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Cite this article: van Schaik CR, Ho D, Hui NY (2015) Comparison of Methylene Blue Distribution in the Nasal Cavity and Paranasal Sinuses Using a Pulsed Versus Non-Pulsed Nebuliser in the Cadaver. Ann Otolaryngol Rhinol 2(12): 1078.

*Corresponding author

Ng Yuk Hui, Department of Otolaryngology, Singapore General Hospital, Academia Building – 5th floor, 20 College Road, 169850 Singapore, Singapore, Tel: 65-6321-4469; Fax: 65 6226 2079; E-mail:

Submitted: 10 October 2015

Accepted: 30 November 2015

Published: 01 December 2015

ISSN: 2379-948X

Copyright© 2015 Ho et al.

OPEN ACCESS

Keywords• Aerosol• Intranasal administration• Methylene blue• Nebulizers• Paranasal sinuses

Research Article

Comparison of Methylene Blue Distribution in the Nasal Cavity and Paranasal Sinuses Using a Pulsed Versus Non-Pulsed Nebuliser in the CadaverCarolina Rebelo van Schaik1, Danielle Ho2* and Ng Yuk Hui1#1Otolaryngology Department, Singapore General Hospital, Singapore2General surgery Department, Singapore General Hospital, Singapore #Both authors contribute equally

Abstract

Background: Topical steroid therapy is first line in treatment of chronic rhino sinusitis. It is often combined with sinus surgery to improve distribution of medication to diseased mucosa. Despite having many delivery methods available, there is no conclusion on the most efficacious mode of penetration. This study aimed to evaluate whether a pulsed nebulised system significantly improves drug distribution to the Paranasal sinuses before and after sinus surgery compared to the conventional nebulizer.

Methods: Intranasal distribution of Methylene blue staining solution delivered with the PARI sinus device with and without pulsation was assessed in the nasal cavities of 4 frozen cadaver heads (8 sides), before and after sinus surgery. 2 independent observers graded the surface area covered by the dye using a scoring system.

Results: There was no significant difference in the surface area of dye distribution to all sites prior to surgical intervention between the pulsed and non-pulsed nebulizer (p = 0.29). This finding was consistent post-surgery within the nasal sinuses and recesses (p = 1) except for a statistically significant increase in dye distributed by the pulsed nebulizer in the posterior ethmoid and sphenoid sinus after surgery (p = 0.046 at both sites).

Conclusion: The pulsation does not significantly alter the distribution of dye within the nasal sinuses pre-operatively. After endoscopic sinus surgery there was significant increase in dye distributed by the pulsed nebulizer within the posterior ethmoid and sphenoid sinus. However, further work is needed to evaluate the impact of different contrast agents in similar studies.

INTRODUCTIONChronic rhino sinusitis (CRS) is a prevalent condition affecting

12.5% of the US population [1]. It has significant adverse effects on the individual’s quality of life and has a billion-dollar economic burden on society [2-4].

Inflammatory dysfunction of the nasal and Paranasal sinus mucosa is a central tenet to the condition and topical steroids have proven to be a safe and effective treatment [5-7]. Steroids down regulate pro-inflammatory gene expression and have been shown to improve symptoms and objective evidence of disease. Topical therapy is an attractive route as it minimizes systemic

complications of the active agent. The main limitation is the delivery of drugs to target tissue which depends on the efficacy of the device. Many studies have been performed to evaluate the performance of drops, sprays, irrigation and Nebulizers [8,9]. However, there is no consensus as to a superior modality.

Nebulizers are one type of device available and it works by vaporising drugs and creating flow patterns in their delivery [10]. Smaller particles are postulated to better penetrate the sinuses while larger droplets are filtered by the nose [11]. Pulsating Nebulizers are a recent addition to the field introduced in the late-2000s. Pulsations are created by sound waves which

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are superimposed onto aerosol stream. These pressure gradients generated may propel the aerosol further within the poorly ventilated Paranasal sinuses to facilitate distribution [12]. Another crucial factor impacting on delivery of topical agents is sinus anatomy. It has been demonstrated that topical therapy has limited access to sinus cavities in the unoperated nose [13].

Sinus surgery is often coupled with medical therapy in patients with symptoms refractory to isolated medical intervention [14]. Remodeling the nasal passage removes anatomical obstruction and theoretically improves access to diseased mucosa. Surgery is often not curative and immune modulation with steroids is continued for symptom control and to minimize disease recurrence.

This study aimed to establish whether the additional feature of pulsation would lead to significant improvement in intranasal deposition of nebulised medication pre- and post-sinus surgery.

MATERIALS AND METHODSThe distribution of dye administered via nebulisation was

studied in the nasal cavities of four fresh frozen cadaver heads in the anatomy laboratory of Singapore General Hospital, Singapore, May 2015. Specimens presenting overt septal deviation, previous sinus surgery, polyposis and mucosal disease were excluded. This study was performed after approval from the institutional review board.

The cadavers were held upright and each nostril was nebulised with the contra lateral nostril plugged as stated in the instructions of the manufacturer. One nostril served as a control while the other was subjected to pulsating nebulisation. Allocation was done randomly. Nebulisation was standardized with 4ml 1:1 ratio of Methylene blue to 0.9% normal saline applied to each nostril for 2 ½ minutes. The pulsating aerosol was produced using the PARI Sinus Pulsating Aerosol System (PARI GmbH, Starnberg, Germany).

Video endoscopy of the nasal cavity was performed with a 0, 30 and 70 degree rigid endoscope to assess the deposition of dye. Two blinded, independent observers graded the deposition of solution immediately after application based on surface area stained with Methylene blue using a 5-point scale: 0 = none, 1 = <1/3, 2 = 1/3, 3 = 2/3, 4 = complete. A consensus was made before assigning the final grade. The following sites were evaluated: inferior (IT), middle (MT) and superior turbinates (ST); uncinate process (UP), olfactory cleft (OC), ethmoidal bulla surface (EB) and sphenoethmoid recess (SER). Each cadaver subsequently underwent bilateral complete ethmoidectomy, type 2 maxillary antrostomy, sphenoidectomy and Draf 2A frontal sinusotomy.

After dissection, deposition of solution was evaluated again using the abovementioned 5-point scale, focusing on distribution in less accessible sites, namely the maxillary sinus (MS), sphenoid sinus (SS), frontal sinus (FS); frontal recess (FR), interior of the anterior (IAE) and posterior ethmoids (IPE). After rinsing the sinonasal cavities with normal saline, bilateral nebulisation was performed post-surgery. There was no re-allocation of pulsation and non-pulsation sides and the distribution of solution by surface area was rated once again. Wilcoxon signed-rank sum test was performed to compare distribution of solution between

different sites pre- and post-surgery. The level of statistical significance was taken to be at p < 0.05. SPSS version 17 (SPSS Inc., Chicago, IL) was used for the analysis.

RESULTS AND DISCUSSIONMean grades for dye deposition in the different sites pre-

operatively are illustrated in Table 1. Pre-surgery comparison between pulsating and non-pulsating nebulisation revealed no significant differences in distribution of dye across all sites of interest (p = 0.285). As expected, anterior nasal sites such as the turbinates, UP and OC were better penetrated than posterior sites in both the pulsating and non-pulsating arm. Both pulsed and non-pulsed nebulisation consistently delivered dye to the olfactory cleft before surgery, with slightly better results seen in the pulsed arm (mean scores 4 ± 0 and 3.25±0.96 respectively). This difference was not statistically significant (p = 0.180).

There was no significant difference in distribution of dye noted with pulsation in the sinuses or recesses of interest collectively (p = 0.76). This finding was consistent between individual Paranasal sinuses with or without pulsation. In addition, it was noted that zero dye was delivered to the PE and SS before surgery.

Table 2 demonstrates post-operative deposition of dye in the sinuses and recesses. Similarly, there was no significant difference in post-operative dye deposition between the pulsed and non-pulsed nebulisation across all sites (p = 1). However, comparisons of mean scores before and after surgery showed an improvement in dye distribution in these less accessible intranasal sites. In particular, PE and SS demonstrated evidence of dye deposition after surgery in both non-pulsating and pulsating arms. A comparison between pre- and post-operative mean scores for dye distribution in the pulsed and non-pulsed arm is illustrated in Table 3. Post-operatively, a statistically significant increase in dye deposition in the PE and SS was found in the pulsed arm (p = 0.046 for both sites). The increase in dye distributed with and without pulsation at other sites did not

Table 1: Pre-operative means scores for all subsites of interest.

Site Pulsation No pulsation p- value

Inferior turbinate 2.75 ± 0.5 2.25 ± 0.96 0.414

Middle turbinate 2.25± 0.5 2.75± 0.5 0.157

Superior turbinate 2.5 ± 1.29 1.75± 0.96 0.257

Uncinate process 3.25± 0.5 2.75± 0.5 0.157

Olfactory cleft 4 ± 0 3.25± 0.96 0.180

Maxillary sinus 0.75± 0.96 0.75± 0.96 1.00

Ethmoidal bulla 1.75± 0.5 1.5 ± 0.58 0.564Interior of anterior ethmoid 0.5 ± 1 0.5 ± 1 1.00

Interior of posterior ethmoid 0 ± 0 0± 0 1.00

Spheno-ethmoid recess 1.75± 0.96 2± 0.82 0.705

Sphenoid sinus 0 ± 0 0± 0 1.00

Frontal sinus 0.25± 0.5 0± 0 0.317

Frontal recess 0.75± 0.96 0.75± 0.96 0.785

Data are expressed as mean ± SD.

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reach statistical significance. This study was designed to assess the advantage of using a pulsed device versus a conventional nebulizer in the distribution of medication to the Paranasal sinuses before and after surgery.

The difference in sinonasal distribution of dye between pulsed and the non-pulsed nebulizer in the pre-operative setting was not statistically significant (Table 1, p = 0.285). The results illustrate that MS, IAE, IPE and SS in particular were equally inaccessible in both arms (p = 1). Of note, there was consistent deposition pre-operatively within the OC with both pulsed and non-pulsed nebulization, with a non-significant improved result in the pulsed arm (4 ± 0 and 3.25 ± 0.96 respectively, p = 0.180). This finding differs from another study where the OC was not well penetrated by nebulisation before and after surgery [15]. This suggests that further studies are needed to ascertain whether nebulization may be advantageous to patients with CRS and prominent olfactory symptoms such as hyposmia.

This study also found no difference in distribution of dye between the two arms in the post-operative setting (Table 2, p = 1). However, this study demonstrates that surgery does enhance deposition of particles within the nasal sinuses. Table 2 illustrates that mean dye deposition across all sites is improved after surgery. Of note, prior to surgery, there was zero dye noted in the IPE and SS. Post-surgery, mean scores at IPE for the pulsating and non-pulsating arm was 2 ± 0 and 1.75 ± 0.96 respectively; at the SS, 1 ± 0 and 1.5 ± 0.58 for pulsating and non-pulsating arm respectively. This finding is in agreement with other studies that suggest that functional endoscopic sinus surgery (FESS) improves distribution of medication to the sinuses [ 9,13,15,16]. Interestingly, the improved deposition of dye in the IPE and SS

post-surgery in the pulsed arm was statistically significant (p = 0.046 at both sites) while the same comparison in the non-pulsed arm was non-significant.

In the non-pulsed model, Manes et al. used fluoresce in to compare pre- and post-FESS distribution of aerosol [15]. The study demonstrated improved intranasal distribution of dye post-surgery across all sites, with statistically significant difference noted in the middle meatus region (p = 0.044). When FESS was combined with endoscopic modified Lothrop procedure, a statistically significant improvement of drug delivered to the frontal neo-ostium was reported (p = 0.001). Despite differing in the extent to which surgery improves dye distribution by non-pulsed devices, the study reinforces that surgery is an important component to disease management and highlights that different types of surgery may afford varying access to diseased mucosa.

Other studies have analyzed the efficacy of pulsed nebulisation for the topical treatment of chronic sinusitis. Valentine et al. compared sinonasal penetration of nasal douching to the pulsed PARI Sinus device using Methylene blue as a stain [10]. The study reported that nasal douching resulted in significantly better outcomes across indices such as intensity of stain, percentage of stain as well as circumference stained (p < 0.001 for all indices). However, other studies using different formulations demonstrate that pulsed nebulizer is a non-inferior device [12,17,18].

Moller et al. evaluated the effect of pulsating airflow on intranasal deposition using 81mKr-gas imaging [12]. Compared to a non-pulsating system which resulted in < 5% total Kr-gas activity detected in the sinuses, pulsation increased penetration to about 48%. The pulsating airflow also resulted in sustained release of 81mKr-gas activity after the device was switched off; the authors hypothesized that this delayed effect could augment aerosol deposition.

The same study assessed deposition efficiency of Tc99m-diethylene triamine pentaacetic acid (99m Tc-DTPA) aerosol dissemination with pulsating airflow compared to a nasal spray [12]. With pulsating aerosol delivery, total deposition in the nasal cavity (including sinuses) was 71 ± 17% of the nebulized dose, and 6.5 ± 2.3% of the total nose activity penetrated to the sinuses. However, there was no significant activity detected in the sinuses with the nasal spray.

In another study, comparing pulsating aerosols and nasal spray, 99mTc-DTPA was used to compare difference in drug delivery in the posterior nasal spaces and Paranasal sinuses [17]. The pulsating aerosol delivered 9.7±2.0% of the nasal dose to the maxillary and sphenoid sinuses while the nasal pump resulted in non-significant sinus deposition. It was similarly concluded that pulsating aerosols may be superior to the nasal spray and is a promising therapeutic option [12,18].

A number of important limitations are present in this study. The small sample size makes this a pilot study and results cannot be generalized to a bigger population. However inferential statistics still has value to detect a large effect size even if the sample size is limited. In our small sized sample, there was no significant advantage in delivery of medication using the pulsed feature before and after surgery, except in the most posterior subsites post-surgery. For that reason we postulate that the

Table 2: Post-operative means scores of Paranasal sinuses and frontal recess.Site Pulsation No pulsation p-value

Maxillary sinus 2± 0.82 1.75 ± 0.96 0.317Interior of anterior ethmoid 2.5 ± 0.58 2.5 ± 0.58 1.00

Interior of posterior ethmoid 2± 0 1.75 ± 0.96 0.564

Sphenoid sinus 1± 0 1.5 ± 0.58 0.157

Frontal sinus 1± 0.82 1 ± 1.41 1.00

Frontal recess 1.75 ± 1.5 1.75 ± 0.96 1.00

Data are expressed as mean ± SD.

Table 3: p-values for comparison of mean scores pre- and post-operatively for pulsed and non-pulsed nebulisation in Paranasal sinuses and frontal recess.Site Pulsation No pulsation

Maxillary sinus 0.102 0.194

Interior of anterior ethmoid 0.102 0.066

Interior of posterior ethmoid 0.046* 0.066

Sphenoid sinus 0.046* 0.063

Frontal sinus 0.083 0.180

Frontal recess 0.285 0.102* p-value < 0.05

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pulsating feature allows for deeper penetration when there is a sufficiently enlarged pathway. We hypothesise that a main limitation in our study was the use of Methylene blue dye as a surrogate to measure drug deposition. Mathematical modeling of aerosolised particle deposition proposes three factors to determine particle deposition within the sinus: particle size, size of the sinus ostium and pressure gradient. It is widely accepted that particle size affects distribution and Hyo et al. concluded that the ideal particle size for delivery to the maxillary sinus would be 3-10μm [19]. The PARI Sinus produces aerosol particles of a mass median aerodynamic diameter (MMAD) of 3.2 μm. This suggests that particle size generated by the device is unlikely to be a limiting factor in particle distribution. Methylene blue has a MMAD of 4.40 μm when nebulized, which may not be ideal in order to be distributed by this device [20]. This study postulates that the choice of dye in this study may confound the assessment of the device. Using another dye that can match the MMAD of the device may be a more accurate representation of its efficacy. If the final concentration of Methylene blue present in the nebulised solution is very low, the presence of the dye may not be detected by the human eye. In addition, this would also hinder the assessment of the pulsation function.

The conflicting results from the study by Valentine et, al which used Methylene blue versus those involving other dyes emphasise that different surrogate markers for intranasal drug deposition may influence the outcome of such studies [10,12,17]. Characterization studies concerning choice of stains in similar study models may be crucial to ensuring accurate conclusions can be drawn from it [21].

The use of cadavers has been a moot point as to whether results derived can be extrapolated to living patients. Hyo et al. compared difference in deposition efficiency in volunteers and a cast model. No significant difference in results was found and it was concluded that ciliary action and respiration are not crucial in drug deposition, particularly in non-ventilated sites such as intranasal sinuses [19]. Furthermore, the use of cadavers may limit potential confounders present in a patient such as extent of disease, concurrent medication and compliance to treatment. The main difference between individual cadavers would only be variation in anatomy.

Subjective observation forms the basis of data collection in this study. To minimize bias, scores were obtained from blinded observers who graded each site independently. Consensus discussion was conducted if there were discrepancies. Radionuclide imaging may offer an alternative, however demarcating delicate anatomical boundaries may prove challenging [22].

Several studies have compared the pulsed nebulizer to other devices such as the nasal spray and the squeeze bottle [10,12,17]. The fundamental benefit of pulsation compared to conventional nebulization has been less explored [12]. This study may help to increase our understanding of the device and further define the role that pulsed Nebulizers may have in the clinical setting.

CONCLUSIONIn conclusion, the pulsed Nebuliser does not significantly

differ in intra-nasal distribution of medication compared to the

conventional Nebuliser in our sample. Surgery enhances dye distributed by the pulsed Nebuliser in certain subsites. However, additional work is needed to assess the use of different surrogate markers in similar studies to better evaluate such devices.

ACKNOWLEDGEMENTSMs. Stephanie Fook- Chong, Institutional Senior Statistician.

PARI GmbH for the loan of the device used in this study. Zayar Min and Team, Anatomy Laboratory of Singapore General Hospital.

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van Schaik CR, Ho D, Hui NY (2015) Comparison of Methylene Blue Distribution in the Nasal Cavity and Paranasal Sinuses Using a Pulsed Versus Non-Pulsed Nebuliser in the Cadaver. Ann Otolaryngol Rhinol 2(12): 1078.

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