Name: design and development of a model to study the effect of supplemental oxygen on the cystic fibrosis airway microbiome
Uploaded: 2021-08-03T14:00:00
Description: Watch this Scientific Journal Video about Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome at JoVE.com

Part of this work was performed at the Marine Biological Lab with support from the Marine Biological Lab, DOE (DE-SC0016127), NSF (MCB1822263), HHMI (grant number 5600373), and a gift from the Simons Foundation.

This study received approval from the Partners Institutional Review Board (Protocol # 2018P002934). Inclusion criterion included adult patients with cystic fibrosis who provided written informed consent for the study. There was no exclusion criterion. According to protocol guidelines, all sputum samples were collected from patients with cystic fibrosis during a scheduled outpatient visit with their clinical provider.
1. Artificial Sputum Medium Preparation
NOTE: Quant...

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bioBakery:+a+meta'omic+analysis+environment.">bioBakery: a meta'omic analysis environment.</a> Bioinformatics. 34 (7), 1235-1237 (2018).</li><li>Truong, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MetaPhlAn2+for+enhanced+metagenomic+taxonomic+profiling.">MetaPhlAn2 for enhanced metagenomic taxonomic profiling.</a> Nature Methods. 12 (10), 902-903 (2015).</li><li>Stammler, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjusting+microbiome+profiles+for+differences+in+microbial+load+by+spike-in+bacteria.">Adjusting microbiome profiles for differences in microbial load by spike-in bacteria.</a> Microbiome. 4 (1), 28 (2016).</li><li>Henke, M. O., Renner, A., Huber, R. M., Seeds, M. C., Rubin, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUC5AC+and+MUC5B+mucins+are+decreased+in+cystic+fibrosis+airway+secretions.">MUC5AC and MUC5B mucins are decreased in cystic fibrosis airway secretions.</a> American Journal of Respiratory Cell and Molecular Biology. 31 (1), 86-91 (2004).</li><li>Henderson, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+airway+secretions+exhibit+mucin+hyper+concentration+and+increased+osmotic+pressure.">Cystic fibrosis airway secretions exhibit mucin hyper concentration and increased osmotic pressure.</a> Journal of Clinical Investigation. 124 (7), 3047-3060 (2014).</li><li>Matthews, L. W., Spector, S., Lemm, J., Potter, J. 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In this study, an in vitro model was developed to study the effect of hyperoxia on lung microbial communities. This model, based on artificial sputum medium and daily sparging of serum bottles, maintains elevated oxygen concentrations and supports the growth of microbes identified in sputum from pwCF.
There are several critical steps of this approach. First is the choice to use filter-sterilization rather than heat-sterilization of the artificial sputum medium. Filter-sterilization pr...

Cystic fibrosis (CF) is a genetic disease characterized by an inability to clear thick mucus from the lungs, leading to repeated infections and progressive lung function decline that often results in the need for lung transplantation or death. The airway microbiome of people with cystic fibrosis (pwCF) appears to track disease activity1, with a reduction in microbial diversity associated with adverse long-term outcomes2,3. In clinical studies of pwCF, supplemental oxygen therapy has been associated with more advanced disease4,5, though traditionally, the use of oxygen therapy has been viewed as simply a marker for disease severity6. Recent studies from a clinical trial of patients with respiratory failure have shown that higher patient oxygen levels are paradoxically associated with an increase in serious bacterial infections and higher mortality7, suggesting that supplemental oxygen may contribute to disease pathogenesis. The effect of supplemental oxygen on the cystic fibrosis lung microbiome and associated lung and airway microbial communities has not been well studied.
Mechanistic studies often cannot be performed directly on human subjects due to logistical difficulties and potential ethical issues associated with interventions of unknown medical benefit or harm. Translational approaches that integrate human biospecimens into model systems can offer important biological insights in these cases. While the ability to use or tolerate oxygen has traditionally been an important component of microbial classification, little is known about how the therapeutic introduction of supplemental oxygen to the environment might perturb airway microbial communities. To shed light on the unknown effects of supplemental oxygen on the airway microbiomes of pwCF, we needed to address two major challenges; first, the creation of a culture medium that physiologically approximates the composition of CF sputum; second, the creation of a model system that allows the maintenance of elevated oxygen concentrations in culture over extended periods of time.
Artificial sputum media (ASM) are widely used to emulate lung sputum ex vivo8,9,10, but there is no clear consensus on a specific recipe. This protocol describes an artificial sputum medium recipe and preparation strategy carefully designed to physiologically approximate sputum from pwCF. Table 1 outlines the chosen recipe values based on published literature. Basic chemical components and pH were matched to values identified by studies of human CF sputum11,12,13. Low concentration physiological nutrients were added using egg yolk, which was included as 0.25% of the final volume10, as well as vitamin and trace metal mixes14,15. Mucin, the key component of sputum16, was included at 1% w/v14. Although more labor-intensive, filter sterilization was chosen over the more conventional practice of heat sterilization to reduce potential problems from heat-induced denaturation of essential media components10. An additional benefit of filter sterilization is that it generates media that are transparent (heat-sterilization can create turbid media due to precipitation and coagulation of salts and proteins), allowing this artificial sputum media to be used to follow microbial growth based on increases in turbidity.
This model system for the hyperoxic culture is based on anaerobic culturing techniques where oxygen is added rather than removed, creating a model for the effect of supplemental oxygen use for pwCF. Figure 1 and the associated oxygen sparging protocol outlines the components of an oxygen sparging system, which can be obtained at low cost from general laboratory and hospital suppliers. This system enables the mixing of compressed oxygen and air to fixed concentrations ranging from 21%-100% oxygen. The integration of an oxygen sensor allows for the verification of the concentration of the output gas mixture, as well as checking the outflow gas composition of previously sparged serum bottles to verify that the oxygen conditions have been maintained within the desired range.
This protocol outlines procedures to create an artificial sputum medium, the construction and use of an oxygen sparging system, and the application of both to culture CF sputum under differential oxygen conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BME Vitamins (100x) Solution</td>
			<td>MilliporeSigma</td>
			<td>B6891</td>
			<td>Concentrated solution of supplemental vitamins.</td>
		</tr>
		<tr>
			<td>Crimper, 30 mm</td>
			<td>DWK Life Sciences</td>
			<td>224307</td>
			<td>Crimper for attaching aluminum seals to serum bottles.</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>MilliporeSigma</td>
			<td>G7021</td>
			<td>Solid glucose powder (dextrorotatory isomer).</td>
		</tr>
		<tr>
			<td>Diaphragm Pump ME 2 NT</td>
			<td>VACUUBRAND</td>
			<td>20730003</td>
			<td>Vacuum pump for vacuum filtration.</td>
		</tr>
		<tr>
			<td>Egg Yolk Emulsion</td>
			<td>HiMedia</td>
			<td>FD045</td>
			<td>Sterile emulsion of 30% egg yolk in saline.</td>
		</tr>
		<tr>
			<td>Ferritin, Cationized from Horse Spleen</td>
			<td>MilliporeSigma</td>
			<td>F7879</td>
			<td>Ferritin (iron-storage protein) solution.</td>
		</tr>
		<tr>
			<td>FIREBOY plus Safety Bunsen Burner</td>
			<td>Integra Biosciences</td>
			<td>144000</td>
			<td>Bunsen burner with user interface and safety features.</td>
		</tr>
		<tr>
			<td>Hydrion pH Paper (1.0&#8211;14.0)</td>
			<td>Micro Essential Laboratory</td>
			<td>94</td>
			<td>pH testing paper for the range of 1.0&#8211;14.0.</td>
		</tr>
		<tr>
			<td>Hydrion pH Paper (4.0&#8211;9.0)</td>
			<td>Micro Essential Laboratory</td>
			<td>55</td>
			<td>pH testing paper for the range of 4.0&#8211;9.0.</td>
		</tr>
		<tr>
			<td>Hydrion pH Paper (6.0&#8211;8.0)</td>
			<td>Micro Essential Laboratory</td>
			<td>345</td>
			<td>pH testing paper for the range of 6.0&#8211;8.0.</td>
		</tr>
		<tr>
			<td>Hypodermic Needle-Pro EDGE Safety Device, 18 G</td>
			<td>Smiths Medical</td>
			<td>401815</td>
			<td>18 G needles with safety caps.</td>
		</tr>
		<tr>
			<td>In-Line Pressure Gauge</td>
			<td>MilliporeSigma</td>
			<td>20469</td>
			<td>Gas pressure gauge for monitoring bottle pressure.</td>
		</tr>
		<tr>
			<td>Innova 42 Incubated Shaker</td>
			<td>Eppendorf</td>
			<td>2231000756</td>
			<td>Combination incubator/orbital shaker.</td>
		</tr>
		<tr>
			<td>Luer-Lok Syringe with Attached Needle</td>
			<td>Becton Dickinson</td>
			<td>309580</td>
			<td>Combination 3 mL syringe and 18 G needle.</td>
		</tr>
		<tr>
			<td>Luer Valve Assortment</td>
			<td>World Precision Instruments</td>
			<td>14011</td>
			<td>Valves for gas flow tubing.</td>
		</tr>
		<tr>
			<td>LSE Orbital Shaker</td>
			<td>ThermoFisher Scientific</td>
			<td>6780-NP</td>
			<td>Orbital shaker to agitate media during filtration.</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate</td>
			<td>MilliporeSigma</td>
			<td>M2773</td>
			<td>Solid epsom salt (magnesium sulfate heptahydrate).</td>
		</tr>
		<tr>
			<td>Medical Air Single Stage Regulator with Flowmeter</td>
			<td>Western Enterprises</td>
			<td>M1-346-15FM</td>
			<td>Air flow rate regulator with 15 L/min meter.</td>
		</tr>
		<tr>
			<td>MEM Amino Acids (50x) Solution</td>
			<td>MilliporeSigma</td>
			<td>M5550</td>
			<td>Concentrated solution of essential amino acids.</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids (100x) Solution</td>
			<td>MilliporeSigma</td>
			<td>M7145</td>
			<td>Concentrated solution of non-essential amino acids.</td>
		</tr>
		<tr>
			<td>Millex-GP Filter, 0.22 &#181;m</td>
			<td>MilliporeSigma</td>
			<td>SLMP25SS</td>
			<td>0.22 &#181;m polyethersulfone membrane sterile syringe filter.</td>
		</tr>
		<tr>
			<td>Milli-Q Academic</td>
			<td>MilliporeSigma</td>
			<td>ZMQS60E01</td>
			<td>Milli-Q sterile water filtration system.</td>
		</tr>
		<tr>
			<td>MiniOX 3000 Oxygen Monitor</td>
			<td>MSA</td>
			<td>814365</td>
			<td>Gas flow oxygen percentage monitor.</td>
		</tr>
		<tr>
			<td>MOPS Buffer (1 M, pH 9.0)</td>
			<td>Boston BioProducts</td>
			<td>BBM-90</td>
			<td>MOPS buffer for adjusting media pH.</td>
		</tr>
		<tr>
			<td>Mucin from Porcine Stomach</td>
			<td>MilliporeSigma</td>
			<td>M2378</td>
			<td>Mucin (glycosylated gel-forming protein) powder.</td>
		</tr>
		<tr>
			<td>Natural Polypropylene Barbed Fitting Kit</td>
			<td>Harvard Apparatus</td>
			<td>72-1413</td>
			<td>Connectors for gas flow tubing.</td>
		</tr>
		<tr>
			<td>Nextera XT DNA Library Preparation Kit</td>
			<td>Illumina</td>
			<td>FC-131-1096</td>
			<td>Library preparation for identification during sequencing.</td>
		</tr>
		<tr>
			<td>NovaSeq 6000 Sequencing System</td>
			<td>Illumina</td>
			<td>770-2016-025-N</td>
			<td>Shotgun sequencing platform for generating sample reads.</td>
		</tr>
		<tr>
			<td>Oxygen Single Stage Regulator with Flowmeter</td>
			<td>Western Enterprises</td>
			<td>M1-540-15FM</td>
			<td>Oxygen flow rate regulator with 15 L/min meter.</td>
		</tr>
		<tr>
			<td>Oxygen Tubing with 2 Standard Connectors</td>
			<td>SunMed</td>
			<td>2001-01</td>
			<td>Tubing for connecting gas system components.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, 10x, pH 7.4</td>
			<td>Molecular Biologicals International</td>
			<td>MRGF-6235</td>
			<td>Concentrated phosphate-buffered saline solution.</td>
		</tr>
		<tr>
			<td>PC 420 Hot Plate/Stirrer</td>
			<td>Marshall Scientific</td>
			<td>CO-PC420</td>
			<td>Combination hot plate/stirrer.</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>MilliporeSigma</td>
			<td>P9541</td>
			<td>Solid potassium chloride salt.</td>
		</tr>
		<tr>
			<td>PTFE Disposable Stir Bars</td>
			<td>ThermoFisher Scientific</td>
			<td>14-513-95</td>
			<td>Disposable magnetic stir bars.</td>
		</tr>
		<tr>
			<td>PTFE Thread Seal Teflon Tape</td>
			<td>VWR</td>
			<td>470042-938</td>
			<td>Teflon tape for reinforcing gas system connections.</td>
		</tr>
		<tr>
			<td>Q-Gard 2 Purification Cartridge</td>
			<td>MilliporeSigma</td>
			<td>QGARD00D2</td>
			<td>Purification cartridge for Milli-Q system.</td>
		</tr>
		<tr>
			<td>Reusable Media Storage Bottles</td>
			<td>ThermoFisher Scientific</td>
			<td>06-423A</td>
			<td>Bottles for mixing and storing culture media.</td>
		</tr>
		<tr>
			<td>Rubber Stopper, 30 mm, Gray Bromobutyl</td>
			<td>DWK Life Sciences</td>
			<td>224100-331</td>
			<td>Rubber stoppers for serum bottles.</td>
		</tr>
		<tr>
			<td>Serum Bottle with Molded Graduations, 500 mL</td>
			<td>DWK Life Sciences</td>
			<td>223952</td>
			<td>Glass serum bottles for sealed culturing.</td>
		</tr>
		<tr>
			<td>Small Bore Extension Set</td>
			<td>Braun Medical</td>
			<td>471960</td>
			<td>Tubing extension with luer lock connectors.</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>MilliporeSigma</td>
			<td>S3014</td>
			<td>Solid sodium chloride salt.</td>
		</tr>
		<tr>
			<td>Spike-in Control I (High Microbial Load)</td>
			<td>ZymoBIOMICS</td>
			<td>D6320</td>
			<td>Spike-in microbes (I. halotolerans and A. halotolerans) for absolute microbial load calculations</td>
		</tr>
		<tr>
			<td>Stericup Quick Release Sterile Vacuum Filtration System</td>
			<td>MilliporeSigma</td>
			<td>S2GPU02RE</td>
			<td>250 mL 0.22 &#181;m vacuum filtration chamber.</td>
		</tr>
		<tr>
			<td>Super Sani-Cloth Germicidal Disposable Wipes</td>
			<td>Professional Disposables International</td>
			<td>H04082</td>
			<td>Disposable germicidal wipes for sterilization.</td>
		</tr>
		<tr>
			<td>Trace Metals Mixture, 1000x</td>
			<td>ThermoFisher Scientific</td>
			<td>NC0112668</td>
			<td>Concentrated solution of physiological trace metals.</td>
		</tr>
		<tr>
			<td>Unlined Aluminum Seal, 30 mm</td>
			<td>DWK Life Sciences</td>
			<td>224187-01</td>
			<td>Aluminum seals crimped over top of rubber stoppers.</td>
		</tr>
		<tr>
			<td>USP Medical Grade Air Tank</td>
			<td>Airgas</td>
			<td>AI USP200</td>
			<td>Compressed air tank for input to sparging system.</td>
		</tr>
		<tr>
			<td>USP Medical Grade Oxygen Tank</td>
			<td>Airgas</td>
			<td>OX USP200</td>
			<td>Compressed oxygen tank for input to sparging system.</td>
		</tr>
	</tbody>
</table>

design and development of a model to study the effect of supplemental oxygen on the cystic fibrosis airway microbiome

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

These protocols were applied to 50 expectorated sputum samples from pwCF presenting for routine care to an outpatient cystic fibrosis clinic at Massachusetts General Hospital in Boston, Massachusetts. Each patient&#39;s sputum was cultured under 21%, 50%, and 100% oxygen conditions using the artificial sputum medium, with 0.5 mL aliquots taken from each culture at 24 h, 48 h, and 72 h of culture time for testing. Cultures were photographed when extractions were made to track visual changes. In addition, a 0.5 mL aliquot ...

Watch this Scientific Journal Video about Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome at JoVE.com

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...

These protocols are designed to model the in vitro effects of supplemental oxygen use on the lung microbiome of cystic fibrosis patients. This media recipe is tailored to mirror the physiological composition of sputum for people living with cystic fibrosis. The sparging process introduces the ability to culture under different oxygen conditions.Alterations to artificial sputum medium allow it to mimic other chronic lung diseases and model microbial communities under other conditions, such as different glucose concentrations in concurrent cystic fibrosis and diabetes patients. An overview of the steps for artificial sputum medium preparation shows the mixing of the prepared ASCM, ASMM and ASBM. The mops buffer is used to titrate the pH to 6.3 and filter sterilization of the medium is carried out in a cold room on an orbital shaker.To begin with, prepare an artificial sputum medium by adding 250 milliliters of ASCM to the one liter bottle containing ASMM. Then add 250 milliliters of ASBM to the medium bottle. Titrate the medium with a basic one molar mops buffer to reach a pH of 6.3 on a pH paper.Refrigerate the artificial sputum medium at four degrees Celsius until filtration. Then to start the filtration process, transfer 200 milliliters of unfiltered artificial sputum medium to a vacuum filtration system with a 0.22 micrometers pore size filter. After connecting the filtration system to the vacuum pump, turn on the vacuum pump, then place the chamber on an orbital shaker, shaking at 90 rotations per minute in a cold room at four degrees Celsius.Change the filter after 350 milliliters of medium gets filtered to overcome the clogging issue. After a considerable amount is filtered, at an extra 150 milliliters medium for filtration. Repeat filtration with additional chambers until all the media is filtered.Overview of oxygen sparging demonstrating the addition of patient sputum to artificial sputum medium in an autoclaved serum bottle. Then sparge the sealed serum bottle with a gas mixture. Finally, the serum bottle is incubated to obtain culture Alec Watts at the 24 hour interval for subsequent meta genomics sequencing.To start serum bottle culture sparging, label 500 milliliter autoclaved serum bottles with sample identifiers, date and time of inoculation and target oxygen percentage. In a biological hood, add 24 milliliters of the artificial sputum medium to each serum bottle being set up. To obtain a sufficient sample volume for each culture condition, if necessary, dilute the sample with sterile phosphate buffered saline, then homogenized sputum with an 18 gauge needle and add one milliliter of the patient sputum to each serum bottle.Next, using sterile tweezers, place the autoclaved rubber stoppers onto the top of each serum bottle, press down the rubber stoppers without touching the underside of the stopper. After removing the bottles from the hood, apply and crimp the aluminum seals. Remove the centerpiece of the seals and wipe down the top of the bottles with an alcohol wipe and pass the bottle seal through a Bunsen burner flame.Now fix a sterile, 18 gauge needle to a plunger list syringe with a filter, insert the affixed gas release syringe into the bottle first. Now fix a sterile, 18 gauge needle to the gas output from the system and insert the gas output needle into the bottle as well. Route the T junctions from the tanks through the oxygen monitor.After verifying the target oxygen concentration flowing through the system, target approximately five liters per minute of gas flow. Reroute the T junctions from the tanks through to the gas output. As the gas starts to flow through the serum bottle, pay close attention to the pressure gauge, and if pressure increases unexpectedly, shut the system off immediately.After running, oxygen sparge through the serum bottle for one minute at five liters per minute, the settings allow for 10 air exchanges and ensure the internal atmosphere reaches the desired concentration of 100%oxygen. Remove the gas release, 18 gauge needle. After allowing the pressure in the serum bottle to build to one atmosphere, immediately remove the gas output needle.Incubate the serum bottles in 37 degrees Celsius incubator shaker at 150 rotations per minute for 3, 24 hour intervals. Alec Watts are taken at each 24 hour interval for downstream analysis and samples are resparged each time. The example here shows what a culture may look like before and after incubation.The outflow oxygen and pH levels were measured over the course of the culture process for sputum samples maintained at 37 degrees Celsius. With both sparging intervals of 12 hours and 24 hours, oxygen concentrations were approximately maintained over time with a moderate drop in oxygen concentration observed for all three conditions. The measured pH levels were within the physiological range with no significant changes over time.The comparison of microbial load, diversity and community composition between uncultured and cultured sputum revealed a 20 fold increase in microbial load in cultured sputum. Diversity metrics indicated preservation of community composition with minimal global differences introduced by the culture process. Comparative analysis of the 120 microbial species in cultured and uncultured sputum was performed by meta genomic sequencing.46 of these species were identified in both uncultured and cultured samples. While 35 were exclusively identified in uncultured samples and 39 were exclusively identified in cultured samples. Absorbent based growth curves were generated for common CF lung pathogens cultured in artificial sputum media from patient sample isolates under normal 21%oxygen concentration.No change over time in the optical density at 600 nanometers in the ASM only negative control indicated contamination free culture. The typical growth curve patterns observed indicating that ASM medium can be used for the generation of growth curves using optical methods. The metagenomic sequencing is used here to describe microbial community composition.Other techniques such as metatranscriptomic sequencing can be used to evaluate gene expression or metabolomics to evaluate metabolite production.

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Research

JoVE Journal

Medicine

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg dimethylnitrosamine (DMN), given intraperitoneally for three consecutive days per week. Intraperitoneal injections were performed in the fume hood as DMN is a known hepatoxin and carcinogen. The model has several advantages. Firstly, liver changes can be studied sequentially or at particular stages of interest. Secondly, the stage of liver disease can be monitored by measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. Thirdly, the severity of liver damage at different stages can be confirmed by sacrifice of animals at designated time points, followed by histological examination of Masson&#39;s Trichome stained liver tissues. After four weeks of DMN dosing, the typical fibrosis score is 5 to 6 on the Ishak scale. The model can be reproduced consistently and has been widely used to assess the efficacy of potential anti-fibrotic agents.

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg ...

Topical Airway Anesthesia for Awake-endoscopic Intubation Using the Spray-as-you-go Technique with High Oxygen Flow

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can jeopardize their spontaneous breathing, topical anesthesia of the airway is a popular technique. The spray-as-you-go technique represents one of the simplest opportunities to anesthetize the airway mucosa. The application of local anesthetic through the working channel of the flexible endoscope is a widespread practice for anesthetists as well as pulmonologists. There is neither need for additional devices nor special training as a pre-requisite to perform this technique. However, a known clinical problem is the coughing and gagging reflex that may occur when the liquid anesthetic strikes the airway mucosa and other sensitive structures like the vocal cords. This can be avoided by the use of oxygen applied through the working channel with the aim of fogging the local anesthetic into finer particles. Furthermore, the oxygen flow provides a higher oxygen supply and contributes to a better view, dispersing mucus secretions and blood away from the lens. Using an atomizer with a high oxygen flow of 10 L/min we maximized these benefits, caused less coughing and had more satisfied and therefore cooperative patients. Possible, but very rare complications of using oxygen flow including gastric insufflation, organ rupture or barotrauma did not arise. We attribute the complication-free use of high oxygen flow to the design of the set, which permits flow and pressure release.

The topical anesthetic lidocaine was atomized using a high oxygen flow through the working channel of a flexible intubating endoscope to achieve topical airway anesthesia for awake endotracheal intubation. We prefer this modified spray-as-you-go technique for endoscopic intubation to classical bolus application because of higher patient satisfaction and better compliance.

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the genotype-directed therapy model currently in use has several limitations. First, rare or understudied mutation groups are excluded from definitive clinical trials. Moreover, as additional modulator drugs enter the market, it will become difficult to optimize the modulator choices for an individual subject. Both of these issues are addressed with the use of patient-derived, individualized preclinical model systems of CFTR function and modulation. Human nasal epithelial cells (HNEs) are an easily accessible source of respiratory tissue for such a model. Herein, we describe the generation of a three-dimensional spheroid model of CFTR function and modulation using primary HNEs. HNEs are isolated from subjects in a minimally invasive fashion, expanded in conditional reprogramming conditions, and seeded into the spheroid culture. Within 2 weeks of seeding, spheroid cultures generate HNE spheroids that can be stimulated with 3',5'-cyclic adenosine monophosphate (cAMP)-generating agonists to activate CFTR function. Spheroid swelling is then quantified as a proxy of CFTR activity. HNE spheroids capitalize on the minimally invasive, yet respiratory origin of nasal cells to generate an accessible, personalized model relevant to an epithelium reflecting disease morbidity and mortality. Compared to the air-liquid interface HNE cultures, spheroids are relatively quick to mature, which reduces the overall contamination rate. In its current form, the model is limited by low throughput, though this is offset by the relative ease of tissue acquisition. HNE spheroids can be used to reliably quantify and characterize CFTR activity at the individual level. An ongoing study to tie this quantification to in vivo drug response will determine if HNE spheroids are a true preclinical predictor of patient response to CFTR modulation.

Here we describe a method to generate three-dimensional spheroid cultures of human nasal epithelial cells. Spheroids are then stimulated to drive Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-dependent ion and fluid secretion, quantifying the change in the spheroid luminal size as a proxy for CFTR function.

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.