1

Introduction

NO is a colorless, odorless gas that is formed in vivo from arginine by three isoforms of the enzyme nitric oxide synthase (NOS). NO acts as a final transmitter in many body systems. In the vascular system, brain and kidneys, NOS enzymes are permanently active and are called constitutive NO synthases (cNOS). In contrast, another isoform of NOS (iNOS) is inducible in response to inflammation and pathophysiological stress.

A large proportion of NO found in exhaled air originates from the upper airways, with only a minor contribution from the lower respiratory tract and the lungs . There is continuous high production of NO in the human nasal passages and paranasal sinuses. Non-invasive measurements of nasal NO are easy to perform and have revealed altered levels in several respiratory disorders albeit with significant discrepancies between studies.

The biological significance of the sinus-derived NO is still uncertain. NO has been proposed as a bronchodilator , a vasodilator , a major neurotransmitter , and to have antimicrobial , antitumor and mucociliary regulating activities and also to act as an airborne messenger with pro-inflammatory actions .

Exhaled NO measurement from the lower airway (FeNO) provides a simple, non-invasive means of measuring airway inflammation in asthma . Levels of FeNO correlate with eosinophilic airway inflammation and raised levels predict steroid responsiveness . FeNO measurements are as accurate as sputum eosinophilia in asthma diagnosis, with a sensitivity of 88% and specificity of 79%, but are simpler and quicker to perform . With the use of FeNO measurements as a marker of inflammation, maintenance doses of inhaled corticosteroids can be reduced without compromising asthma control . Conversely, elevated FeNO levels are associated with daily lung function declines and increased medication usage in urban children with moderate to severe asthma, despite use of inhaled corticosteroids and long-acting beta agonists , and predict relapse in children after discontinuation of steroids .

The situation in the upper respiratory tract is more complex. Nasal NO is altered in several respiratory disorders—for example, primary ciliary dyskinesia (PCD) , cystic fibrosis (CF) , and allergic rhinitis , and this has led to the proposal that a nasal NO test could be clinically useful in diagnosis and monitoring of these diseases.

The American Thoracic Society and European Rhinologic Society have agreed on a highly standardized procedure for measurements of lower respiratory tract FeNO . Such guidelines are extremely helpful for researchers when comparing results and they have been of great value in the process of moving FeNO measurements into clinical application.

However, for nasal NO measurements, one single standardized measurement procedure has not yet been defined. There are several reasons for this as follows: (1) there has been no well-defined major disease indication (such as asthma for FeNO) where routine measurements of nasal NO can be easily foreseen, (2) the methodology is quite complex, and (3) measurement variability is often substantial. Because of these reasons, the nasal NO field has been driven mostly by scientific curiosity especially in North America.

However, because nasal NO is altered in certain airway disorders, the measurements could be clinically beneficial to aid in the diagnosis and monitoring of therapy. Especially in the diagnosis and monitoring of sinus disease, where objective criteria such as sinus-specific symptoms are absent and the use of imaging is limited by several factors, an objective diagnostic method defined through a human study model as presented in this application has the potential to prove invaluable. Today, even the differential diagnoses between the common cold, allergic or non-allergic rhinitis, acute sinusitis and chronic sinusitis remain a challenge, and in many clinical encounters, the diagnostic algorithm resembles an exercise in the practice of educated guessing.

There is a great difference in background NO output between the upper and the lower airways . Background lower airway NO output is normally low, which makes it easy to find increases (e.g., asthma) but more difficult to detect decreases. In the upper airways, there is a high background output, and so an increase (e.g., in allergic rhinitis) can be obscured, whereas a decrease is usually easy to reveal. Therefore, at present, nasal NO measurements are definitive only in the diagnosis of primary ciliary dyskinesia (PCD) because of the near-complete absence of NO production in PCD.

As in the lower airway, epithelial cells lining the tract contain iNOS and will form NO in response to inflammatory stimuli. In the sinuses, however, NO is continually formed at levels around 20–25 parts per million (ppm), which are toxic to bacteria, viruses, fungi and tumor cells and may form part of the upper airway defense. Theoretically, nasal NO levels have been postulated to depend mainly on the amount of NO produced by the ciliated epithelium of the paranasal sinuses and the size of the paranasal sinus ostia . The relative importance of the size of the individual sinus (volume–surface area) has not been addressed, since differential measurements of sinus NO have not been systematically studied or performed.

Adding to the complexity is the fact that swelling of the mucosa or accumulated secretions during inflammation may lead to decreased transport of NO from the paranasal sinuses to the nasal cavity where it is measured. In such cases, the net change in nasal NO will be variable and difficult to predict. The following studies reflect this complexity.

Nasal NO has been reported as decreased in acute rhinosinusitis , nasal polyposis , and cystic fibrosis , and as normal or increased in allergic rhinitis (AR) . NO production in the nasal mucosa of patients with AR may be up-regulated. On the other hand, this increase could be counteracted by swelling of the mucosa and secretions resulting in impaired NO diffusion from the paranasal sinuses, where particularly high levels of NO have been found. Also, the high background levels of NO from constitutive sources in the nose may blunt smaller increases in mucosal NO output.

In chronic rhinosinusitis, Lindberg et al. found lower nasal NO levels than in healthy controls; Arnal et al. found no significant difference. Ragab et al. found that nasal NO correlated well with computed tomography changes, and the percentage rise in nasal NO seen on medical and surgical treatment of chronic rhinosinusitis correlated with changes in symptom scores, saccharine clearance time, endoscopic changes, polyp grades and surgical scores. The authors suggested that nasal NO provides a valuable non-invasive objective measure of the response of chronic rhinosinusitis to therapy, but that topical nasal corticosteroids may be needed to reduce the contribution of nasal epithelial NO and allow that emanating from the sinuses to be measured. However this may not be sufficient since nasal steroids reduce nasal measurements of NO by 20% and this effect may also be confounded by the vasoconstriction and improved ostial patency caused by the steroid. NO measurements obtained from individual sinuses addresses these variables.

Several techniques for measuring nasal NO have been used. The most common way to measure nasal NO is to sample nasal air directly from one nostril. Using the intrinsic sampling flow of a chemiluminescent analyzer or an external pump, air is aspirated from (or insufflated into) one nostril . Guidelines for FeNO and nasal NO measurement have recently been produced . Stark et al. suggested that the measurement should be made at the same time of day. Struben et al. found no significant diurnal variation. The discrepancy here may be due to the effects of the nasal cycle, introducing a high-level variable by nasal mucosal congestion preventing sinus NO from entering the nasal cavity.

The production of NO by different sinuses and the effect of intravenous arginine have been investigated in one volunteer who was the researcher himself. This study was however not designed to explore parameters contributing to differential sinus and nasal NO production and mainly investigated the effects of gaseous carbon dioxide and intravenous arginine on NO measurements. The need for differential measurements of sinus NO production in defining NO exchange dynamics in the upper airway is unmet.

One of the more recent findings is the discovery that nasal NO increases dramatically (5- to 15-fold) during humming compared with silent nasal exhalation . The current explanation for this phenomenon is that oscillating sound waves speeding up the exchange of gases over the sinus ostium, resulting in a rapid washout of NO from the sinuses . However, this theory appears weak with no explanation of how sound waves accomplish this. The theory also does not take into account that humming primarily is slow exhalation. NO levels during slow exhalation have not been studied. The humming-induced NO peak is transient and the levels decrease gradually during repeated humming maneuvers. Nasal NO levels fully recover after a short period of silence, which allows sinus NO to accumulate again. In a model of the nose and sinus, it was found that ostium size was the main determinant of the humming-induced transient increase in NO. In addition, in patients with complete sinus ostial obstruction (bilateral nasal polyposis), the nasal NO increase during humming was abolished . This suggests that the increase in nasal NO during humming correlates with ostial function. Ostial obstruction is central in the pathogenesis of sinusitis, and one goal in medical and surgical therapy of chronic sinusitis is to improve sinus ventilation. Therefore humming has been suggested as a test of patency of the ostiomeatal complex ; it improves discrimination between healthy controls and children with cystic fibrosis better than static NO measurements . Here again demonstrated is the importance of the dynamics of NO exchange between the sinuses and the nasal cavity, affecting the diagnostic accuracy of nasal NO measurements. Differential measurements are needed to assess the fractional contribution of sinus NO to nasal NO measurements, in variable conditions, including humming, which are in turn, likely to improve our understanding of NO dynamics in the upper airway.

A human study model for research on sinonasal disease using NO, a non-invasive objective marker, is the key to advancing our knowledge about the physiology, function and pathology of the sinuses. This study model can help describe and classify inflammation of the nose and sinuses in acute, chronic, bacterial, viral and other inflammatory diseases such as polyps. The study model can be used to design studies testing various hypotheses and will help to describe and validate objective diagnostic criteria for the differential diagnosis of different forms of rhinosinusitis and to describe key inflammatory pathways leading to the development of rhinosinusitis. New and advanced treatment methods may be developed based on our enhanced understanding of sinonasal pathophysiology. As a direct result of our observations during the study, a new aerodynamic theory of the nose is developed that explains sinonasal gas exchange dynamics, lends support to the role of NO as an aerocrine messenger, and has the potential to shape sinonasal surgical techniques (IFAR Int Forum on All Rhinol. Presented at COSM 2012 in San Diego and ERS-ISIAN 2012, Toulouse, France. Accepted for publication) .

2

Materials and methods

The study was supported by a grant from the Scott & White Research Grants Program at the Texas A&M Health Science Center as a Research Advancement Award for the primary investigator Anil A. Gungor MD (A Human Study Model for Research in Sinonasal Disease—Scott & White Institutional Review Board Registration #IRB00000706 Project ID#: 90344) Protocol and consent forms were IRB approved and study was monitored by the IRB.

Subject inclusion criteria: Twelve adult volunteers; healthy subjects with no sinonasal disease, no anatomic sinonasal deformity, non-smoker; male or female; and age 18 to 65 years.

Subject selection methods: Health history questionnaire, nasal symptom score questionnaire, physical and ENT exam, nasal endoscopic exam, and in vitro allergy testing by RAST method.

Research procedures: CT scan of the sinuses, nasal NO measurement, placement of sinus catheters under endoscopic guidance, with topical and local anesthesia, and sinus and nasal NO measurements. NO measurements are made with a chemiluminescence analyzer in real time.

Data collection: History and physical exam, sinonasal symptom questionnaire, allergy test results, and CT scans for delineating anatomy, planning for catheter placement, and calculation of individual sinus volumes.

NO measurements for nasal NO and sinus NO from individual sinuses during quiet breathing, breath-holding and humming maneuvers were performed. In addition to breath-holding, a Foley balloon catheter was used to seal the choana while sinus NO measurements were performed.

Nasal NO production was measured as ppb, with 3 l/min airflow through nasal passages in parallel and 200 ml/min flow rate of sampling. Sinus NO was measured as ppb through 200 ml/min flow rate of sampling. Sinus volumes and mucosal surface area of each sinus are calculated from a 3D CT model of the sinuses by digitization software (iplannet).

Subjects were instructed to have a light breakfast and to avoid caffeinated beverages or food and to present at 8:00 AM on the study day. After registration subjects were brought to the lab to acclimatize. A baseline nasal NO measurement was performed as described previously . Topical spray mix of 2 cc (15 cc of oxymetazoline with 2 cc of 1% lidocaine) was applied. Ten minutes later a sterile injection of 1% lidocaine plus 1/100000 epinephrine solution into the uncinate, lateral nasal wall submucosa and attachment of middle turbinate to the lateral nasal wall were performed. A total of 7 cc was used. After 20 min, based on the pre-determined sinus of interest, with the help of either a 0° or a 30° Hopkins telescope, a Relieva Flex S-0, F-70C or M-110 guide (Acclarent, Inc) attached to a Sidekick (Acclarent Inc) (a handle) was used to approach the sinus ostium. Relieva Luma Light probe (LUMA) (Acclarent Inc) is used to enter the sinus through the guide, providing verification of sinus entry through illumination. A sterile polyethylene catheter tube (PEC) (Clay Adams, Intramedic) with an outer diameter of 1.27 mm was introduced into the sinus over the LUMA. The LUMA was carefully withdrawn and the PEC was secured to the facial skin with tape. All procedures were performed with sterile instruments. The NO analyzer collection line was attached to the polyethylene tubing through a disposable liquids filter to prevent contamination of analyzer sampling line.

2

Materials and methods

The study was supported by a grant from the Scott & White Research Grants Program at the Texas A&M Health Science Center as a Research Advancement Award for the primary investigator Anil A. Gungor MD (A Human Study Model for Research in Sinonasal Disease—Scott & White Institutional Review Board Registration #IRB00000706 Project ID#: 90344) Protocol and consent forms were IRB approved and study was monitored by the IRB.

Subject inclusion criteria: Twelve adult volunteers; healthy subjects with no sinonasal disease, no anatomic sinonasal deformity, non-smoker; male or female; and age 18 to 65 years.

Subject selection methods: Health history questionnaire, nasal symptom score questionnaire, physical and ENT exam, nasal endoscopic exam, and in vitro allergy testing by RAST method.

Research procedures: CT scan of the sinuses, nasal NO measurement, placement of sinus catheters under endoscopic guidance, with topical and local anesthesia, and sinus and nasal NO measurements. NO measurements are made with a chemiluminescence analyzer in real time.

Data collection: History and physical exam, sinonasal symptom questionnaire, allergy test results, and CT scans for delineating anatomy, planning for catheter placement, and calculation of individual sinus volumes.

NO measurements for nasal NO and sinus NO from individual sinuses during quiet breathing, breath-holding and humming maneuvers were performed. In addition to breath-holding, a Foley balloon catheter was used to seal the choana while sinus NO measurements were performed.

Nasal NO production was measured as ppb, with 3 l/min airflow through nasal passages in parallel and 200 ml/min flow rate of sampling. Sinus NO was measured as ppb through 200 ml/min flow rate of sampling. Sinus volumes and mucosal surface area of each sinus are calculated from a 3D CT model of the sinuses by digitization software (iplannet).

Subjects were instructed to have a light breakfast and to avoid caffeinated beverages or food and to present at 8:00 AM on the study day. After registration subjects were brought to the lab to acclimatize. A baseline nasal NO measurement was performed as described previously . Topical spray mix of 2 cc (15 cc of oxymetazoline with 2 cc of 1% lidocaine) was applied. Ten minutes later a sterile injection of 1% lidocaine plus 1/100000 epinephrine solution into the uncinate, lateral nasal wall submucosa and attachment of middle turbinate to the lateral nasal wall were performed. A total of 7 cc was used. After 20 min, based on the pre-determined sinus of interest, with the help of either a 0° or a 30° Hopkins telescope, a Relieva Flex S-0, F-70C or M-110 guide (Acclarent, Inc) attached to a Sidekick (Acclarent Inc) (a handle) was used to approach the sinus ostium. Relieva Luma Light probe (LUMA) (Acclarent Inc) is used to enter the sinus through the guide, providing verification of sinus entry through illumination. A sterile polyethylene catheter tube (PEC) (Clay Adams, Intramedic) with an outer diameter of 1.27 mm was introduced into the sinus over the LUMA. The LUMA was carefully withdrawn and the PEC was secured to the facial skin with tape. All procedures were performed with sterile instruments. The NO analyzer collection line was attached to the polyethylene tubing through a disposable liquids filter to prevent contamination of analyzer sampling line.