- Original research article
- Open Access
Corrosion casting of the subglottis following endotracheal tube intubation injury: a pilot study in Yorkshire piglets
© Kus et al.; licensee BioMed Central Ltd. 2013
Received: 9 May 2013
Accepted: 14 September 2013
Published: 14 October 2013
Subglottic stenosis can result from endotracheal tube injury. The mechanism by which this occurs, however, is not well understood. The purpose of this study was to examine the role of angiogenesis, hypoxia and ischemia in subglottic mucosal injury following endotracheal intubation.
Six Yorkshire piglets were randomized to either a control group (N=3, ventilated through laryngeal mask airway for corrosion casting) or accelerated subglottic injury group through intubation and induced hypoxia as per a previously described model (N=3). The vasculature of all animals was injected with liquid methyl methacrylate. After polymerization, the surrounding tissue was corroded with potassium hydroxide. The subglottic region was evaluated using scanning electron microscopy looking for angiogenic and hypoxic or degenerative features and groups were compared using Mann–Whitney tests and Friedman’s 2-way ANOVA.
Animals in the accelerated subglottic injury group had less overall angiogenic features (P=.002) and more overall hypoxic/degenerative features (P=.000) compared with controls. Amongst angiogenic features, there was decreased budding (P=.000) and a trend toward decreased sprouting (P=.037) in the accelerated subglottic injury group with an increase in intussusception (P=.004), possibly representing early attempts at rapid revascularization. Amongst hypoxic/degenerative features, extravasation was the only feature that was significantly higher in the accelerated subglottic injury group (P=.000).
Subglottic injury due to intubation and hypoxia may lead to decreased angiogenesis and increased blood vessel damage resulting in extravasation of fluid and a decreased propensity toward wound healing in this animal model.
Subglottic stenosis is a potential complication of endotracheal tube (ETT) intubation. The degree of injury can range from mild mucosal erythema or ulceration to granuloma formation and airway stenosis[1, 2]. Injury is believed to be caused by pressure from the ETT on the mucosa overlying the cricoid cartilage. Although the risk of injury usually increases with prolonged intubation, airway injury can occur even within a few hours of intubation and is exacerbated by hypoxic conditions[4, 5]. One hypothesis is that when ETT cuff pressure exceeds tissue capillary perfusion pressure, mucosal blood flow is impaired and the resulting edema and ischemic necrosis can lead to formation of fibrotic scar tissue and subglottic stenosis[3, 6]. Our group previously developed an animal model of ETT cuff injury that used hypoxia to accelerate the formation of an ischemic subglottic mucosal injury caused by an ETT cuff. We then investigated histopathological changes in the subglottis with varying degrees of ETT cuff pressure and found that constant pressure leads to significant epithelial loss, extensive subepithelial and glandular necrosis, and acute inflammation. Though these studies and others have investigated indirect measures of vascular injury, none have directly examined the microscopic vascular features of the subglottic mucosa following ETT-related injury[3, 6].
Corrosion casting is an experimental technique that allows for the study of vascular structures. This method involves the injection of methylmethacrylate, a liquid plastic polymer, into an organ’s blood supply. As the polymer hardens, a three-dimensional cast of the blood vessels is formed. The overlying tissues are then corroded in basic solution and the resulting vascular cast can be analyzed in fine detail using imaging techniques such as scanning electron microscopy (SEM). The purpose of this study was to analyze the microscopic vascular changes that occur in the subglottis following injury secondary to ETT intubation using corrosion casting and SEM.
Materials and methods
This study was approved by the Animal Care Committee at the Hospital for Sick Children. Six Yorkshire piglets (Sus scrofa domesticus) were randomized to two groups:
Three control animals were ventilated through a laryngeal mask airway SpO2 = 100%) while undergoing immediate corrosion casting (described below) and three experimental animals were exposed to a 4 hour accelerated intubation injury described elsewhere prior to corrosion casting. The mean age of control piglets was 7.0 +/- 1.0 weeks (range 6–8 weeks) and their mean weight was 14.7 +/- 0.30 kg (range 14.2 - 14.8 kg). The mean age of intubated piglets was 7.0 +/- 0.5 weeks (range 6.5 - 7.5 weeks) and their mean weight was 15.3 +/- 1.67 kg (range 13.4 - 16.4 kg). There was no difference across groups with respect to age or weight (P>.05).
Accelerated intubation injury model
Animals were intubated and exposed to accelerated ETT cuff injury conditions as described previously elsewhere[3, 7, 8]. Briefly, animals were sedated with an intramuscular injection of Akmezine (0.2 mL/kg) and were placed under anaesthesia using Isoflurane (5%) delivered by facemask. Animals were intubated with a 6.0 mm or 6.5 mm internal diameter cuffed Sheridan endotracheal tube (ETT; Teleflex Medical, Research Triangle Park, North Carolina) with the cuff directly below the vocal cords and cuff pressure maintained at 25 cm H2O using a Magnehelic manometer (Dwyer Instruments, Michigan City, Indiana). When inflated to 25 cm H2O, the length of contact between the ETT cuff and the subglottic mucosa extended for 2.0 cm or 2.5 cm below the vocal cords for 6.0 mm or 6.5 mm internal diameter Sheridan ETT’s, respectively. All intubations were atraumatic. Animals were placed in the supine position and the ETT was secured to the snout. Ventilation was maintained using a volume-cycled ventilator (Air Shields Ventimeter; Narco Health Company, Hatboro, Pennsylvania). Intravenous fluid was administered through an auricular vein. Hypoxia was induced for 4 hours by ventilating the animal with a mixture of oxygen and nitrous oxide to maintain a target SpO2 of 70% and end-tidal CO2 less than 40 mmHg. Heart rate, respiratory rate, oxygen saturation, end-tidal carbon dioxide levels, and body temperature (rectal) were monitored throughout the procedure. All animals were intubated and maintained under hypoxic conditions by the same investigator (LHK). After 4 hours, the ETT cuff pressure was decreased to 20 cm H2O and the SpO2 was gradually increased to 100% over 15 minutes prior to vascular casting.
Casting reagent consisted of 200 mL Batson’s #17 monomer base solution (Polysciences, Warrington, Pennsylvania), 80 mL ProBase Cold Monomer (Ivoclar Vivadent, Schaan, Liechtenstein), 30 mL Batson’s #17 catalyst (Polysciences, Warrington, Pennsylvania), 24 drops Batson’s #17 promoter (Polysciences, Warrington, Pennsylvania), and 3 g phthalocyanine blue dye (Polysciences, Warrington, Pennsylvania). This blue casting reagent (310 mL) was perfused into the aortic cannula until it was seen extravasating from the venous cannula, indicating that it had perfused the entire head and neck. The animal’s skin colour changed from pink to blue indicating complete perfusion to the capillary level (Figure 1). The animal was placed on ice for 3 hours while the casting resin solidified.
Scanning Electron Microscopy (SEM)
Data were analyzed using SPSS version 21 (IBM, Armonk, New York). Based on analysis for normality, equality of variance and the relatively small sample size, non-parametric descriptive statistics were used (median and inter-quartile range).
Comparisons between control and experimental groups was performed using Mann–Whitney U (aka Wilcoxon Rank Sum) test. Within a given group, comparison of two imaging features was performed using the related-samples Wilcoxon Signed Rank test and comparison of three imaging features was performed using the related-samples Friedman's 2-way analysis of variance (ANOVA) by ranks. The limit of significance was taken to be 0.625% (p<0.00625) for all comparisons after Bonferroni correction for multiple comparisons. Interrater reliability was assessed using intra-class correlation (ICC) coefficient with an ICC greater than 0.8 considered acceptable (high reliability).
Gross and microscopic vascular anatomy of control subglottis and trachea
Quantitative analysis of control and accelerated subglottic injury animals
Angiogenic and hypoxic/degenerative features in the subglottis of control and intubated animals
Control group (Features/FOV)
Intubation group (Features/FOV)
Endotracheal intubation is a life-saving measure that can cause varying degrees of injury to the subglottis, ranging from mild mucosal ulceration to complete stenosis[1, 2]. Subglottic stenosis is believed to result from ETT cuff pressures that exceed capillary perfusion pressure leading to ischemia, necrosis, perichondritis, chondritis, and fibrotic scar formation[3, 12]. Hypoxia is also believed to predispose subglottic mucosa to injury[4, 13]. While the link between ETT cuff pressure and mucosal necrosis is well established, the precise vascular etiology of these changes is not clearly understood. The purpose of the present study was to analyze the microscopic vascular changes that occur in the subglottis following injury secondary to ETT intubation using corrosion casting and SEM.
The gross vascular anatomy of control tracheas in our study revealed two large longitudinal vessels (LV) on either side of the posterior aspect of the trachea, regularly-spaced circumferential branches between the longitudinal vessels, and smaller vertical branches connecting the circumferential vessels that penetrated deeply to form a dense plexus of fine capillaries on the lumenal side. Even though our study is the first to describe this vascular network in pigs, it has been described previously in human fetuses, guinea pigs, sheep, and dogs[14–17]. The regularly spaced circumferential branches in humans traverse the intercartilaginous spaces, where they provide small branches to the superficial perichondrial vascular bed and then further ramify to pierce the tracheal wall and supply the microcirculation of its mucosal lining. Anatomical similarities between porcine and human tracheal vasculature suggest that results from our study may be translatable to human patients.
Animals in the accelerated subglottic injury group had less overall angiogenic features (P=.002) compared with controls. Amongst angiogenic features, there was decreased budding (P=.000) and a trend toward decreased sprouting (P=.037) in the accelerated subglottic injury group with an increase in intussusception (P=.004)Angiogenesis occurs in two ways, namely sprouting and intussusception. In sprouting angiogenesis, existing blood vessels develop a bud or outgrowth that narrows and extends into a new branch or sprout. This occurs by proteolytic degradation of extracellular matrix followed by chemotactic migration of endothelial cells, formation of a lumen, and endothelial maturation. In intussusceptive angiogenesis, a pre-existing vascular plexus divides internally into mature capillaries. This phenomenon is mediated by bridges of interstitium that widen until a perforation forms through their center. These perforations elongate until a groove is formed between two adjacent rows of endothelial cells, creating the walls of two distinct capillary vessels. The dominant type of angiogenesis varies by organ, although both types may occur concurrently. Maturation of the vasculature involves pruning of the newly formed vascular tree and remodeling of blood vessels. Interestingly, results from the present study demonstrated both sprouting and intussusceptive angiogenesis in control and experimental piglets, suggesting continuous revascularization even without being subjected to injury. This may be a feature of tracheas in general or may be due to the rapid growth of the Sus scrofa piglet trachea at a young age. The finding that animals in the accelerated subglottic injury model showed fewer angiogenic features of budding and sprouting with increased intussusceptions than controls suggests an attempt at rapid revascularization. Angiogenesis is required to restore oxygenation and allow new tissue to grow to fill a wound space. Decreased angiogenesis likely leads to delayed growth of new tissue over the wounded area of subglottis, leaving cartilage exposed and at risk for infection, granulation and scar formation.
Animals in the accelerated subglottic injury group had more overall hypoxic or degenerative features than controls (P<.001). However, extravasation was the only feature that attained significance (P<.001). Extravasations, circular constrictions and corrugations have been found in corrosion casts of brains of rats subjected to cerebral ischemia. Extravasations were thought to reflect damaged microvessels that leaked plasma or incurred a small hemorrhage, and these increased in frequency with increasing duration of ischemia. A greater number of resin extravasations in our accelerated subglottic injury group suggests that the subglottic injury likely resulted from vascular injury leading to extravasation of fluid from leaky blood vessels in the form of edema or hemorrhage. Inflammatory mediators released in this extravasated fluid could contribute to subglottic injury. Corrugations are thought to be related to vasospasm caused by convolutions of the internal elastic lamina in constricted arterioles and are not seen in capillaries due to their decreased contractility compared to arterioles[11, 21, 22]. Circular constrictions are also believed to be due to vasoconstriction and have been seen in rat studies investigating cerebral hemorrhage and vasoconstrictive neurotransmitters[21, 22]. The present study found no significant difference across groups with respect to circular vessel constrictions or corrugations. One possible explanation is that vasospasm does not play a major role in subglottic injury caused by an ETT cuff. Alternatively, the casting procedure itself may have negated these vascular changes. However, circular constrictions and corrugations were seen in both control and experimental groups, supporting the former hypothesis.
The main limitation of our study is its small sample size and the limited number of angiogenic and hypoxic or degenerative features that can be counted visually. In addition, artifacts from corrosion casting could have skewed the results. However, such artifacts would not likely preferentially affect one group over the other. Future studies investigating a larger number of animals and a greater variety of vascular features using computerized software may yield more detailed results. Future studies investigating the role of mechanical or pharmacological interventions in preventing vascular injury in the subglottis are required.
We studied the role of vascular injury in subglottic stenosis using an animal model of accelerated subglottic intubation injury in combination with corrosion casting and SEM imaging. Results suggest that subglottic injury due to intubation and hypoxia may lead to decreased angiogenesis and increased blood vessel damage resulting in extravasation of fluid and a decreased propensity toward wound healing in this animal model. Future studies investigating a larger number of animals subjected to a prolonged period of intubation are required.
This material has never been published and is not currently under evaluation in any other peer-reviewed publication. None of the authors have any conflicts of interest to declare.
This work was funded by a Hospital for Sick Children Surgical Services Innovation Grant.
This manuscript was presented at the Poliquin competition at the 67th Canadian Society of Otolaryngology Annual Meeting, Banff, Alberta, June 2–4, 2013.
- Santos PM, Afrassiabi A, Weymuller EA: Risk factors associated with prolonged intubation and laryngeal injury. Otolaryngol Head Neck Surg. 1994, 111: 453-459.PubMedGoogle Scholar
- Allen TH, Steven IM: Prolonged endotracheal intubation in infants and children. Br J Anaesth. 1965, 37: 566-573. 10.1093/bja/37.8.566.PubMedView ArticleGoogle Scholar
- Chadha NK, Gordin A, Luginbuehl I, Patterson G, Campisi P, Taylor G, Forte V: Automated cuff pressure modulation: a novel device to reduce endotracheal tube injury. Arch Otolaryngol Head Neck Surg. 2011, 137: 30-34. 10.1001/archoto.2010.228.PubMedView ArticleGoogle Scholar
- Whited RE: A prospective study of laryngotracheal sequelae in long-term intubation. Laryngoscope. 1984, 94: 367-377.PubMedView ArticleGoogle Scholar
- Weymuller EA: Laryngeal injury from prolonged endotracheal intubation. Laryngoscope. 1988, 98: 1-15.PubMedView ArticleGoogle Scholar
- Donnelly WH: Histopathology of endotracheal intubation. An autopsy study of 99 cases. Arch Pathol. 1969, 88: 511-520.PubMedGoogle Scholar
- Gordin A, Chadha NK, Campisi P, Luginbuehl I, Taylor G, Forte V: An animal model for endotracheal tube-related laryngeal injury using hypoxic ventilation. Otolaryngol Head Neck Surg. 2011, 144: 247-251. 10.1177/0194599810392894.PubMedView ArticleGoogle Scholar
- Gordin A, Chadha NK, Campisi P, Luginbuehl I, Taylor G, Forte V: Effect of a novel anatomically shaped endotracheal tube on intubation-related injury. Arch Otolaryngol Head Neck Surg. 2010, 136: 54-59. 10.1001/archoto.2009.195.PubMedView ArticleGoogle Scholar
- Hammel JM, Deptula J, Hunt PW, Lang H, Duncan KF: Anoxic ventilation improves systemic perfusion during extracorporeal circulation with uncontrolled systemic-to-pulmonary shunt. Asaio J. 2007, 53: 238-240. 10.1097/MAT.0b013e31802f501f.PubMedView ArticleGoogle Scholar
- Macchiarelli G, Jiang JY, Nottola SA, Sato E: Morphological patterns of angiogenesis in ovarian follicle capillary networks. A scanning electron microscopy study of corrosion cast. Microsc Res Tech. 2006, 69: 459-468. 10.1002/jemt.20305.PubMedView ArticleGoogle Scholar
- Ohtake M, Morino S, Kaidoh T, Inoue T: Three-dimensional structural changes in cerebral microvessels after transient focal cerebral ischemia in rats: scanning electron microscopic study of corrosion casts. Neuropathology. 2004, 24: 219-227. 10.1111/j.1440-1789.2004.00560.x.PubMedView ArticleGoogle Scholar
- Seegobin RD, van Hasselt GL: Endotracheal cuff pressure and tracheal mucosal blood flow: endoscopic study of effects of four large volume cuffs. Br Med J (Clin Res Ed). 1984, 288: 965-968. 10.1136/bmj.288.6422.965.View ArticleGoogle Scholar
- Karhausen J, Ibla JC, Colgan SP: Implications of hypoxia on mucosal barrier function. Cell Mol Biol (Noisy-le-grand). 2003, 49: 77-87.Google Scholar
- Strek P, Nowogrodzka-Zagorska M, Litwin JA, Miodonski AJ: The lung in closeview: a corrosion casting study on the vascular system of human foetal trachea. Eur Respir J. 1994, 7: 1669-1672. 10.1183/09031936.94.07091669.PubMedView ArticleGoogle Scholar
- Miodonski A, Kus J, Tyrankiewicz R: Scanning electron microscopical study of tracheal vascularization in guinea pig. Arch Otolaryngol. 1980, 106: 31-37. 10.1001/archotol.1980.00790250033007.PubMedView ArticleGoogle Scholar
- Hill P, Goulding D, Webber SE, Widdicombe JG: Blood sinuses in the submucosa of the large airways of the sheep. J Anat. 1989, 162: 235-247.PubMedPubMed CentralGoogle Scholar
- Laitinen A, Laitinen LA, Moss R, Widdicombe JG: Organisation and structure of the tracheal and bronchial blood vessels in the dog. J Anat. 1989, 165: 133-140.PubMedPubMed CentralGoogle Scholar
- Folkman J: Tumor angiogenesis. Adv Cancer Res. 1985, 43: 175-203.PubMedView ArticleGoogle Scholar
- Risau W: Mechanisms of angiogenesis. Nature. 1997, 386: 671-674.PubMedView ArticleGoogle Scholar
- Burri PH, Tarek MR: A novel mechanism of capillary growth in the rat pulmonary microcirculation. Anat Rec. 1990, 228: 35-45. 10.1002/ar.1092280107.PubMedView ArticleGoogle Scholar
- Nakai K, Naka Y, Yokote H, Ikatura T, Imai H, Komai N, Maeda T: Vascular "sphincter" and microangioarchitecture in the central nervous system: constriction of intraparenchymal blood vessels following a treatment of vasoconstrictive neurotransmitter. Scanning Microsc. 1989, 3: 337-341.PubMedGoogle Scholar
- Ono S, Date I, Nakajima M, Onoda K, Ogihara K, Shiota T, Asari S, Ninomiya Y, Yabuno N, Ohmoto T: Three-dimensional analysis of vasospastic major cerebral arteries in rats with the corrosion cast technique. Stroke. 1997, 28: 1631-1637. 10.1161/01.STR.28.8.1631.PubMedView ArticleGoogle Scholar
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