in rabbits with cessation of steroid therapy
Hüseyin FİDAN1, Önder ŞAHİN2, Fatma FİDAN3, Yüksel ELA1, Ahmet SONGUR4, Murat YAĞMURCA5
1Afyon Kocatepe Üniversitesi Tıp Fakültesi, Anesteziyoloji Anabilim Dalı,
2 Afyon Kocatepe Üniversitesi Tıp Fakültesi, Patoloji Anabilim Dalı,
3Afyon Kocatepe Üniversitesi Tıp Fakültesi, Göğüs Hastalıkları Anabilim Dalı,
4Afyon Kocatepe Üniversitesi Tıp Fakültesi, Anatomi Anabilim Dalı,
5Afyon Kocatepe Üniversitesi Tıp Fakültesi, Histoloji ve Embriyoloji Anabilim Dalı, Afyon.
ÖZET
Steroid tedavisi kesilen tavşanlarda endotrakeal entübasyonun ödem riski
Dokulardaki rebound ödem, steroid tedavisinin kesilmesinin iyi tanımlanmış bir komplikasyonudur. Kronik obstrüktif ak- ciğer hastalığı akut alevlenmesinde, kısa süreli steroid tedavisinde sistemik steroidin kademeli olarak azaltılması çoğu du- rumlarda gereksiz görülür, bu durumda larengeal rebound ödemin varlığı araştırılmamıştır. Biz steroid tedavisinin kesilme- sinden sonra entübasyon uygulandığında entübasyonun larengeal ödemi arttırıp arttırmadığını araştırdık. Randomize ola- rak 36 tavşan altı gruba bölündü. Steroid uygulanan dört gruba 10 gün boyunca intraperitoneal 1 mg/kg metilprednizo- lon verildi. Bir gruba 10 gün boyunca serum fizyolojik verildi ve son kontrol grubu sadece entübe edildi. Steroid tedavisi verilen tavşanlar steroid tedavisinin kesilmesinden sonra birinci gün, birinci hafta, ikinci hafta ve birinci ayda entübe edi- lerek gruplara ayrıldı. Hava yolu alanı ve larenks lümeninin tiroid kartilaj ve özefagus ile çevrili larenks dokularına kesit- sel yüzdesi stereolojik yöntemle çalışıldı. Birinci hafta steroid grubunun kontrol grubuna göre larenks lümen alanları an- lamlı olarak daha dardı ve larenks lümeninin tiroid kartilaj ve özefagus ile çevrili larenks dokularına kesitsel yüzdesi an- lamlı olarak daha büyüktü. Tavşanlarda steroid tedavisinin ani kesilmesinden bir hafta sonra larenkste rebound ödem olu- şur. Steroid tedavisinin kesilmesinden sonra entübasyon için klinik olarak güvenli zaman çalışmamızla tanımlanmıştır. Bu sonuçlar steroid tedavisinin kesilmesinden sonraki bir haftalık sürecin entübasyon için riskli bir zaman olabileceğini dü- şündürmektedir.
Anahtar Kelimeler: Larenks ödemi, steroid, rebound ödem, entübasyon.
Yazışma Adresi (Address for Correspondence):
Dr. Fatma FİDAN, Hattat Karahisar Mahallesi 4. Sokak Kaya Apartmanı Daire: 7 03200 AFYON - TURKEY e-mail: drffidan@yahoo.com
Endotracheal intubation is one of the most fre- quently used methods to ventilate patients in critical care units and operating rooms. Altho- ugh life saving, this invasive method may cause laryngeal damage (1,2). Inflammatory response can be seen as early as an hour following extu- bation and characterized with glottic or subglot- tic oedema, ulceration, and stenosis (3). After short intubation periods, inflammatory cell ac- cumulation was shown at submucosal vessels resembling ischemia-reperfusion injury in other organs (4).
Laryngeal oedema manifests itself by respira- tory distress and inspiratory whistling called
“stridor”. Such oedema formation can develop as early as 6 hour following intubation, so that prolonged intubation is not a prerequisite to its development (5-7). By decreasing the upper respiratory tract diameter, laryngeal oedema
increases airway resistance and, consequently, the respiratory work (6).
Corticosteroids are potent anti-inflammatory agents. The mode of action, however, is not completely understood. It has been shown that steroids restore ground substance and basement membranes of connective tissue, probably res- toring normal function. Besides, steroids have also an unpleasant effect. Rebound oedema forms with abrupt cessation of steroid therapy.
Rebound oedema has been shown to form in cornea, macula, unstable asthma and dermal flaps (8-10). However, tapering of systemic cor- ticosteroid regimens in short course steroid the- rapy is considered unnecessary in most cir- cumstances, evidence of laryngeal rebound edema is uncertain (11). Global Initiative for Chronic Obstructive Lung Disease (GOLD) sug- gests to apply 30 to 40 mg of oral prednisolone SUMMARY
Oedema risk of endotracheal intubation in rabbits with cessation of steroid therapy
Hüseyin FİDAN1, Önder ŞAHİN2, Fatma FİDAN3, Yüksel ELA1, Ahmet SONGUR4, Murat YAĞMURCA5
1Department of Anesthesiology, Faculty of Medicine, Afyon Kocatepe University, Afyon, Turkey,
2Department of Pathology, Faculty of Medicine, Afyon Kocatepe University, Afyon, Turkey,
3Department of Chest Diseases, Faculty of Medicine, Afyon Kocatepe University, Afyon, Turkey,
4Department of Anatomy, Faculty of Medicine, Afyon Kocatepe University, Afyon, Turkey,
5Department of Histology and Embriology, Faculty of Medicine, Afyon Kocatepe University, Afyon, Turkey.
Rebound oedema of tissues is a well defined complication of cessation of steroid therapy. Tapering of systemic corticostero- id regimens in short course steroid therapy is considered unnecessary in most circumstances in acut exacerbation of chro- nic obstructive pulmonary diseases, presence of laryngeal rebound edema is obscure in this situtation. We studied whet- her or not laryngeal oedema increases after intubation when intubation is established after cessation of steroid therapy.
Thirty-six rabbits were randomly divided into six groups. We administered 1 mg/kg methyl prednisolone intraperitoneally to four steroid groups for ten days. Another group received serum physiologic for ten days and last group was sham cont- rol that was intubated only. Rabbits that received steroid therapy were intubated and separated into groups one day, one week, two weeks, and a month after the cessation of steroid therapy. Airway area and percentage of cross sectional area of larynx lumen to their own larynx tissues surrounded by thyroid cartilage and oesophagus were studied by stereologi- cal methods. Larynx lumen area of one week steroid group was significantly narrower and percentage of cross sectional area of larynx lumen to their own larynx tissues surrounded by thyroid cartilage and oesophagus was significantly larger than sham control. Rebound oedema forms in larynx with abrupt cessation of steroid therapy in rabbits. Clinical safe time for intubation after abrupt cessation of steroid therapy is also defined with our study. These results suggest that one week after the cessation of steroid therapy may be a hazardous time for tracheal intubation.
Key Words: Larynx oedema, steroid, rebound oedema, intubation.
to Chronic Obstructive Pulmonary Disease (COPD) patients as a short course steroid the- rapy for 10 to 14 days when COPD exacerbates (12). 0.5-1.0 mg/kg prednisolone is administe- red as the treatment of acute asthma excacerba- tion (13).
We studied whether or not laryngeal oedema increases after intubation when intubation is es- tablished after cessation of steroid therapy.
MATERIALS and METHODS
This study was approved by Animal Committee of Ethics of our institution. Thirty six female New Zealand rabbits were enrolled into the study.
Rabbits were divided into six groups.
Surgical Procedures
Anesthesia was induced by Xylazin HCl (15 mg/kg) and ketamine (25 mg/kg) intramuscu- larly (IM). We have placed an arterial line from one ear and venous line from the other. We intu- bated rabbits with uncuffed polivinylchloride en- dotracheal tube (I.D. 2.5, Kendall) by the help of laryngoscope that has Millar blade. Anesthesia was maintained by 1-2% sevoflurane and rabbits were ventilated with volume-controlled ventila- tor (CWE Inc. SAR 830/AP, Ardmore, USA) for an hour. During anesthesia, invasive arterial ten- sion, heart rate and peripheral oxygen saturati- on were monitored (Datex Ohmeda Type F- CU8, Helsinki, Finland).
All rabbits were extubated and kept on sponta- neous respiration for two hours with oxygen sup- port. Finally, all rabbits were sacrificed with 50 mg/kg pentothal Na intravenously (IV). Immedi- ately their necks were dissected to reach larynx.
Larynx was harvested from the level of hyoid cartilage above and fifth to sixth tracheal carti- lages below. Harvested tissue was put into for- malin for further histological examination.
Groups
1stgroup (sham control group): Anesthesia was induced. We intubated rabbits and kept them on mechanical ventilation for an hour. Rabbits were extubated and went through above procedures.
2ndgroup (SP control group): We have adminis- tered 2 mL of 0.9% NaCl (SP) intraperitoneally
(IP) to the rabbits in this group for ten days. One day after the final injection of ten injections, rab- bits were intubated and went through above pro- cedures.
3rdgroup (st 1d group): We have administered methyl prednisolone (1 mg/kg) in 2 mL of SP IP to the rabbits in this group for ten days. One day after the final injection of ten injections, rabbits were intubated and went through above proce- dures.
4thgroup (st 1w group): We have administered methyl prednisolone (1 mg/kg) in 2 mL of SP IP to the rabbits in this group for ten days. Rabbits were fed for one week after the final injection.
One week after the final injection, rabbits were intubated and went through above procedures.
5thgroup (st 2w group): We have administered methyl prednisolone (1 mg/kg) in 2 mL of SP IP to the rabbits in this group for ten days. Rabbits were fed for two weeks after the final injection.
Two weeks after the final injection, rabbits were intubated and went through above procedures.
6thgroup (st 1m group): We have administered methyl prednisolone (1 mg/kg) in 2 mL of SP IP to the rabbits in this group for ten days. Rabbits were fed for one month after the final injection.
One month after the final injection, rabbits were intubated and went through above procedures.
Histopathologic Examinations
We have harvested the larynx 0.5 cm proximally and distally to cricoid’s process which was our reference point.
Anatomical examination of rabbit larynx showed us that the level of vocal cords was its narrowest place and its width was about 1.0 mm horizon- tally (Figure 1). We sectioned whole larynx transversely every 150 µm. We obtained 25 to 35 sections with 10 µm width from every larynx.
We chose two sections as target sections not to miss a target section if a technical accident hap- pens.
All obtained transverse sections were processed with haematoxylin and eosin. The sections that resemble human vocal cords transverse secti- ons were chosen (14). We have chosen the sec-
tion that had the most anterior protruding ary- tenoid cartilage, while arytenoid cartilage was protruding anteriorly and drawing back in secti- ons that follow one after the other. We suggest that maximum level difference that one can make with our targeting method between diffe- rent rabbits is about 75 to 85 µm according to the choice of the first or the second target sec- tion at the target level.
Two different measurements were performed on target sections. Firstly we measured the airway area because airway gets narrower when laryn- geal oedema forms. We put a microfilm consis- ting of squares with 100 µm wall thickness on histological target sections. We photographed them and magnified (Figure 2). We counted the squares that matched the airway. The half squ- ares that match the right side of the larynx were counted but the squares on the other side were not counted. This measurement was done for all target sections. The counted number for target section was divided by 100 to give the airway area in mm2because 100 squares form an area of 1 mm2.
The above measurement gave us the quantitati- ve airway area. Furthermore, we examined the airway narrowing in each individual rabbit. This meauserement was done to rule out faulty analysis because rabbits may have different air- way area although they may have the same we- ight. We graded the proportion of airway to lar- ynx tissue surrounded by the cartilages (a/b ra-
tio) while tissue becomes swollen and airway narrows when laryngeal oedema forms. The a/b ratio was arbitrarily graded to reflect varying degrees of airway narrowing with higher grades indicating more extreme airway narrowing.
Target sections were magnified 100 times and printed. We counted a/b ratios by the help of in- dependent counting measures stereologically.
Independing counting measures have points on the measure, and points are equally distant to each other. “a” was defined as the counts that matched airway when independent counting measure was placed over the target section. “b”
was defined as the counts that matched larynx tissues surrounded by thyroid cartilage and oe- sophagus when independent counting measure was placed over the target section (Figure 3). In other words; “a” is the cross-sectional area of the airway and “b” is the cross-sectional area of the laryngeal tissues surrounding the airway lu- men and internal to the cartilaginous skeleton of the airway. The “a/b ratio” measures airway lu- men relative to the surrounding soft tissues; a decrease in the ratio is hypothesized to result from airway edema.
We have graded a/b ratios from zero to five to measure them statistically. We have graded a/b Figure 1. Inferior view of vocal cords in rabbit larynx.
Figure 2. View of target cross section of larynx with microfilm (wall thickness of every square is 100 µm) placed on the preparation.
ratios from the lowest ratio to the highest ratio.
The highest ratio was 0.5035 and the lowest ra- tio was 0.2485. The difference between the hig- hest and lowest ratios were divided into six gra- des; grade 0 (range: 0.5035-0.4610), grade 1 (range: 0.4610-0.4185), grade 2 (range:
0.4185-0.3760), grade 3 (range: 0.3760- 0.3335), grade 4 (range: 0.3335-0.2910) and grade 5 (range: 0.2910-0.2485). When airway narrows and tissue is swollen, a/b ratio decre- ases, and consequently gets a higher grade.
Statistical Analysis
Statistical analysis was carried out using the Sta- tistical Package for Social Sciences SPSS 10.0.
Comparison between groups was performed using one-way ANOVA and posthoc Tukey test. Data
were expressed as mean ± standard deviation. p<
0.05 is considered as statistically significant.
RESULTS
Mean weight of rabbits between groups were not significantly different (Table 1). Larynx airway areas and a/b ratios are also presented in Table 1. Airway areas were not statistically different between sham control group and SP control gro- up. Airway areas in st 1d group and st 2w group were not significantly different than any other groups. Airway areas in st 1w group were signi- ficantly narrower than airway areas of control group, and st 1m groups but not than SP control group. Airway areas in st 1w group were not sig- nificantly different than airway areas of st 1d group and st 2w group.
Figure 3. “a” was defined as the counts that matched airway (encircled with dots in Figure 3.1 above) when inde- pendent counting measure was placed over the target section (Figure 3.2). “b” was defined as the counts that matched larynx tissues surrounded by thyroid cartilage and oesophagus (area between two dotted circles above) when independent counting measure was placed over the target section.
Table 1. Weight, larynx area and a/b ratioes of rabbits.
Larynx area (mm2) a/b ratio (grade)
Groups Weight (grams) (mean ± SD) (mean ± SD)
Sham control 2126.67 ± 317.17 12.5 ± 0.5 0.83 ± 0.41
SP control 2132.50 ± 267.32 11.9 ± 0.7 1.50 ± 1.05
St 1d group 2223.33 ± 404.85 10.8 ± 0.7 3.33 ± 1.63c
St 1w group 2225.83 ± 319.49 10.5 ± 1.2a 3.67 ± 1.21d,e
St 2w group 2214.17 ± 262.00 11.6 ± 1.0 2.17 ± 1.47
St 1m group 2186.67 ± 283.45 12.6 ± 1.8b 1.83 ± 0.98
a Compared to sham control group, p= 0.038, b Compared to St 1w group, p= 0.030, c Compared to sham control group, p= 0.012, d Compared to sham control group, p= 0.003, e Compared to SP control group, p= 0.039.
a/b ratios were not statistically different between sham control and SP control groups. a/b ratios in st 1d group had significantly higher grades than a/b ratios of control group but not SP cont- rol groups. a/b ratios in st 1d group were not sig- nificantly different than a/b ratios of st 1w gro- up, st 2w group and st 1m group. However, a/b ratios in st 1w group had significantly higher grades than a/b ratios of control group and SP control group. a/b ratios in st 1w group were not significantly different than a/b ratios of st 1d group, st 2w group and st 1m group. a/b ratios in st 2w group and st 1m group were not signifi- cantly different than a/b ratios of sham control group and SP control group.
DISCUSSION
This study shows that rebound oedema forms in larynx with abrupt cessation of steroid therapy in rabbits. Although rebound oedema formation was shown in many tissues before, this is the first study to show rebound oedema in larynx tissue after intubation. Clinical safe time for intu- bation after abrupt cessation of steroid therapy is also defined with our study.
Tapering of systemic corticosteroid regimens are used in long course steroid therapy, howe- ver, tapering of corticosteroids in short course steroid therpy is considered unnecessary in most circumtances in treating exacerbation of COPD (12). Laryngeal rebound oedema secon- dary to cessation of steroid therapy wasn’t stu- died before. We wanted to study rebound oede- ma in a clinical scenario by applying the steroid therapy model that is suggested for exacerbati- on of COPD is used as steroid therapy because we suggest that patients with this model, are one of the most frequent ones that anaesthesiolo- gists may meet (13). Although elective surgeri- es may be postponed during exacerbation peri- od, emergent surgeries and elective surgeries af- ter acute exacerbation period may be met by anaesthesiologists. Again, COPD patients may need intubation during acute exacerbation peri- od in intensive care units.
When oedema is present in airways, re-intubati- on or tracheostomy may be required if the pati-
ent is not able to sustain the increase in respira- tory work (15). The development of laryngeal oedema requires close monitoring, and someti- mes the application of non-invasive respiratory assistance, aerosolized epinephrine and perhaps corticosteroids, although the latter are controver- sial (6,16,17). If not rapidly recognized and ade- quately treated, laryngeal oedema can be fatal.
Laryngeal oedema can also be formed by endot- racheal intubation and this injury is influenced by many reasons. Serious laryngeal injuries like ha- ematoma, laceration of mucous membrane and muscles and subluxation of arytenoid cartilage are reported in 6.2% of patients in a study by Kambic et al. (1). Endotracheal intubation may generate local complications, including mecha- nical lesions like friction, compressions between the tube and the anatomic structures, and also biochemical reactions between the plastic or sili- cone tube material and the upper airway muco- sa (18). It is shown that the pressure of the en- dotracheal tube balloon should be lower than 20 mmHg to allow tracheal mucosa blood flow (19).
Even though their incidence has decreased with the use of far more flexible polyvinyl chloride tu- bes generating less pressure on the anatomical structures and high volume low pressure cuffs in- ducing fewer mucosal lesions, these complicati- ons remain frequent (20,21).
Oedema in the airway can have disastrous con- sequences if airway compromise occurs. Treat- ment of airway oedema with steroids has been shown to be effective (22). They have also been shown to decrease migration of inflammatory cells, decrease vascular permeability, and redu- ce exudation (23). Intravascular and perivascu- lar leukocyte aggregation is diminished with cor- ticosteroid therapy, improving microvascular flow (23,24). The production of phospholipase A2 is inhibited, which leads to inhibition of the arachidonic acid cascade and platelet activating factor synthesis. However, it was demonstrated that dexamethasone significantly impaired the healing of tracheal anastomoses in rats (25).
It is not easy to monitorize laryngeal oedema and perform a randomized study with equal ste- roid therapy and cessation times in humans so we decided to perform the study in rabbits. The
rabbit vocal folds are similar to human in struc- ture although the lamina propria is less well de- veloped (26). We used rabbit larynx in our expe- rimental model because rabbit was used by ot- her researchers for human larynx airway area models (27,28).
Quantification of oedema in tissues may be argu- ed. Rebound graft oedema was quantified by Od- land et al. with weighing grafts previously (11).
We suggested that this was not an appropriate way to show laryngeal oedema because it would not be possible to determine the exact tissue de- finitely. Also oedema may not be in the same ex- tend with the previous study because we have used 1 mg/kg instead of 100 mg/kg. Tissue oe- dema may be restricted to muscular area or sub- mucosal area so all tissue with the oedematous tissue should be weighed so this would have dec- reased the sensitivity of measurement. We used histopathology to identify oedema.
We studied the oedema risk of tracheal intubati- on that can form laryngeal oedema itself. Rebo- und oedema that form secondary to steroid the- rapy cessation, may exaggerate due to tracheal intubation and lead to respiratory distress. Sham control and SP control groups were planned to rule out the effect of just intubation and intuba- tion for one hour.
Although study times of larynxes can be argued, we let rabbits to breathe spontaneously for two hours after one hour of intubation because Kil et al. reported that subglottic pressures declined gradually after two hours following extubation (29). We wanted to sacrifice rabbits when laryn- geal oedema was excessive.
Although present study confirms that a week af- ter cessation steroid therapy is an risky time for endotracheal intubation, we haven’t experienced stridor or peripheral oxygen desaturation in no- ne of the rabbits. We consider that laryngeal oe- dema that is formed in a week after cessation of steroid therapy, does not cause stridor and res- piratory distress but these results should also be revealed for humans as well. However, the re- sults of st 1d group were not as worse as the re- sults of st 1w group and results of larynx area and a/b ratio of st 1d group were not different
than the results of SP control group. These re- sults may suggest that laryngeal oedema was not formed in st 1d group and may be the result of continuation of steroid effect. However, result of a/b ratio of st 1w group was significantly hig- her than both sham control and SP control gro- ups. Larynx area was significantly narrower than sham control group. These results suggest that laryngeal oedema occurred in st 1w group.
Although we have used a clinical scenario, we haven’t traumatized the larynx during tracheal intubation in rabbits. Because we have used un- cuffed endotracheal tubes to limit traumatizati- on. Traumatization during tracheal intubation may lead to further oedema to cause respiratory distress. A study should also be performed in humans to show whether rebound oedema that will form secondary to either traumatized or non traumatized tracheal intubation in a week after cessation of steroid therapy lead to stridor and respiratory distress. It is difficult to state laryn- geal oedema with ethical issues and without in- vasive measurements in humans. Therefore, in- cidence of stridor and respiratory distress se- condary to traumatic or non-traumatic tracheal intubation with the history of cessation of stero- id therapy should be sought.
We suggest that one week after cessation of ste- roid therapy may be a risky time for endotrache- al intubation. Since we are not often experiencing such problems in COPD patients in the clinical practice, to translate these results to human be- ing may not be suitable. However, the results of this study showed that abrupt cessation of syste- mic steroid treatment following 10 day course causes rebound oedema in the upper airways of rabbits and this may increase the risk for postex- tubation upper airway oedema if intubation ne- cessities one week following steroid cessation.
ACKNOWLEDGEMENT
Our study was presented in Annual meeting of European Society of Anaesthesiology, Vienna, Austria, 2005.
This study has been supported by The Commis- sion of Scientific Research Projects of Afyon Ko- catepe University, Turkey.
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