NARs). An average of three spore-forming microorganisms per cage were counted from AR. The disinfection time to reach 1 log reduction for Bacillus atrophaeus spores varied from 138 s (100 ppm MB-10) to 290 (Virkon™) to <20 s for S. epidermidis (100 ppm MB-10). AR management protocols lead to a microbial load that is 1000 times lower than that found in NARs. Data comparing the microbial load in SPF and axenic facilities can be used to improve the effectiveness of their microbial control procedures.

Computed Tomography Myelosimulation Versus Magnetic Resonance Imaging Registration to Delineate the Spinal Cord During Spine Stereotactic Radiosurgery.

BACKGROUND: Underestimation of the spinal cord's volume or position during spine stereotactic radiosurgery can lead to severe myelopathy, whereas overestimation can lead to tumor underdosage. Spinal cord delineation is commonly achieved by registering a magnetic resonance imaging (MRI) study with a computed tomography (CT) simulation scan or by performing myelography during CT simulation (myelosim). We compared treatment planning outcomes for these 2 techniques. METHODS: Twenty-three cases of spine stereotactic radiosurgery were analyzed that had both a myelosim and corresponding MRI study for registration. The spinal cord was contoured on both imaging data sets by 2 independent blinded physicians, and Dice similarity coefficients were calculated to compare their spatial overlap. Two treatment plans (16 Gy and 18 Gy) were created using the MRI and CT contours (92 plans total). Dosimetric parameters were extracted and compared by modality to assess tumor coverage and spinal cord dose. RESULTS: No differences were found in the partial spinal cord volumes contoured on MRI versus myelosim (4.71 ± 1.09 vs. 4.55 ± 1.03 cm3; P = 0.34) despite imperfect spatial agreement (mean Dice similarity coefficient, 0.68 ± 0.05). When the registered MRI contours were used for treatment planning, significantly worse tumor coverage and greater spinal cord doses were found compared with myelosim planning. For the 18-Gy plans, 10 of 23 MRI cases (43%) exceeded the spinal cord or cauda dose constraints when using myelosim as the reference standard. CONCLUSIONS: Significant spatial, rather than volumetric, differences were found between the MRI- and myelosim-defined spinal cord structures. Tumor coverage was compromised with MRI-based planning, and the high spinal cord doses were a concern. Future work is necessary to compare thin-cut, volumetric MRI registration or MRI simulation with myelosim.

Avian influenza viruses that cause highly virulent infections in humans exhibit distinct replicative properties in contrast to human H1N1 viruses.

Avian influenza viruses present an emerging epidemiological concern as some strains of H5N1 avian influenza can cause severe infections in humans with lethality rates of up to 60%. These have been in circulation since 1997 and recently a novel H7N9-subtyped virus has been causing epizootics in China with lethality rates around 20%. To better understand the replication kinetics of these viruses, we combined several extensive viral kinetics experiments with mathematical modelling of in vitro infections in human A549 cells. We extracted fundamental replication parameters revealing that, while both the H5N1 and H7N9 viruses replicate faster and to higher titers than two low-pathogenicity H1N1 strains, they accomplish this via different mechanisms. While the H7N9 virions exhibit a faster rate of infection, the H5N1 virions are produced at a higher rate. Of the two H1N1 strains studied, the 2009 pandemic H1N1 strain exhibits the longest eclipse phase, possibly indicative of a less effective neuraminidase activity, but causes infection more rapidly than the seasonal strain. This explains, in part, the pandemic strain's generally slower growth kinetics and permissiveness to accept mutations causing neuraminidase inhibitor resistance without significant loss in fitness. Our results highlight differential growth properties of H1N1, H5N1 and H7N9 influenza viruses.

Assessing the Dosimetric Accuracy of Magnetic Resonance-Generated Synthetic CT Images for Focal Brain VMAT Radiation Therapy.

PURPOSE: The purpose of this study was to assess the dosimetric accuracy of synthetic CT (MRCT) volumes generated from magnetic resonance imaging (MRI) data for focal brain radiation therapy. METHODS AND MATERIALS: A study was conducted in 12 patients with gliomas who underwent both MR and CT imaging as part of their simulation for external beam treatment planning. MRCT volumes were generated from MR images. Patients' clinical treatment planning directives were used to create 12 individual volumetric modulated arc therapy (VMAT) plans, which were then optimized 10 times on each of their respective CT and MRCT-derived electron density maps. Dose metrics derived from optimization criteria, as well as monitor units and gamma analyses, were evaluated to quantify differences between the imaging modalities. RESULTS: Mean differences between planning target volume (PTV) doses on MRCT and CT plans across all patients were 0.0% (range: -0.1 to 0.2%) for D(95%); 0.0% (-0.7 to 0.6%) for D(5%); and -0.2% (-1.0 to 0.2%) for D(max). MRCT plans showed no significant changes in monitor units (-0.4%) compared to CT plans. Organs at risk (OARs) had average D(max) differences of 0.0 Gy (-2.2 to 1.9 Gy) over 85 structures across all 12 patients, with no significant differences when calculated doses approached planning constraints. CONCLUSIONS: Focal brain VMAT plans optimized on MRCT images show excellent dosimetric agreement with standard CT-optimized plans. PTVs show equivalent coverage, and OARs do not show any overdose. These results indicate that MRI-derived synthetic CT volumes can be used to support treatment planning of most patients treated for intracranial lesions.

Impact of the H275Y and I223V Mutations in the Neuraminidase of the 2009 Pandemic Influenza Virus In Vitro and Evaluating Experimental Reproducibility.

The 2009 pandemic H1N1 (H1N1pdm09) influenza virus is naturally susceptible to neuraminidase (NA) inhibitors, but mutations in the NA protein can cause oseltamivir resistance. The H275Y and I223V amino acid substitutions in the NA of the H1N1pdm09 influenza strain have been separately observed in patients exhibiting oseltamivir-resistance. Here, we apply mathematical modelling techniques to compare the fitness of the wild-type H1N1pdm09 strain relative to each of these two mutants. We find that both the H275Y and I223V mutations in the H1N1pdm09 background significantly lengthen the duration of the eclipse phase (by 2.5 h and 3.6 h, respectively), consistent with these NA mutations delaying the release of viral progeny from newly infected cells. Cells infected by H1N1pdm09 virus carrying the I223V mutation display a disadvantageous, shorter infectious lifespan (17 h shorter) than those infected with the wild-type or MUT-H275Y strains. In terms of compensating traits, the H275Y mutation in the H1N1pdm09 background results in increased virus infectiousness, as we reported previously, whereas the I223V exhibits none, leaving it overall less fit than both its wild-type counterpart and the MUT-H275Y strain. Using computer simulated competition experiments, we determine that in the presence of oseltamivir at doses even below standard therapy, both the MUT-H275Y and MUT-I223V dominate their wild-type counterpart in all aspects, and the MUT-H275Y outcompetes the MUT-I223V. The H275Y mutation should therefore be more commonly observed than the I223V mutation in circulating H1N1pdm09 strains, assuming both mutations have a similar impact or no significant impact on between-host transmission. We also show that mathematical modelling offers a relatively inexpensive and reliable means to quantify inter-experimental variability and assess the reproducibility of results.

Effects of intracerebroventricular and intra-accumbens melanin-concentrating hormone agonism on food intake and energy expenditure.

The brain melanin-concentrating hormone (MCH) system represents an anabolic system involved in energy balance regulation through influences exerted on the homeostatic and nonhomeostatic controls of food intake and energy expenditure. The present study was designed to further delineate the effect of the MCH system on energy balance regulation by assessing the actions of the MCH receptor 1 (MCHR1) agonism on both food intake and energy expenditure after intracerebroventricular (third ventricle) and intra-nucleus-accumbens-shell (intraNAcSH) injections of a MCHR1 agonist. Total energy expenditure and substrate oxidation were assessed following injections in male Wistar rats using indirect calorimetry. Food intake was also measured. Pair-fed groups were added to evaluate changes in thermogenesis that would occur regardless of the meal size and its thermogenic response. Using such experimental conditions, we were able to demonstrate that acute MCH agonism in the brain, besides its orexigenic effect, induced a noticeable change in the utilization of the main metabolic fuels. In pair-fed animals, MCH significantly reduced lipid oxidation when it was injected in the third ventricle. Such an effect was not observed following the injection of MCH in the NAcSH, where MCH nonetheless strongly stimulated appetite. The present results further delineate the influence of MCH on energy expenditure and substrate oxidation while confirming the key role of the NAcSH in the effects of the MCH system on food intake.

A central thermogenic-like mechanism in feeding regulation: an interplay between arcuate nucleus T3 and UCP2.

The active thyroid hormone, triiodothyronine (T3), regulates mitochondrial uncoupling protein activity and related thermogenesis in peripheral tissues. Type 2 deiodinase (DII), an enzyme that catalyzes active thyroid hormone production, and mitochondrial uncoupling protein 2 (UCP2) are also present in the hypothalamic arcuate nucleus, where their interaction and physiological significance have not been explored. Here, we report that DII-producing glial cells are in direct apposition to neurons coexpressing neuropeptide Y (NPY), agouti-related protein (AgRP), and UCP2. Fasting increased DII activity and local thyroid hormone production in the arcuate nucleus in parallel with increased GDP-regulated UCP2-dependent mitochondrial uncoupling. Fasting-induced T3-mediated UCP2 activation resulted in mitochondrial proliferation in NPY/AgRP neurons, an event that was critical for increased excitability of these orexigenic neurons and consequent rebound feeding following food deprivation. These results reveal a physiological role for a thyroid-hormone-regulated mitochondrial uncoupling in hypothalamic neuronal networks.

A randomized cross over trial of tolerability and compliance of a micronutrient supplement with low iron separated from calcium vs high iron combined with calcium in pregnant women [ISRCTN56071145].

BACKGROUND: Prenatal micronutrient combinations with high iron content are associated with high rates of gastrointestinal symptoms. This coupled with nausea and vomiting of pregnancy results in women often discontinuing their multivitamins. A new prescription supplement (PregVit) that separates iron from calcium in two tablets--morning and evening, has lower elemental iron content (35 mg), but results in similar extent of iron absorption when compared to another supplement containing (60 mg) of elemental iron (Materna). The objectives of this study were to compare tolerability and compliance with PregVit vs. a supplement with high iron content (Materna), in pregnant women. METHODS: Randomized, crossover open labeled study in 135 pregnant women attending outpatient clinics in Ontario and Quebec. RESULTS: Use of PregVit was associated with a 30% reduction in constipation rate as compared to Materna. Both products demonstrated similar compliance rates. Compliance of Materna was negatively associated with the severity of nausea and vomiting of pregnancy. No such correlation was found for PregVvit. CONCLUSION: PregVit, a supplement with lower iron content (35 mg), has significantly decreased constipation rates as compared to 60 mg iron--Materna and has similar compliance rates. High iron content in multivitamin supplements is associated with adverse effects in pregnancy.

Increased infarct size and lack of hyperphagic response after focal cerebral ischemia in peroxisome proliferator-activated receptor beta-deficient mice.

Peroxisome proliferator-activated receptors (PPARs) are involved in energy expenditure, regulation of inflammatory processes, and cellular protection in peripheral tissues. Among the different types of PPARs, PPARbeta is the only one to be widely expressed in cortical neurons. Using PPARbeta knockout (KO) mice, we report here a detailed investigation of the role of PPARbeta in cerebral ischemic damage, associated inflammatory and antioxidant processes as well as food intake regulation after middle cerebral artery occlusion (MCAO). The PPARbeta KO mice had a two-fold increase in infarct size compared with wild-type (WT) mice. Brain oxidative stress was dramatically enhanced in these KO mice, as documented by an increased content of malondialdehyde, decreased levels of glutathione and manganese superoxide dismutase, and no induction of uncoupling protein 2 (UCP2) mRNA. Unlike WT mice, PPARbeta KO mice showed a marked increase of prooxidant interferon-gamma but no induction of nerve growth factor and tumor necrosis factor alpha after MCAO. In WT mice, MCAO resulted in inflammation-specific transient hyperphagia from day 3 to day 5 after ischemia, which was associated with an increase in neuropeptide Y (NPY) mRNA. This hyperphagic phase and NPY mRNA induction were not observed in PPARbeta KO mice. Furthermore, our study also suggests for the first time that UCP2 is involved in MCAO food intake response. These data indicate that PPARbeta plays an important role in integrating and regulating central inflammation, antioxidant mechanisms, and food intake after MCAO, and suggest that the use of PPARbeta agonists may be of interest for the prevention of central ischemic damage.

A role for lipoprotein lipase during synaptic remodeling in the adult mouse brain.

Lipoprotein lipase (LPL) is a member of a lipase family known to hydrolyze triglyceride molecules found in lipoprotein particles. This particular lipase also has a role in the binding of lipoprotein particles to different cell-surface receptors. LPL has been identified in the brain but has no specific function yet. This study aimed at elucidating the role of LPL in the brain in response to injury. Mice were subjected to hippocampal deafferentation using the entorhinal cortex lesion and mRNA and protein expression were assessed over a time-course of degeneration/reinnervation. Hippocampal LPL levels peaked at 2 days post-lesion (DPL) both at the mRNA and protein levels. No change was observed for receptors of the LDL-receptor family or RAP at DPL 2 in the hippocampus but the glia-specific syndecan-4 was found to be significantly upregulated at DPL 2. These results suggest that LPL is involved in the recycling of cholesterol and lipids released from degenerating terminals after a lesion through a syndecan-4-dependent pathway.

Requirement for enzymatically active lipoprotein lipase in neuronal differentiation: a site-directed mutagenesis study.

Lipoprotein lipase (LPL) is well known for its role in the catabolism of plasma triglyceride (Tg)-rich lipoproteins, such as very low density lipoproteins (VLDL) and chylomicrons. The action of LPL on Tg-rich lipoproteins provides free fatty acids to skeletal muscle and adipose tissues, the main sites of LPL synthesis. Several studies have demonstrated that LPL is widely expressed in the parenchyma of brain tissues. We have recently shown that LPL expression is essential for promoting VLDL-stimulated differentiation of Neuro-2A cells. In the present study, we have generated stably transfected Neuro-2A cell lines expressing either wild-type LPL or various LPL mutants, including three enzymatically inactive variants (Asp156Asn, Gly188Glu and Pro207Leu), an enzymatically defective variant (Asn291Ser) and a variant known to express increased LPL activity (Ser447Ter). In Neuro-2A cells expressing enzymatically inactive LPL variants, VLDL-stimulated differentiation and neurite extension were not observed. However, in Neuro-2A cells expressing partially active or overactive LPL variants, VLDL added to the cultured medium was able to induce the phenotypic differentiation similar to that observed in Neuro-2A cells expressing wild-type LPL. In summary, these data show that the availability of fatty acids, resulting from the catabolism of VLDL by LPL, is required to promote the phenotypical differentiation of neuroblastoma cells. These findings may have significant relevance to lipoprotein metabolism in the brain as well as to the maturation and regeneration of nervous tissues in carriers of mutant LPL.

Kainic acid upregulates uncoupling protein-2 mRNA expression in the mouse brain.

Intraperitoneal injection of kainic acid (KA) in C57BL/6J and 129T2SvEmsJ mice led to a transient induction of uncoupling protein-2 (Ucp2) mRNA expression in several brain regions, which included the CA1 subfield of the hippocampus, the dorsal endopiriform nucleus and the piriform cortex in both strains. In all those regions, levels of Ucp2 mRNA expression, as determined by in situ hybridization, peaked at 24 h and returned to basal levels within 72 h post-injection. The increase in mRNA expression was mainly observed in neurons, with microglial cells displaying only scattered expression of the gene. The neuronal induction of Ucp2 in response to KA was stronger in 129T2SvEmsJ mice than in C57BL/6J, which suggests a role for Ucp2 in excitotoxic challenges and neuroprotection.

Lipoprotein lipase affects the survival and differentiation of neural cells exposed to very low density lipoprotein.

Lipoprotein lipase (LPL) is a key enzyme involved in the metabolism of lipoproteins, providing tissues like adipose tissue or skeletal muscle with fatty acids. LPL is also expressed in the brain, fulfilling yet unknown functions. Using a neuroblastoma cell line transfected with a NEO- or a LPL-expression vector, we have developed a model to study the function of LPL in neurons exposed to native or copper-oxidized lipoproteins. The addition to the culture media of VLDL with 10 microm copper sulfate led to a significant reduction in the viability of NEO transfectants whereas LPL-transfectants were protected from this injury. In the presence of VLDL and CuSO(4), LPL transfectants were even able to display significant neurite extension. This neuritogenic effect was also observed in LPL transfectants exposed to native lipoproteins. However, addition of VLDL particles oxidized with CuSO(4) prior to their addition to the culture media resulted in neurotoxic effects on LPL transfectants. These findings suggest that the presence of LPL in cultured neuronal cells modulates the physiological response of neurons following exposure to native or oxidized lipoproteins. LPL could thus play a key role in the differentiation of Neuro-2A cells and in the pathophysiological effects of oxidative stress in several neurodegenerative disorders.