Name: a novel inhalation mask system to deliver high concentrations of nitric oxide gas in spontaneously breathing subjects
Uploaded: 2021-05-04T13:00:00
Description: Watch this Scientific Journal Video about A Novel Inhalation Mask System to Deliver High Concentrations of Nitric Oxide Gas in Spontaneously Breathing Subjects at JoVE.com

This study was supported by the Reginald Jenney Endowment Chair at Harvard Medical School to L.B., by L.B. Sundry Funds at MGH, and by laboratory funds of the Anesthesia Center for Critical Care Research of the Department of Anesthesia, Critical Care and Pain Medicine at MGH.

NOTE: See the Table of Materials for the materials needed to assemble the delivery system. Sources of medical air, O2, and NO gases should also be available on site. The device has been developed for investigation use in research protocols that underwent rigorous review by the local Institutional Review Board (IRB). Under no circumstances should providers operate solely based on the indications included in this manuscript, assembling and using this device without seeking prior appropriate inst...

<ol>
	<li>Roberts, I. D., Fineman, J. F., Zapol, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhaled+nitric+oxide+and+persistent+pulmonary+hypertension+of+the+newborn.">Inhaled nitric oxide and persistent pulmonary hypertension of the newborn.</a> Pneumologie. 52 (4), 239 (1998).</li><li>Rossaint, R., 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=Inhaled+nitric+oxide+for+the+adult+respiratory+distress+syndrome.">Inhaled nitric oxide for the adult respiratory distress syndrome.</a> New England Journal of Medicine. 328 (6), 399-405 (1993).</li><li>Robinson, J. N., Banerjee, R., Landzberg, M. J., Thiet, M. 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A., Sacco, O., Mancino, E., Cristiani, L., Midulla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+and+similarities+between+SARS-CoV+and+SARS-CoV-2:+spike+receptor-binding+domain+recognition+and+host+cell+infection+with+support+of+cellular+serine+proteases.">Differences and similarities between SARS-CoV and SARS-CoV-2: spike receptor-binding domain recognition and host cell infection with support of cellular serine proteases.</a> Infection. 48 (5), 665-669 (2020).</li><li>Berra, L., 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=Protocol+for+a+randomized+controlled+trial+testing+inhaled+nitric+oxide+therapy+in+spontaneously+breathing+patients+with+COVID-19.">Protocol for a randomized controlled trial testing inhaled nitric oxide therapy in spontaneously breathing patients with COVID-19.</a> medRxiv. , (2020).</li><li>Lei, C., 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=Protocol+for+a+randomized+controlled+trial+testing+inhaled+nitric+oxide+therapy+in+spontaneously+breathing+patients+with+COVID-19.">Protocol for a randomized controlled trial testing inhaled nitric oxide therapy in spontaneously breathing patients with COVID-19.</a> medRxiv. , (2020).</li><li>. Nitric oxide inhalation therapy for COVID-19 infections in the ED Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT04338828">https://clinicaltrials.gov/ct2/show/NCT04338828</a> (2020)</li><li>Gianni, S., 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=Nitric+oxide+gas+inhalation+to+prevent+COVID-2019+in+healthcare+providers.">Nitric oxide gas inhalation to prevent COVID-2019 in healthcare providers.</a> medRxiv. , (2020).</li><li>Safaee Fakhr, B., 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=High+concentrations+of+nitric+oxide+inhalation+therapy+in+pregnant+patients+with+severe+coronavirus+disease+2019+(COVID-19).">High concentrations of nitric oxide inhalation therapy in pregnant patients with severe coronavirus disease 2019 (COVID-19).</a> Obstetrics &amp; Gynecology. , (2020).</li><li>Gianni, S., 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=Ideation+and+assessment+of+a+nitric+oxide+delivery+system+for+spontaneously+breathing+subjects.">Ideation and assessment of a nitric oxide delivery system for spontaneously breathing subjects.</a> Nitric Oxide. 104-105, 29-35 (2020).</li><li>. Nitric oxide gas inhalation therapy for mild/moderate COVID-19 Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT04305457">https://clinicaltrials.gov/ct2/show/NCT04305457</a> (2020)</li><li>. NO prevention of COVID-19 for healthcare providers Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT04312243?term=Berra&amp;draw=2&amp;rank=7">https://clinicaltrials.gov/ct2/show/NCT04312243?term=Berra&amp;draw=2&amp;rank=7</a> (2020)</li><li>. 1988 OSHA PEL Project-Nitrogen Dioxide|NIOSH|CDC Available from: <a target="_blank" href="https://www.cdc.gov/niosh/pel88/10102-44.html">https://www.cdc.gov/niosh/pel88/10102-44.html</a> (2020)</li><li>Yu, B., Zapol, W. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhaled+nitric+oxide.">Inhaled nitric oxide.</a> British Journal of Pharmacology. 176 (2), 246-255 (2019).</li><li>INO Therapeutics. INOMAX - nitric oxide gas. Food and Drug Administration (FDA) Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/label/2013/020845s014lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/label/2013/020845s014lbl.pdf</a> (2013)</li><li>Klinger, J. R., 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=Therapy+for+pulmonary+arterial+hypertension+in+adults:+Update+of+the+CHEST+Guideline+and+Expert+Panel+Report.">Therapy for pulmonary arterial hypertension in adults: Update of the CHEST Guideline and Expert Panel Report.</a> Chest. 155 (3), 565-586 (2019).</li><li>Cornfield, D. 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Given the increasing interest in NO gas therapy for non-intubated patients, including those with COVID-198, the present report describes a novel custom device and how to assemble its components to deliver NO at concentrations as high as 250 ppm. The proposed system is built out of inexpensive consumables and safely delivers a reproducible concentration of NO gas in spontaneously breathing patients. The ease of assembly and use, together with the safety data published elsewhere16<...

NO inhalation therapy is regularly used as a life-saving treatment in several clinical settings1,2,3. In addition to its well-known pulmonary vasodilator effect4, NO displays a broad antimicrobial effect against bacteria5, viruses6, and fungi7, particularly if administered at high concentrations (&#62;100 ppm).8 During the 2003 Severe Acute Respiratory Syndrome (SARS) outbreak, NO showed potent antiviral activity in vitro and demonstrated therapeutic efficacy in patients infected with the SARS-Coronavirus (SARS-CoV)9,10. The 2003 strain is structurally similar to SARS-Cov-2, the pathogen responsible for the current Coronavirus Disease-2019 (COVID-19) pandemic11. Three randomized controlled clinical trials are ongoing in patients with COVID-19 to determine the potential benefits of breathing high-concentration NO gas to improve outcomes12,13,14. In a fourth ongoing study, the prophylactic inhalation of high concentrations of NO is being investigated as a preventive measure against the development of COVID-19 in healthcare providers exposed to SARS-CoV-2-positive patients15.
The development of an effective and safe treatment for COVID-19 is a priority for the healthcare and scientific communities. To investigate the administration of NO gas at doses &#62; 80 ppm in non-intubated patients and volunteering healthcare workers, the need to develop a safe and reliable non-invasive system became apparent. This technique aims to administer high NO concentrations at different fractions of inspired oxygen (FiO2) to spontaneously breathing subjects. The methodology described here is currently in use for research purposes in spontaneously breathing COVID-19 patients at the Massachusetts General Hospital (MGH)16,17. Following the guidelines of MGH&#39;s human research ethics committee, the proposed system is currently in use to conduct a series of randomized controlled trials to study the following effects of high concentrations of NO gas. First, the effect of 160 ppm NO gas is being studied in non-intubated subjects with mild-moderate COVID-19, admitted either in the Emergency Department (IRB Protocol #2020P001036)14 or as inpatients (IRB Protocol #2020P000786)18. Second, the role of high-dose NO is being examined to prevent SARS-CoV-2 infection and the development of COVID-19 symptoms in healthcare providers routinely exposed to SARS-CoV-2-positive patients (IRB Protocol # 2020P000831)19.
This simple device can be assembled with standard consumables routinely used for respiratory therapy. The proposed apparatus is designed to non-invasively deliver a mixture of NO gas, medical air, and oxygen (O2). Nitrogen dioxide (NO2) inhalation is minimized to reduce the risk of airway toxicity. The current NO2 safety threshold set by the American Conference of Governmental Industrial Hygienists is 3 ppm over an 8-h time-weighted average, and 5 ppm is the short-term exposure limit. Conversely, the National Institute for Occupational Safety and Health recommends 1 ppm as a short-term limit of exposure20. Given the increasing interest in high-dose NO gas therapy, the present report provides the necessary description of this novel device. It explains how to assemble its components to deliver a high concentration of NO for research purposes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>90&#176; ventilator elbow connector without ports 22 mm ID x 22 mm OD</td>
			<td>Teleflex, Wayne, PA, USA</td>
			<td>1641</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol tee connector: horizontal ports 22 mm OD, vertical port 11 mm ID/22 mm OD</td>
			<td>Teleflex, Wayne, PA, USA</td>
			<td>1077</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexible patient connector for endotracheal or tracheostomy tube (15 mm OD x 22 mm OD/15 mm ID, length 5 cm to 6.5 cm)</td>
			<td>Vyaire Medical Inc., Mettawa, IL, USA</td>
			<td>3215</td>
			<td></td>
		</tr>
		<tr>
			<td>High-efficiency particulate air (highly hydrophobic bacterial/viral filter,&#160; HEPA class 13) filter (22 mm ID/15 mm OD x 22 mm OD/15 mm ID connector)</td>
			<td>Teleflex, Wayne, PA, USA</td>
			<td>28012</td>
			<td></td>
		</tr>
		<tr>
			<td>Latex-free 3-L breathing reservoir bag</td>
			<td>CareFusion, Yorba Linda, CA, USA</td>
			<td>5063NL</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitric Oxide tank 800 ppm medical-grade (size AQ aluminum cylinders containing 2239 L at STP of 800 ppm NO gas balanced with nitrogen, volume 2197 L)</td>
			<td>Praxair, Bethlehem PA, USA</td>
			<td>MM NO800NI-AQ</td>
			<td></td>
		</tr>
		<tr>
			<td>One-way valve 22 mm male/female (arrow pointing towards female end)</td>
			<td>Teleflex, Wayne, PA, USA</td>
			<td>1664</td>
			<td>N=2 inspiratory limb (upward arrow)</td>
		</tr>
		<tr>
			<td>One-way valve 22 mm male/female (arrow pointing towards male end)</td>
			<td>Teleflex, Wayne, PA, USA</td>
			<td>1665</td>
			<td>N=1 expiratory limb (downward arrow)</td>
		</tr>
		<tr>
			<td>Rad-57 Handheld Pulse Oximeter with Rainbow SET Technology</td>
			<td>Masimo Corporation, Irvine, CA, USA</td>
			<td>3736</td>
			<td>Including SpMet Option</td>
		</tr>
		<tr>
			<td>Scavenger (ID = 60 mm, internal length = 53 mm, volume = 150 mL) containing 100 g of calcium hydroxide</td>
			<td>Spherasorb, Intersurgical Ltd, Berkshire, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon rubber flexible connectors 22 mm F x 22 mm F</td>
			<td>Tri-anim Health Services, Dublin, OH, USA</td>
			<td>301-9000</td>
			<td></td>
		</tr>
		<tr>
			<td>Snug-fit standard face mask of appropriate size</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Star Lumen standard medical grade vynil oxygen tubing with universal connectors</td>
			<td>Teleflex, Morrisville, NC, USA</td>
			<td>1115</td>
			<td>Variable length according to distance from source of gas. 2.1 m length used in protocol</td>
		</tr>
		<tr>
			<td>Straight connector with a 7.6 mm sampling port (15 mm OD x 15 mm ID/22 mm OD)</td>
			<td>Mallinckrodt, Bedminster, NJ, USA</td>
			<td>502041</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-step adapter (15 mm to 22 mm)</td>
			<td>Airlife Auburndale, FL, USA</td>
			<td>1824</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-piece connector with 7.6 mm ports (22 mm to 22 mm and 15 F)</td>
			<td>Vyaire Medical Inc., Mettawa, IL, USA</td>
			<td>1831</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel inhalation mask system to deliver high concentrations of nitric oxide gas in spontaneously breathing subjects

Nitric Oxide (NO) is administered as gas for inhalation to induce selective pulmonary vasodilation. It is a safe therapy, with few potential risks even if administered at high concentration. Inhaled NO gas is routinely used to increase systemic oxygenation in different disease conditions. The administration of high concentrations of NO also exerts a virucidal effect in vitro. Owing to its favorable pharmacodynamic and safety profiles, the familiarity in its use by critical care providers, and the potential for a direct virucidal effect, NO is clinically used in patients with coronavirus disease-2019 (COVID-19). Nevertheless, no device is currently available to easily administer inhaled NO at concentrations higher than 80 parts per million (ppm) at various inspired oxygen fractions, without the need for dedicated, heavy, and costly equipment. The development of a reliable, safe, inexpensive, lightweight, and ventilator-free solution is crucial, particularly for the early treatment of non-intubated patients outside of the intensive care unit (ICU) and in a limited-resource scenario. To overcome such a barrier, a simple system for the non-invasive NO gas administration up to 250 ppm was developed using standard consumables and a scavenging chamber. The method has been proven safe and reliable in delivering a specified NO concentration while limiting nitrogen dioxide levels. This paper aims to provide clinicians and researchers with the necessary information on how to assemble or adapt such a system for research purposes or clinical use in COVID-19 or other diseases in which NO administration might be beneficial.

A 33-year-old respiratory therapist working at the ICU at MGH during the surge of ICU admission for COVID-19 volunteered to receive NO as part of the trial involving healthcare workers15,19. The trial tested the efficacy of 160 ppm of NO as a virucidal agent, thereby preventing disease occurrence in lungs at risk for viral contamination. The first session of the inhalation prophylaxis was administered before starting a shift throu...

Watch this Scientific Journal Video about A Novel Inhalation Mask System to Deliver High Concentrations of Nitric Oxide Gas in Spontaneously Breathing Subjects at JoVE.com

A Novel Inhalation Mask System to Deliver High Concentrations of Nitric Oxide Gas in Spontaneously Breathing Subjects

This simple and highly adaptable system device for the inhalation of high-concentration nitric oxide (NO) gas does not require mechanical ventilators, positive pressure, or high gas flows. Standard medical consumables and a snug-fitting mask are used to safely deliver NO gas to spontaneously breathing subjects.

Nitric Oxide (NO) is administered as gas for inhalation to induce selective pulmonary vasodilation. It is a safe therapy, with few potential risks even if ...

Inhaled nitric oxide can improve oxygenation in patients with severe hypoxemia. At high concentrations, nitric oxide displays broad antimicrobial effects. Several trials are testing the effect of nitric oxide on COVID-19.To date, there's no device available that is able to administer inhaled nitric oxide at concentrations higher than 80 parts per million without the need for dedicated heavy and costly equipment. This device will allow for high-dose inhaled nitric oxide to be delivered outside the hospital setting. Patients with lung colonization due to multi-resistant bacteria, like cystic fibrosis patients, will be able to benefit from this treatment.Demonstrating the procedure will be Stefano Gianni and Bijan Safaee Fakhr, two postdoc research fellows from my laboratory. To set up the patient interface, connect the built-in elbow port of a snug fitting standard non-invasive ventilation face mask of the appropriate size for the subject to a high efficiency particulate air filter through the 22 millimeter outer diameter 15 millimeter inner diameter connector. To build a Y-piece, use two opposite sense, low resistance 22 millimeter male-female one-way valves to create expiratory and inspiratory limbs on the two distal ends of a 22 millimeter to 22 millimeter and 15 French Y-piece connector with 7.6 millimeter ports.Position the one-way valve connector to the expiratory limb with the arrow pointing downward to allow a proximal to distal flow only and position the connector for the inspiratory limb with the arrow pointing upward to allow a distal to proximal flow only. Then use a 15 to 22 millimeter two-step adapter to attach the scavenging chamber and tubing assembly to the inspiratory limb of the Y-piece. To prepare the scavenging chamber, connect a 22 by 22 millimeter flexible silicon rubber connector adapter to the proximal end of a scavenger chamber containing 100 grams of calcium hydroxide and connect another 22 by 22 millimeter flexible silicon rubber connector adapter to the distal end of the scavenger and use standard kink-resistant vinyl gas tubing with universal adapters at both ends to connect the oxygen source to the inspiratory limb.To prepare the nitric oxide reservoir system, connect an aerosol T-piece in a 90 degree ventilator elbow connector without ports and connect the other end of the elbow to three liter latex-free breathing reservoir bag. Attach another one-way inspiratory valve with the arrow pointing up at the distal end of two consecutive 15 millimeter outer diameter by 22 millimeter outer diameter 15 millimeter inner diameter connectors with 7.6 millimeter sampling ports and flip top caps and connect a 15 to 22 millimeter two-step adapter to the proximal end of the system and connect the proximal two-step adapter to the remaining free inlet of the green T-piece from the nitric oxide reservoir system. To attach the air and nitric oxide gas flow lines, use standard kink-resistant Star Lumen vinyl oxygen gas tubing to connect the medical air flow to the most distal gas inlet port and connect the nitric oxide gas flow from an 800 parts per million medical-grade nitric oxide tank to the next port downstream.To administer nitric oxide to a spontaneously breathing subject, set the air, oxygen, and nitric oxide gas flow according to the table and position the tight fitting mask onto the subject's face similar to a noninvasive ventilation interface setup. Then start the inhalation session for the desired duration. The nitric oxide and nitrogen dioxide concentrations can be traced to assess the stability of the gas delivery during the treatment.A 33-year-old respiratory therapist working at the ICU at Mass General Hospital during the surge of ICU admission for COVID-19 volunteered to receive nitric oxide as part of the trial involving healthcare workers. The resulting nitric oxide concentration was 160 parts per million at a total gas flow rate of 19.5 liters per minute as measured by three standard 15 liter per minute flow meters and remained stable throughout the entire period of inhalation. Nitrogen dioxide peaked at 0.77 parts per million and was therefore safely below the recommended toxicity threshold.The possibility of safely delivering high-concentration inhaled nitric oxide opens the field for the application of this therapy to other diseases in which nitric oxide can be an effective treatment.

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Research

JoVE Journal

Medicine

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth has solicited growing clinical interest among physicians in the emergency department for various indications: (1) surveillance et monitoring of the intubated patient; (2) verification of the correct positioning of an endotracheal tube; (3) monitoring of a patient in cardiac arrest; (4) achieving normocapnia in intubated head trauma patients; (5) monitoring ventilation during procedural sedation. The video allows physicians to familiarize themselves with the use of capnography and the text offers a review of the theory and principals involved. In particular, the importance of CO2 for the organism, the relevance of measuring expired CO2, the differences between arterial and expired CO2, the material used in capnography with their artifacts and traps, will be reviewed. Since the main reluctance in the use of expired CO2 measurement is due to lack of correct knowledge concerning the physiopathology of CO2 by the physician, we hope that this explanation and the video sequences accompanying will help resolve this limitation.

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

This video describes how to perform CO2 measurement in intubated as well as spontaneously breathing patients. The main clinical indications refer to emergency situations: (1) verifying adequate positioning of an endotracheal tube; (2) achieving normocapnia in trauma patients; (3) monitoring ventilation in the case of procedural sedation.

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth ...

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. Therefore, the ability of the blood vessels to dilate or constrict, known as Cerebral-Vascular-Reactivity (CVR), represents an important domain of vascular function. An imaging marker representing this dynamic property will provide new information of cerebral vessels under normal and diseased conditions such as stroke, dementia, atherosclerosis, small vessel diseases, brain tumor, traumatic brain injury, and multiple sclerosis. In order to perform this type of measurement in humans, it is necessary to deliver a vasoactive stimulus such as CO2 and/or O2 gas mixture while quantitative brain magnetic resonance images (MRI) are being collected. In this work, we presented a MR compatible gas-delivery system and the associated protocol that allow the delivery of special gas mixtures (e.g., O2, CO2, N2, and their combinations) while the subject is lying inside the MRI scanner. This system is relatively simple, economical, and easy to use, and the experimental protocol allows accurate mapping of CVR in both healthy volunteers and patients with neurological disorders. This approach has the potential to be used in broad clinical applications and in better understanding of brain vascular pathophysiology. In the video, we demonstrate how to set up the system inside an MRI suite and how to perform a complete experiment on a human participant.

Non-invasive imaging of the brain vasculature&#8217;s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI).

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. The potential adverse health effects of electronic cigarette vapor (E-vapor) exposure are poorly defined. While several animal models of E-vapor exposure have been developed, few models expose rodents to clinically relevant quantities of nicotine and make direct comparisons to cigarette smoke within the same exposure system. Here, we present a method for constructing and operating an E-vapor chamber and cigarette smoke chamber. The chambers are constructed by outfitting anesthesia chambers with a computer controlled pumping system that delivers consistent amounts of E-vapor or cigarette smoke to rodents. Nicotine exposure is measured indirectly by quantifying pre and post-exposure serum cotinine levels. This exposure system can be modified to accommodate various types of E-cigarettes&#160;and tobacco cigarettes, and can be used to compare the effects of E-vapor&#160;and cigarette smoke in vivo.

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

This protocol describes a method for exposing rodents to electronic cigarette vapor (E-vapor) and cigarette smoke. Exposure chambers are constructed by modifying anesthesia chambers with an automated pumping system that delivers E-vapor or cigarette smoke to rodents. This system can easily be modified to accommodate many experimental endpoints.

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. ...