INTRODUCTION

The lamina propria helps viscosity, contractility and formation of mucous wave of vocal fold. Its good performance is essential for the beginning and maintenance of vibration supported by Bernoulli's principle(1). Humans can produce sound within an ample band of frequency and intensity, varying in tone quality. To do that, they have to use their vocal folds, which hold histological and structure features that are suitable for phonation activity. From viewpoint of phonation, vocal fold forms a structure of multiple layers with different mechanical properties. Vocal folds do not work as a single structure vibrator like a musical instrument string, but as a vibrator of different multiple layers. Such laminar structure can be adjusted on frequency and intensity of human voice(2). Friedrich et al (1993) support the idea the complexity of vocal fold, which is the base of understanding of human vocal capacity(3).

Microflap techniques were developed to help dissection and removal of mature cysts, polyps and nodules on the superficial layer of lamina propria while there is a preservation of adjacent normal mucosa layer and subjacent vocal ligament(4,5,6). It aims scars development and hardening with synechiae formation on mucosa covering that is responsible for damages on vocal fold vibration.

Clinical studies aiming to evaluate effects of injected corticoid use on lamina propria of vocal fold with the help of a fiberscope on larynx surgery under local anesthesia without causing injury on the structure reported its useful effect on therapy of phonotraumatic injury, such as vocal nodules and Reinke's oedema, leading to vocal quality improvement, reduction on phonotherapy and on injuries, in most of cases(7,8).

There is also improvement report on glottic closure and vocal quality after corticoid injection followed by microflap technique(9). However, a study on dogs, did not relate corticoid injection before microflap technique with scar reduction on experimental group, or did not reveal difference on vocal fold activity measured by videoestroboscopy(10).

Scars on lamina propria layer of vocal folds produce an abnormal relation between body and mucous covering, affecting its sliding and then leading to a reduction on mucosa wave. In case scar gets worse, segment with no mucous wave gets greater and dysphonia gets inflamed. Some report this is the most common reason of dysphonia (35%) followed by endolarynx surgery11.

Systemic or injected corticosteroids on vocal folds are usually associated with laryngeal microflap in order to prevent scars, then assuring vocal quality. Systemic corticosteroids delay cicatrisation leading to a better organization of scar tissue; therefore, their effects over vocal folds are unclear(10). Other types of therapy have been under observation in order to adjust scar process on vocal fold after removing its mucous covering. Systemic or topic(12) corticosteroids, cooling of the vocal cord before the surgery, fibrin adhesive and other methods have been used in order to improve cicatrisation of mucous covering after phonosuergery procedure(13).


OBJECTIVES

Having revising the literature aimed the following:

a. To macroscopically observe the presence of synechiae on vocal cords after mucous portion removal by using cold instrument and with no injected corticoid on vocal fold.

b. To comparatively quantify total deposition of collagen fibers on pig vocal fold submitted to mucous portion removal with and without using injected corticotherapy.


METHOD

This study was carried at Fazenda Experimental da Universidade Federal do Paraná - UFPR (Experimental Farm of the Federal University) after being approved by the Research Ethics Committee of the Hospital Angelina Caron - protocol #008-2005. Then, it was followed the principles of the Colégio Brasileiro de Experimentação Experimental- COBEA (Brazilian Institution of Experiment) and recommendations of euthanasia for experimental animals recommended by Close and by Internacional Comitee on Veterinary Gross Anatomical Nomenclature.

12 large white pigs (Suideo Sus) were used for such experiment. There were seven males and five females, aging from 27 to 31 years, weighing between 8.7 Kg and 11.5 kg (average: 10.1kg), and divided into two equally numbered groups. (Chart 1)

Control group underwent surgery on vocal fold with no injected corticoid. Surgery of mucous portion removal from the right vocal fold was performed with laryngeal scissors, by beginning from prehension of the mucosa of the anterior third of the free edge, of the right vocal fold with Bouchayer's tweezers (Microfrance^®). Afterwards 2mm of mucosa was cut with laryngeal scissors to the left. Left vocal fold was not touched.

Experimental group underwent injection of 01ml of sodium phosphate salt form of dexamethasone by using a 23g Venescalp needle on each focal fold, guided by Bouchayer's tweezers (Microfrance_®) before removal surgery of the mucous portion on right vocal fold. Venescalp was introduced in the anterior third of right and left vocal folds in their superficial layers of the lamina propria. Removal of mucous portion of right vocal fold was done with laryngeal scissors after corticoid injection with prehension of the mucosa from the anterior third of the free edge of right vocal fold, by using Bouchayer Microfrance^® prehension tweezers. Afterwards, it was performed a 2mm cut of the apprehend mucosa with laryngeal scissors to the left. Mucous fragment was not removed from the left vocal fold, which was only corticoid injected.

Euthanasia was carried 30 days after surgery in the slaughterhouse at Fazenda Experimental da UFPR, followed by total larynx removal, so vocal folds could be analyzed. Macroscopic analysis of the glottis on the anterior third of vocal folds aimed researching presence and absence of postoperative synechiae; after that, a ventral longitudinal incision was performed in the larynx with #15 razor scapel to expose vocal fold, to analyze macroscopically the presence or absence of postoperative synechiae for the second time and another removal of mucous portion of vocal fold near thyroid cartilage.

The laminae analysis was done through Pro-image-plus 4.5_® for Windowsâ in a Pentium IV_® attached to a Olympus_® BX50 camera and Sony_® video, calibrated in micrometers in 20 times zoom lenses. Such program read picrosirius red stained tissues, by starting from the analysis of microscopic features of tissues as epithelium and submucosa. The same program performed image polarization to analyze collagen deposition through tissue colors, and then measures were transferred to Windows Excelâ.

Fibroplasy was also measured in square micrometers, which consists of collagen deposition on surface of mucosa from scar process.

Statistical analysis was done through paired comparison (right and left side) with the help of nonparametric Wilcoxon test. When comparing results from groups, parametric Mann-Whitney test was applied. In all comparisons, null hypothesis met the same results from paired groups and alternative hypothesis met different ones (p < 0.05).


RESULTS

Macroscopic analysis showed absent synechiae on the larynx mucous after surgery in both groups.

Once the image polarization is performed, the collagenous tissue which has been presently studied may be differentiated from the non-collagenous tissue, through the observation of the collagen birefringence and of its orange and red coloring, when compared to the non-collagenous substance which has a darken color (Picture 1); (Picture 2).

Picture 1. Example of animal laminae (sample/E-D3) colored by Picrosirius Red technique showing conjunctive tissue of the lamina propria before polarization (400x larger).


Picture 2. Example of animal laminae (sample/E-D3) colored by Picrosirius Red technique showing conjunctive tissue of the lamina propria before polarization (400x larger).

In the control group, the deposition area average on submucosa of left vocal folds which were not submitted to surgery was 3116.33 square micrometers; and the one of the right vocal folds submitted to surgery was 2353.29 square micrometers (Table 1).

In the experimental group the same areas were 3526.05 square micrometers and 2167.92 square micrometers respectively (Table 1).

P = 0.0464 was found when comparing collagen deposition of control group with no injury X control group with injury, and that showed the difference between total quantity of collagen in the two groups was statistically significant (Chart 2).

The comparison between control group with injury X experimental group with injury showed P=0.0043, also showing that total quantity of collagen on the vocal folds was statistically significant (Chart 2). When comparing experimental group with no injury X control group with injury, P = 0.0065; comparing experimental group with no injury X experimental group with injury, p = 0.0277, what showed statistical significance, or that, there was a difference between total collagen quantity in the two groups (Chart 2).


DISCUSSION

Only one paper, during literature revision, has compared endolaryngeal surgery in animals. It compared vocal folds in dogs, pigs and apes by observing quantity of collagen deposition, elastin fibers and amorphous tissues, concluding that pig's and dog's vocal folds present a larger amount of elastic fibers and collagen in profound layers of lamina propria, which is similar to histological features of human vocal folds, than ape's. Dissection process of microdflap on dog's and pig's vocal folds are very similar to the human's(14).

It was also found two papers reporting the use of corticoid on vocal folds injuries. A clinical study reported improvement on glottic closure and on vocal quality after corticoid injection followed by microflap on vocal folds. And an experimental study on dogs, in which corticosteroids injection was used before microflap, concluded that, despite the fact that the corticoids delay scar process in both inflammatory infiltrate and neovascularization, and that did not cause quantity improvement at stroboscopic analysis of the larynx(9,10).

Systemic corticoids delay cicatrisation process by providing a better organization on scar tissue and they are also often used in association with laryngeal microflap in order to prevent scars, providing then a better vocal quality. There is still very little information on the use of injected corticoids on vocal fold in the literature(10).

This study reports less deposition of total collagen in the group operated with corticoid than in the one operated with no corticoid, though this difference was not expressive according to the literature(10).

An expressive difference could be seen when comparing groups regarding surgery with or without injury, which was observed in both groups the experimental and control ones. In the group with injury, there was a lesser deposition of total collagen.

The lack of statistical importance on total collagen decrease in the corticoid-operated group makes one aware of the need of more experimental studies with the purpose of evaluating the activity of such substances in scar areas of the vocal folds, however many clinical studies confirm its use by vocal fold improvement in patients with phonotraumatic injuries (vocal nodules, polyps and Reinke oedema), with no scar injuries (removable ones)(7,8).


CONCLUSION

Before the results from this study, one might conclude that:

a. Presence of synechiae was not observed in experimental examples of mucosa fragment removal on the free edge with cold instrument with or without injected corticoid use on vocal fold at the macroscopic analysis.

b. The use of injected corticoid on lamina propria of the vocal folds which were submitted to mucosa fragment removal with cold instrument provide a non-expressive difference of total collagen deposition on submucosa which was quantified by optical microscopy assisted by Pro-image-plus 4.5^® program.


REFERENCES

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3. Friedrich G, Kainz J, Freidl W. Zurfunktionellen struktur der menschlichen stimmlippe. Laryngorhinootologie. 1993;72:215-24.

4. Courey MS, Gardener GM, Stone RE, Ossoff RH. Endoscopic vocal fold microflap: a three-year experience. Ann. Otol. Rhino. Laryngol.. 1995; 104: p.267-73.

5. Sataloff RT, Spiegel JR, Heuer RJ. Laryngeal minimicroflap: a new technique and reassessment of microflap saga. J. Voice. 1995; 9:198-204.

6. Courey MS, Garrett CG, Ossoff RH. Medial microflap excision of benign vocal fold lesions. Laryngoscope. 1997; 107(3):340-4.

7. Hirano S, Tateya I, Omori K. Steriod injection for Reinke`s edema using fiberoptic laryngeal surgery. Acta Otolaryngol.. 2003; 123(3):417-20.

8. Hirano S, Tateya I, Omori K. Steroid injection to vocal nodules using fiberoptic laryngeal surgery under topical anesthesia. Eur. Arch. Otorhinolaryngol. 2004; 261(9):489-92.

9. Bouchayer M, Cornut G. Microsurgery for benign lesions of the vocal folds. Ear Nose Throat J.. 1988; 67 (6): 446-66.

10. Coleman JR Jr, Smith S, Reinisch L. Histomorphometric and laryngeal videostroboscopic analysis of the effects of corticosteroids on microflap healing inthe dog larynx. Ann. Otol. Rhinol. Laryngol. 1999; 108(2):119-27.

11. Woo P, Casper J, Colton R, Brewer D. Diagnosis and treatment of persistent dysphonia after laryngeal surgery: a retrospective analysis of 62 pacientes. Laryngoscope. 1994; 104:1084-91.

12. Sheppard LM. Effect of steroids or precooling on edema and tissue thermal coagulation after CO2 laser impact. Lasers Surg. Med.. 1992; 12:137-46.

13. Garrett CG, Reinisch L. New-generation pulse carbon dioxide laser: comparative effects on vocal fold wound healing. Ann. Otol. Rhinol. Laryngol. 2002; 111:471-6.

14. Garrett CG, Coleman JR, Reinisch L. Comparative histology and vibration of the vocal folds: implications for experiemntal studies in microlaryngeal surgery. Laryngoscope. 2000; 110:814-24.














1. ENT Doctor and Master in Surgery by IPEM.
2. ENT Doctor at the Centro Médico Especializado Baptistella and residence preceptor in ENT at Hospital Angelina Caron.
3. Professor at Faculdade Federal do Paraná UFPR - Scientific Head of the Científico do Colegio Brasileiro de Cirurgia Digestiva (Institution of Digestive Surgery); coordenator of Medical Research Institute at HUEC and professor of discipline of Surgical Clinic at FEPAR.
4. PhD in Health Science by UFPR - Member of Administration State Office - Paraná - Doctor at HUEC - professor at Faculdade Evangélica do Paraná - 4th Adjunt Professor at UFPR)
5. 1st year Resident Doctor in ENT at Hospital Universitário Evangélico de Curitiba.
6. 12th semester/senior Medical School student at Faculdade Evangélica do Paraná.
7. 10th semester/senior Medical School student at Faculdade Evangélica do Paraná.
8. 10th semester/senior Medical School student at Pontifícia Universidade Católica do Paraná.

UFPR Faculdade Federal do Paraná - Federal University - Paraná

Dr. Eduardo Baptistella
Mail Address: Av. João Gualberto, nº 1795, conj 01 Juvevê, Curitiba/Pr - CEP: 80030-001 - Fax: (41) 3039-8878 - E-mail: fabiano_trotta@yahoo.com.br

This article was submitted to SGP (Sistema de Gestão de Publicações - Publication Management system) of R@IO on October 13th, 2007 and approved on November 2nd, 2007 at 10:43:01.