Subglottic stenosis after endolaryngeal intubation in infants and children: result of wound healing processes

International Journal of Pediatric Otorhinolaryngology 62 (2002) 1 – 9 www.elsevier.com/locate/ijporl

Subglottic stenosis after endolaryngeal intubation in infants and children: result of wound healing processes M.L.G. Duynstee a,*, R.R. de Krijger b,1, Ph. Monnier c,2, C.D.A. Verwoerd a, H.L. Verwoerd-Verhoef a a

Department of Otorhinolaryngology, Erasmus Uni6ersity Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands b Department of Pathology, Erasmus Uni6ersity Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands c Department of Otolaryngology, Head and Neck Surgery, Uni6ersity Hospital, CH-1011 Lausanne, Switzerland Received 22 March 2001; received in revised form 7 July 2001; accepted 7 July 2001

Abstract Objecti6e: to study the histopathology of subglottic stenosis in children of different ages after treatment during different periods of time, with or without laser application. Partial resection of the anterior cricoid with adhering stenotic subglottic area in the live young patient provides unique material for studying wound healing and scarring processes. Methods: 25 specimens obtained from partial cricotracheal resection (PCTR) in children, were histologically processed and stained with Haematoxylin and Eosin, Resorcin and Fuchsin (for elastic fibers), and immunohistochemical staining (for the presence of macrophages). Results: all specimens were found to have severe and sclerotic scarring with squamous metaplasia of the epithelium, loss of glands and elastic mantle fibers (tunica elastica), and dilation of the remaining glands with formation of cysts. Also, the cricoid cartilage was affected on the internal and external side, with irreversible loss of perichondrium on the inside and resorption by macrophages of cartilage on both sides. Detrimental effects of laser therapy were demonstrated in four cases. The normal intercellular matrix was completely destroyed and the number of chondrocytes in the cartilage structure diminished. Conclusion: wound healing after laryngeal injury is a process of intense restoration and reorganization of the various tissues involved. This process, however, does not guarantee complete repair. In the severe cases irreversible scarring has replaced normal tissues. There seems to be no direct relationship between the length of the post-lesional period, the age of the patient and the severity of the stenosis. When subglottic stenosis has developed and the majority of the tissues is replaced by dense fibrous tissue, PCTR is strongly indicated to achieve renewed patency of the airway. © 2002 Published by Elsevier Science Ireland Ltd. Keywords: Endolaryngeal intubation; Subglottic stenosis; Partial cricotracheal resection; Pediatric larynx; Perichondrium

* Corresponding author. Tel.: + 31-10-408-7661; fax: + 31-10-408-9441. E-mail addresses: [email protected] (M.L.G. Duynstee), [email protected] [email protected] (P. Monnier), [email protected] (C.D.A. Verwoerd). 1 Tel.: + 31-10-408-7901; fax: +31-10-408-9487. 2 Tel.: + 41-21-314-1111; fax: +41-21-314-2706. 0165-5876/02/$ - see front matter © 2002 Published by Elsevier Science Ireland Ltd. PII: S 0 1 6 5 - 5 8 7 6 ( 0 1 ) 0 0 5 4 5 - 6

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1. Introduction The most common and severe complication of endolaryngeal intubation in infants and children, especially in pre-term neonates, is subglottic stenosis (SGS). Its incidence in intubated infants was found to range from 1 to 10% [1,2]. The subglottis is particularly vulnerable to intubation trauma because of two anatomical features. It is the narrowest part of the larynx, and secondly, the only part surrounded by a complete cartilaginous ring not allowing insertion of a tube that is too wide, without damage to the laryngeal wall. Histopathology of SGS after endolaryngeal intubation has been studied in post-mortem specimens of infants and children [3– 5]. Ulceration was frequently observed. The depth varied from superficial to full-thickness ulceration penetrating the cartilage [3–5]. Regeneration of the epithelium was mostly observed as squamous metaplasia [5]. The formation of fibrous scar tissue caused stenosis of the airway. Furthermore, cartilage injury was reported with fragmentation or distortion of the cricoid [3,4]. In this study, we investigated the resected surgical specimens obtained by partial cricotracheal resection (PCTR) in 25 infants and children. PCTR followed by thyrotracheal anastomosis has appeared to be a safe and effective method of treatment of SGS in children [6– 8]. All specimens were derived from SGS, which had developed after previous endolaryngeal intubation. Five patients also underwent one or more treatments with laser surgery. Study of the histologic features of these surgical specimens was especially focussed on wound healing processes of the perichondrium, cartilage, and specific structures in the mucosa. Moreover, the results of this study, next to clinical observations and endoscopic findings, should help the surgeon to have a clear understanding of the patient’s severe SGS and to plan the rational therapy [9].

2. Materials and methods The material for this study was obtained from 24 infants and children that were referred to the

Centre Hospitalier Universitaire Vaudois Lausanne, Switzerland, between 1997 and 2000, and were treated by PCTR according to Monnier et al. [6,7]. Additionally one specimen was included that was obtained from a PCTR performed in Great Ormond Street Hospital for Children London, UK, of which the medical records were not available. The ages of the 14 male and 11 female infants and children ranged from 0 to 14 years (mean age 4 years and 2 months) (Table 1). Seventeen infants and children were intubated in the first year after birth. The number of intubations per patient prior to PCTR varied between one and nine times (mean number three times) and the duration of intubation between 1 day and 2 months. The period between intubation and PCTR ranged from 1 month to 14 years (mean 6 years and 9 months). AII patients had severe dyspnoea and were clinically diagnosed as SGS Cotton grade III or IV [10]. The studied specimens contain the complete stenotic area inclusive of the anterior arch of the cricoid and one or more tracheal rings. The specimens were fixated in 4% buffered formalin, embedded in paraffin and transversally sectioned at 5 mm. Sections, cut 60 mm apart, were mounted on slides. In larger specimens, sections at various levels through the stenosis, were studied. Sections were stained with hematoxylin and eosin; to demonstrate elastin in the tunica elastica and glands, sections were stained with resorcin and fuchsin. Immunohistochemical staining for expression of CD-68 was performed to identify macrophages.

3. Results The normal subglottic airway in children is lined with ciliated cylindrical epithelium (Fig. 1). The subepithelial layer contains loose connective tissue, blood vessels and glands. Staining with resorcin and fuchsin demonstrates in this layer a network of elastic fibers creating a subepithelial elastic tube, the conus (tunica) elastica. This tunica elastica is continuous with the conus elasticus originating from the vocal membrane. It supports the epithelium, surrounds the glands and is con-

M.L.G. Duynstee et al. / Int. J. Pediatr. Otorhinolaryngol. 62 (2002) 1–9

nected to the various parts of the cartilaginous skeleton of larynx and trachea. The cricoid cartilage is composed of a basophilic intercellular substance and chondrocytes, which are grouped in chondrons. In histologic sections characteristics of the cartilage are the same throughout all parts of the cricoid ring. The cartilage is covered by perichondrium on both the internal as well as the external side. Previous studies have demonstrated the chondrogenic potential of perichondrium [11– 13]. Within the group were five children with Down syndrome, 13 had congenital anomalies. Four were premature, which is reported to be an important factor in the development of SGS [14]. Seventeen infants and children had a clinically diagnosed intubation-induced SGS without any therapeutic surgical procedure prior to PCTR. Five patients had previously undergone laser

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surgery to alleviate their acquired SGS and one child had been treated unsuccessfully by laryngotracheoplasty and dilation. The stenoses in the various specimens, all clinically classified as Cotton III–IV, have several histological features in common (Table 2). 1. The circumferential wall of the stenosis has no uniform composition and shows loco-regional variation in histopathology, e.g. the amount of fibrous scar tissue and the injury-induced changes of the cartilage. 2. The luminal surface of the stenotic ring demonstrates occasionally remnants of normal ciliated cylindrical epithelium, but more often (19 specimens) squamous metaplastic epithelium. In addition focal areas without epithelium (17 specimens) were observed. Denuded soft tissue with ulcers is a common finding in combination with squamous metaplastic and/ or ciliated epithelium (Figs. 2 and 3).

Table 1 Clinical data of the patients Patient

Sex

Age at PCTR

No. intub

Duration of intubation

Age at tracheotomy

Anomalies

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

AW MP WG JE AB MH MB NR TF LW EK LV MF KK MH BL CS KS JW SS GF SN

F M M M F M F M M F F F M F M M F F M M M M

12.6 2.2 9.11 14.5 3.8 7.9 2.9 2.9 4 0.7 2.2 2.4 1.2 1.2 5.1 2.3 0.8 3.11 6.8 2.11 1.2 2.1

8 1 9 3 9 5 6 5 3 2 2 3 1 1 1 1 1 3 1 2 1 2

1–2 weeks 3 days ? ? 2, 21 days 2–5 days 1–3 weeks 2–10 days ?, ?, 15 dats 42, 26 days 2, 1 months 7, 7, 10 days 5 days 6 days 30 days 10 days 9 days 6, 4, 2 weeks 7 days 6, 1 days 9 days 12, 25 days

0.8 0.1 1.1 – 0.7 6.5 0.3 1.5 1.6 0.7 0.8 – – 0.7 0.1 0.3 0.7 0.3 – 1.7 0.8 2.5

23 24 25

JF AD M

F M F

7.7 12.9

1 1

8 weeks 8 days

– 1.2

– – VonRecklinghausen M. Down, cardiac malformations Prematurity (24 weeks) – Trisomy chr. 22 Fallot tetralogy Apert syndrom (cricoid hypoplasia) – Prematurity (27 weeks) di George+Fallot (Catch 22) Fallot tetralogy – Esophageal atresia type III B Cardiac malformations M. Down, cardiac malformations Cardiac malformations M. Down Head trauma M. Down Premature (30 weeks), cardiac malformation Premature (35 weeks), M. Down –

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Fig. 1. Histological section of the normal subglottic airway of a child. Resorcin and fuchsin is specifically staining the fibers of the tunica elastica, × 125.

3. The stenotic wall shows a folded—gyrated — surface in 22 specimens, with deep epitheliumlined indentations into the subepithelial layer, predominantly composed of scar tissue (Fig. 3). Here, cystically dilated structures have the appearance of dilated mucous glands or glandular ducts (20 specimens). Other cysts lined with a similar epithelium, look like extensions of the epithelial indentations (Fig. 4). In all specimens considerably less than 50% of the normal glandular tissue remains. In the dilated cystic structures CD68 positive cells are frequent, indicating the presence of many macrophages (Fig. 5). No relation could be demonstrated between the duration of intubation and the percentage of normal glandular tissue present. 4. ln all stenotic specimens the wall of the stenosis shows a thick layer of cell-poor, firm fibrous sclerotic scar tissue, causing substantial thickening of the layer between epithelium and cartilage. Sometimes, scar tissue demonstrates a circular orientation. Within this sclerotic tissue small areas of ectopic cartilage or bone are found. Immediately underneath the epithelium, focal lymphoid aggregates, at times with true lymph follicles, are observed (Fig. 3). Sparsely, CD68 positive cells were also noted in the subepithelium. Blood vessels were also demonstrated in the inflammatory areas.

Staining with resorcin and fuchsin (Fig. 6) revealed that hardly any fibers of the tunica elastica were left in the sections. 5. A remarkable feature is the partial or total absence of perichondrium. Where the perichondrium is absent the fibrous scar tissue abuts directly on the cartilage. Furthermore, in four cases ectopic bone can be found near the border of the cricoid cartilage where the perichondrium is absent (Fig. 7). In one specimen bone formation can be observed externally to the cricoid cartilage (Fig. 8). Microfissures and indentations are extending from the internal (21 specimens) as well as from the external (20 specimens) circumference into the cartilage of the cricoid ring (Fig. 9). These fissures and indentations are filled with blood vessels, plasma cells, fibroblasts and CD68 positive macrophages (Fig. 9). Even in the larynx of a child that was intubated only once for the short period of 3 days, these indentations and fissures were observed. Necrosis of the cartilage was found in 16 specimens. The specimens of infants and children who underwent laser surgery prior to PCTR demonstrated the most severe cartilage destruction. Necrotic cartilage is replaced by fibrous tissue with vast ingrowth of blood vessels. In one specimen no normal cartilage is left. AII specimens of this group contain large areas of denuded mucosa and smaller fields of squamous metaplasia of the epithelium with abundant keratinisation. In one specimen of the study Langerhans cell histiocytosis was diagnosed; this case was presented as a separate report [15].

4. Discussion and conclusion The wall of the subglottic stenosis (SGS) of 25 surgical specimens, obtained after partial cricotracheal resection (PCTR), was studied. Prior to surgery each stenosis was classified by endoscopy, as Cotton III–IV. In agreement with this classification the prepared specimens did show a severe stenosis, with a small remaining lumen and only minor inter-specimen differences. In all cases, the wall of the stenosis demonstrated essentially simi-

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ing from the edges of the wound. The maturation of these granulations includes the formation of scar tissue. Retraction of the circular and sclerotic scar adds to narrowing of the lumen. The luminal surface of the stenotic ring frequently shows irregular protrusions. It may be hypothesized that these protrusions originate from irregular granulations, and later, become more prominent during retraction of the underlying scar. Submucosal mucous gland hyperplasia as reported by Harrison [19] and Chen and Holinger [3], was not observed. Ductal cysts showing glandular lining were regularly found. They are not associated with hyperplasia but with gland atrophy. Gland hyperplasia may occur in the initial phase of traumatisation whereas ductal cysts are the result of subsequent atrophy. Supportive of

lar and specific histopathological features. In the laryngeal tissues reactive changes like squamous metaplasia and regressive changes were found. Squamous metaplasia of the epithelium has been previously reported in humans [16] as well as in experimental animals [17,18]. It is evident that this abnormal epithelium has severely diminished mucociliary function, and is more vulnerable to renewed injury, like re-intubation. The narrowing of the airway lumen is mainly due to a thick layer of cell-poor scar tissue between epithelium and cricoid. Scar tissue is formed by differentiation of initially less differentiated cells generated in the wound area. Clinically, this phase in the wound healing process is recognized as the formation of granulations, which are gradually covered by epithelium migratTable 2 Histopathologic data of the patients

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Patient

Sex

SME

DE

EF

DCS

FI

MG

EL

PI

PE

EB

CFI

CFE

CN

BV

AW MP WG JE AB MH MB NR F LW EK LV MF KK MH BL CS KS JW SS GF SN JF AD M

F M M M F M F M M F F F M F M M F F M M M M F M F

+ + + + np − + + + − + + + + + + − + + + + − + + −

+ + + + np − − − + + + − + + + + + + − − − + + + +

+ + + − np + + + + + + + + + + + + + − + + + + + +

− − − + − + + + + + + + + + + + + + − + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + +

+ − + + + − + + + − + − − − + − − − + − − − + − −

nd + nd nd nd − nd + + + nd nd + nd nd nd nd nd md nd nd + nd + nd

− + − + + + + + + + + + + − + + + + + + − + + + +

− + − − + + + + + + − − + − − + − + − − − − − + +

− − − − + − − − − + − − − − − − − − − + − − − + −

+ + + + + − − + + + + + + − + + + + + + + + + + −

+ + − + + − − + + + + + + + + + + + − − + + + + +

+ + + + + − + − + − + + − − + − − + + − − + + + −

− + + + − − − + + − + + + + + + + + + + + + + + +

SME, squamous metaplastic epithelium; DE, denuded epithelium; EF, epithelial folding; DCS, dilated cystic structures; FI, fibrosis; MG, mucous glands absent; EL elastin absent; PI, perichondrium internal side absent; PE, perichondrium external side absent; EB, ectopic bone; CFI, cartilage fissurae internal side; CFE, cartilage fissurae external side; CN, cartilage necrosis; BV, blood vessels in cartilage; np, not present; nd, not done.

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Fig. 2. Histological section of the larynx at subglottic level after 8 days intubation (case 24). The luminal surface demonstrates remnants of normal ciliated epithelium (CE) and squamous metaplasia (SME). Haematoxylin and eosin stain, ×500.

Fig. 4. Histological laryngeal section of a child intubated twice for 1 and 2 months, respectively (case 11). The stenotic wall demonstrates a folded lumen surface, with dilated cystic structures (DCS) that are identified as extensions of the epithelial indentations. Haematoxylin and eosin stain, × 500.

this hypothesis is that giant cells and macrophages are frequently seen in the cysts, indicating a reaction to the debris. Another type of cysts, found near the luminal surface and lined with epithelium, is probably formed when the duct opening is filled with scar tissue, obstructing glandular secretion. These epithelial inclusions and the ductal cysts can both contribute to considerable lumen reduction.

In literature the conus elasticus is described as important factor for the proper functioning— elasticity and flexibility— of the vocal membrane. The conus elasticus not only runs from vocal cord to cricoid but also continues as an elastic mantle (tunica elastica) supporting the epithelium and connecting with the cartilage rings of the larynx and trachea. An intact tunica elastica has been described in experimental studies in animals after

Fig. 3. Histological section demonstrates remnants of ciliated epithelium (CE); the partly denuded soft tissue (DE) contains massive fibrous tissue (FI), a mixed inflammatory infiltrate with lymphoid aggregates (LA) and dilated cystic structures (DCS) (case 9). Haematoxylin and eosin stain, × 500.

Fig. 5. Histological section of tissue mass causing severe stenosis of the subglottis (case 24). The fibrous tissue (FI) contains many CD68 positive cells (arrows) indicating the presence macrophages in the dilated cystic structures. CD68 antibody stain, ×500.

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Fig. 6. Specific elastic fiber staining shows a few remnants of the tunica elastica in the subglottis of a child intubated for 5 days (case 13). Resorcin and fuchsin stain, × 500.

short duration of intubation [20]. The elastic mantle, however, is almost completely destroyed in our specimens with severe subglottic stenosis. This observation and our earlier findings in experiments [21] support the hypothesis that as a consequence of inferior wound healing, the tunica elastica, and thus elasticity and flexibility of that part of the larynx, will never recur. Animal experiments have shown that lesions restricted to the soft tissue lining result in stenosis, which is membranous, and at a microscopic level

Fig. 7. Subglottis of an infant who was intubated twice for 1 and 6 days, respectively (case 20). Ectopic bone (EB) is found near the border of the cricoid cartilage where the perichondrium is absent. Haematoxylin and eosin stain, × 500.

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Fig. 8. Bone formation could also be observed on the exterior side of the cricoid cartilage (EB) (case 24). Haematoxylin and eosin stain, ×500.

appears to be cell-poor and to contain only a thin circular zone of fibrous tissue immediately under the epithelium [21]. Compact stenosis with abundant scar formation, however, occurs after an endolaryngeal injury, which also involves perichondrium and cartilage. Injured perichondrium, in particular, seems capable of producing large amounts of cells resulting in a compact type of stenosis. This study demonstrated that in stenoses classified as Cotton III and IV the perichondrium over most of the inner contour of the cricoid ring cannot be identified as such. The fibrous scar tissue is in immediate contact with the remaining and partial necrotic cartilage. This observation leads to the conclusion that just as in the previously mentioned experimental animals, in children also the bulk of the scarring formed in severe subglottic stenosis finds its origin in wound reaction of injured perichondrium and/or cartilage, and not in deep and extensive mucosal ulceration, as reported by Chen et al. [3]. Also their suggestion that regeneration of perichondrium may occur with reactional new cartilage formation is not in agreement with our findings. New cartilage seems to be formed at random throughout the soft tissue at places where loose perichondrial cells have survived. In the literature not much attention has yet been paid to the role of the perichondrium. Sasaki described that in tracheostomized dogs perichon-

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drium appeared disrupted by the 14th day, resulting in subsequent underlying chondritis and new cartilage formation [22]. Our observations suggest that injured perichondrium plays an active role in aberrant wound healing of the subglottic area. On the one hand, the perichondrial cells contribute to abundant fibrosis by differentiation into fibroblasts, and on the other hand to the formation of ectopic cartilage and bone [11–13]. Most important, however, is that injury of the perichondrium finally results in loss of perichondrium. Perichon-

Fig. 9. (a) Microfissures and indentations extending from the internal (CFI) as well as from the external circumference (CFE) into the cricoid ring have severely affected the cartilage (case 13). Haematoxylin and eosin stain, × 500. (b) In higher magnification many macrophages in the microfissures are positively stained with (arrows) (case 13). CD68 antibody stain, ×1000.

dritis was suggested in several studies on endolaryngeal intubation [3,5,23]. Yet, inflammation of the perichondrium was never observed in our cases. Preservation of perichondrium seems therefore of paramount importance for the prevention of severe stenosis. Further investigation into the specific contribution of perichondrium in wound healing, is needed. In all specimens, the inner contour of the remaining cartilage shows local indentations invaded by fibroblasts, blood cells, lymphocytes, and macrophages, indicating an ulcerative inflammatory response. Probably these indentations are the result of local loss of cartilage induced by endolaryngeal trauma and further effectuated by necrosis of chondrocytes, the release of growth factors, and intercellular matrix components. The outer contour of the cricoid ring, however, demonstrated the same type of irregularities. Microfissures extending to the center of the cartilage are filled with fibroblasts, macrophages, and blood vessels. It is suggested that in particular the lesions on the outer side of the cartilage are caused by forceful expansion at the time of intubation, more than by local infection. The five specimens with a SGS recurring after (repeated) laser surgery, show essentially the same features as the other specimens. However, much more intense scar formation and cartilage injury are present. Large parts of the cricoid ring are lost and replaced by fibrous tissue. This suggests that this laser treatment caused serious damage to the cartilage, which induced vigorous wound healing reactions and abundant scar formation. It means that treating SGS with the laser bears a special risk while it is difficult to predict the depth of the laser-induced injury during surgery. More extensive data are required to assess the impact of laser surgery on the larynx. In this study no direct relationship was found between the length of the post-lesional period, the length of the intubation period, the age of the patient and the severity of the stenosis [5]. Based on our observations, and supported by earlier experimental work of our group, it can be concluded that the development of severe SGS is a process of intense reorganization of the various tissues involved. The majority of the laryngeal

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tissues has been replaced by dense fibrous tissue, producing a laryngeal wall of poor quality. No adequate repair is achieved. In these cases PCTR with complete removal of the abundant scar tissue has been an excellent surgical therapy to attain renewed and lasting patency of the airway.

Acknowledgements The authors would like to thank Professor Wolter Mooi for his contribution in the pathological assessment, and to Dr Gerjo van Osch for her constructive criticism. They are indebted to Frank van der Panne and Huib de Bruin for microscopic photography. They are grateful to Dr C. Martin Bailey from Great Ormond Street Hospital for Children in London for adding the 25th specimen to the series.

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