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From: Methods in Molecular Medicine, vol. 78: Wound Healing: Methods and ProtocolsEdited by: Luisa A. DiPietro and Aime L. Burns © Humana Press Inc., Totowa, NJ

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Wound Healing in Airways In Vivo

Steven R. White

1. IntroductionThe airway epithelium is a target of inflammatory, environmental, and

physical stimuli in diseases such as asthma and bronchopulmonary dysplasia. Damage to the epithelium may compromise both the physical barrier and key metabolic functions. Repair involves the migration and spreading of cells over the basement membrane and the proliferation of new epithelial cells. Each step can be modulated actively by growth factors secreted by constitutive cells within the airway, or suppressed by mediators secreted by inflammatory cells that have migrated into the airway. Understanding these steps is essential to gaining insight into the repair process in airway epithelium.

Much of what is known about epithelial repair in vivo is derived from studies in which the epithelium is wounded either with a mechanical probe or with a chemical agent, and the subsequent proliferation, migration, and differentiation into subtype cells is observed over time (reviewed in refs. 1and 2). The removal of epithelial cells from the mucosa, whether by trauma or inflammation, induces prompt extravasation of plasma from the microvascular bed beneath and surrounding the immediate area. Gaps open in and along the junctional stretches between venular endothelial cells, and plasma moves into the injury site and onto the airway surface overlying the basement membrane (3). Immediately after injury and after plasma leakage occurs, epithelial cells both adjacent to and remaining in the injury site become activated. Basal cells spread and migrate over the provisional gel until the injured site is completely covered. This process reconstitutes the epithelial barrier to fluid and plasma transport (3). Small wounds can be covered completely in <24 h by migration and spreading alone (1). Cell proliferation within and adjacent to the site of

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epithelial injury begins within 24 h and produces a multilayered metaplastic epithelium after 48–72 h. Mitosis and division of normally arrested cells within the epithelium usually begins only after cell migration and spreading have completely covered the site of injury (4,5), although we have demonstrated a regional reflex in which proliferation is also seen in epithelial cells away from the injury site (6). Mitosis continues among the newly migrated cells, producing immature, “indifferent” cells, which differentiate into functional, subtype cells usually within 4–7 d of the original injury (1).

From this sequence of events, it follows that repair after injury involves the coordination and integration of several separate processes. A number of in vitro methods have been developed to examine epithelial cell repair after injury. These generally involve proliferation assays; chemotaxis assays using blind-well chambers; adhesion assays in which cells adhere to extracellular matrix proteins; two-dimensional wound closure assays of cells on a culture dish or cover slip, generally assessed by time lapse video microscopy; and three-dimensional growth and cell activation assays in which two different cell types are grown separated by a gel matrix or a membrane. These methods are extremely useful to examine specific mechanisms under standardized conditions but are limited in their ability to increase the understanding of the in vivo environment, particularly when examining cell-to-cell interactions.

Our laboratory has developed a method that examines airway epithelial cell repair after injury in the in vivo setting (6,7). This method utilizes a standardized mechanical injury to the tracheal mucosal surface in a small mammal such as a mouse, rat, or guinea pig. At time points selected by the investigator, animals can be killed and tracheal epithelial cell wound closure can be assessed by histological techniques. Additionally, the expression of selected genes or proteins can be examined by appropriate in situ methods;inflammatory cells can be counted and typed; and the response of other constitutive cells within the airway wall, such as fibroblasts, can be examined. Finally, these responses can be examined in the setting of experimental treat-ments to the animal given immediately prior to or at selected time points after the tracheal wounding. Thus, the in vivo method may be a useful additional tool by which to examine wound repair and gain insight into basic mechanisms of cell proliferation and migration.

The method of intubation we describe in this chapter is similar to a variety of techniques used by other investigators. Each has, as its core, the direct visualization of the vocal cords. Fiberoptic illumination of the vocal cords with a flexible guide can be used if such a tool is available (8). Costa et al. (9)described the fabrication of rodent fiberoptic laryngoscopes specific for each species. Such a light source could be adapted for the method we provide but requires additional fabrication. The use of an otoscope and otoscopic speculum,

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as we describe, provides ample light at the base of the pharynx and a wide channel through which to pass a metal wand. More important, these tools are inexpensive and can be used within a confined space such as a glove box. Jou et al. (10) note the use of a wedge fabricated from the barrel of a 3-mL syringe to aid in intubating small rodents such as rats. Such a wedge facilitates a direct view of the vocal cords but does not provide additional light. Alpert et al. (11)note the use of a spatula to elevate the tongue of a rodent and provide direct visualization. With our method, or those methods previously published, a clear view of the vocal cords is required prior to placing a wand into the trachea to create the mechanical injury to the epithelium.

Adaptations of our method can be derived to create tracheal mucosal injury by chemical rather than mechanical means, to instill radioopaque or marker dyes, and for endotracheal intubation followed by mechanical ventilation.

The in vivo method we describe in this chapter is as follows: After proper anesthesia in the test animal, the hypopharynx and vocal cords of the animal are visualized directly using a human otoscope with a medium-sized, disposable speculum. Once seen, a metal wand of suitably small diameter is passed between the vocal cords into the cervical trachea. This wand has a hook at the distal tip; when withdrawn from the trachea under gentle torsion, the hook will remove the epithelial layer without significant damage to the basement membrane and underlying submucosa. Once the tracheal injury is created, the wand and speculum are removed and the animal is allowed to recover from anesthesia. At selected time points, tracheas can be collected and images obtained. Such images (as illustrated in Fig. 1, a representative series of images at different time points after tracheal mucosal injury in guinea pigs) then can be processed to meet the goals of the project.

2. MaterialsNote that it is incumbent on the investigator to obtain proper approval for the

use of animals from the institutional review board of his or her institution.

2.1. Tracheal Injury1. Appropriate animals for study (mouse, rat, guinea pig).2. Appropriate anesthetic agents for the animals to be used.3. Welch-Allyn otoscope, human adult size: The otoscope may have an open

(conventional) or closed (fiberoptic) head. In either case, it will have a removable (sliding) magnifying lens. We have used both designs and prefer the closed head.

4. Speculums for otoscope: For guinea pigs, either a 3- or 4-mm speculum may be used.

5. Stylette, appropriately sized for the animal: This is a metal wand that flexes a little to facilitate applying the necessary torsion in the injury process. For guinea

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pigs, the wand used is 1.5 mm in diameter. The tip is shaped as shown in Fig. 2and is designed to catch the tracheal surface when the wand is pulled backward under torsion. It should be long enough to fit comfortably in one’s hand—about 20 cm. For our work, we had these wands fabricated from a surgical grade stainless steel rod in a university machine shop. The length of the wand is milled so that the cross-section, from the hook to the back, is a semicircle. This facilitates the flexing required to torque the wand.

6. Cotton swabs and 2 × 2 cotton sponges.

Fig. 1. Representative model in guinea pig using methods described herein for sequence of repair of airway epithelium. The trachea was abraded at time 0 using a metal wand, and tracheas were collected at the indicated time points. In the first phase of repair, there is migration of flattened cells into the injury site. Over time a multiple layer of flattened cells develop. Subsequently, these cells shift their phenotype into the needed terminal cells within the mucosa, such as ciliated and secretory cells. (From ref. 6, with permission of the publisher.)

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2.2. Digital Imaging of Tracheal Wounds

1. NIH Image software (Wayne Rasband, National Institutes of Health).2. Macintosh computer capable of running NIH Image (any G3 or G4 Macintosh

or Power Macintosh).3. Good-quality bright-field microscope with an adapter appropriate to the digital

camera used.4. Digital camera with appropriate adapter for microscope (at least 1.3 megapixels,

and preferably ≥2.0 megapixels), with appropriate interface to the computer (e.g., USB port or memory card reader).

5. Hemacytometer (for calibration).

3. MethodsWe describe the method in guinea pigs. This method can be adapted to

either smaller or larger animals using the same principles as described in Notes 1–6.

3.1. Tracheal Injury

1. Anesthetize Hartley guinea pigs, 400–700 g in mass, with 40 mg/kg of ketamine and 5 mg/kg of xylazine intramuscularly.

Fig. 2. Schematic of wand used to create tracheal injury. The wand is made from stainless steel. (A) The wand is about 20 cm long and 1.5 mm in diameter. (B) Thewand is milled along its length so that the cross-section is a semicircle down to a hook. The hook is designed to catch the tracheal surface when the wand is pulled backward under torsion. The tip of the wand is rounded so as not to injure the vocal cords or trachea on insertion.

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2. Confirm the depth of anesthesia at periodic intervals by monitoring pedal withdrawal and corneal reflexes. To check pedal withdraw, grasp the foot pad and squeeze gently. To check the corneal reflex, use a soft tissue or cotton swab and gently touch the cornea. If the animal withdraws or blinks, more anesthesia is required.

3. Lay the animal on its back on a work table. No restraints should be required with proper anesthesia. You may find it useful to brace the animal with a rolled towel or with foam rubber to prevent the animal from sliding on the table when you intubate it. An alternate method is to have an assistant present the animal to you, abdomen up, cradling the neck.

4. Intubate the animal using the otoscope to visualize the vocal cords. In guinea pigs, we use a 3- or 4-mm disposable speculum on the otoscope. With a gloved hand, open the mouth and grasp the tongue gently. Pull the tongue outward and up; this also lifts the mandible. Using the other hand, insert the tip of the otoscope speculum with the handle of the otoscope facing upward (Fig. 3). The tip needs to enter the hypopharynx, and thus go beyond the back of the tongue. Pivot and lift the scope in your hand such that the mandible is lifted. Insert the speculum farther (gently) until the vocal cords come into view. The magnifying lens on the otoscope helps substantially in viewing the vocal cords. At this point you can release the tongue. Continue to hold the otoscope firmly throughout the entire procedure.

5. Guinea pigs many times retain food products in the hypopharynx, thus blocking the view of the vocal cords. We find it useful to swab out the hypopharynx using cotton-tipped applicators. Either pull the applicator tip through the speculum or withdraw the speculum and pass the applicator tip by feel to the hypopharynx.

6. If you are still unable to visualize the vocal cords, have an assistant press down gently on the larynx after you have inserted the otoscope speculum. This will help bring the vocal cords into view.

7. Through the otoscope, insert the metal stylette with the shaped tip going first(Fig. 3B). This needs to pass between the vocal cords without causing trauma to the cords or the aretinoid cartilage on either side. Trauma can elicit edema and constriction of the larynx and cords with subsequent asphyxiation of the animal.

8. Once the tip is past the vocal cords, insert it about 1–1.5 cm farther into the trachea. Orient the shaped hook of the tip so that it faces downward (dorsally).

9. Torque the wand downward in your hand as you pull back gently to the trachea. The goal is to shave the tracheal mucosa. We generally do this three times. You may rotate the stylette after each attempt if you wish to cause a larger injury. Too much torque can damage not only the epithelium but also the underlying submucosa.

10. Withdraw the stylette and the otoscope. Monitor the animal until it has recovered completely from anesthesia. With appropriate anesthesia and due care to avoid injury, the mortality rate is very low (review Notes 1 and 2 for some of the complications that may occur with this method).

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3.2. Digital Imaging of Tracheal Wounds

At appropriate time points, animals may be killed and tracheas collected for histological and morphometric analysis. The details of tissue harvest and various staining methods are not covered here. It is appropriate to consider

Fig. 3. Drawings of technique for in vivo tracheal injury. (A) Lateral view of guinea pig, demonstrating important structures of pharynx. (B) Lateral view of guinea pig during intubation with otoscope. An assistant holds the animal, belly up. The tongue is grasped using cotton gauze, and the tongue and jaw are lifted upward. The otoscope speculum is inserted to bring the vocal cords and epiglottis into view. The metal stylette then can be passed through the speculum into the cervical trachea. (C) View of vocal cords and epiglottis from pharynx. The inset shows the view of the vocal cords and epiglottis through the magnifying lens of the otoscope. The metal wand is inserted through the otoscope speculum, between the vocal cords, and into the cervical trachea.

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the technique of measuring tracheal injury and repair. We provide a method that will serve as a starting point. This method assumes the prior collection, fixation, and sectioning of an entire trachea in cross-section. While any staining method that provides adequate contrast of the epithelial layer and basement membrane will do, our preference is to work with paraffin-embedded, 5-μmsections stained with hematoxylin and eosin. With such a system, one can collect images of the quality shown in Fig. 4.

We use a photomicrography system that employs a good consumer-quality digital camera to obtain images of these airways. This is preferred to either 35-mm photography, owing to the ease and lower cost of image collection, or to video microscopy, owing to the superior resolution of most digital cameras compared to comparably priced, industrial-model video cameras that have been adapted to a microscope (see Notes 7 and 8). Measurements are made using NIH Image, a popular, free image analysis program. A complete manual is provided with the software; however, the methods given here provide supplemental information on using the program.

1. Calibrate the photomicrography system for morphometric analysis. This is critical to making appropriate measurements of tracheal injury. Using the photomicrography system, photograph the squares of a hemacytometer (shown in Fig. 5) at different magnifications. Transfer these images to the computer, and open them in NIH Image. Using the rectangular trace tool, trace the outline of a hemacytometer square and measure its perimeter length and area in pixels. Knowing the millimeter length of this perimeter, one then can derive for that magnification the number of pixels per millimeter and square millimeter. This value is used later to convert measurements made in pixels to absolute units and will provide the investigator with the limit of resolution at a particular magnification.

2. Photograph the tracheal wound slides at appropriate magnifications. We provide examples in Fig. 4 of how a larger injury may be photographed and montaged to provide a complete image at suitable magnification. Transfer these images to the computer, and open them in NIH Image. Using the freehand trace tool, trace the length of the basement membrane around the tracheal ring or along the airway segment of interest and measure its length, and the area of the airway lumen plus epithelium, in pixels.

3. The tracheal wound should be clearly visible. Using the freehand trace tool, trace the length of the basement membrane at points at which the epithelium is completely or partially denuded. Measure each of these lengths in pixels. Wound width is expressed in pixels and as the percentage of denuded basement membrane for the tracheal ring. Additional measurements relevant to the ques-tions the investigator might ask can be made on the same image, such as the height of the epithelium and the thickness of the basement membrane (Fig. 4).

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Fig. 4. Representative images collected from typical tracheal wound experiment in guinea pig. (A) Tracheal mucosal wound 24 h after creation in normal guinea pig. This low-power view was collected by an imaging system, and individual frames were montaged to create the final image. The tracheal mucosal wound in this particular animal was approximately one-fourth of the total circumference. When measured by the techniques described in this chapter, the tracheal injury measures approx 1705 pixels, or 407 μm, in length between the two arrows. (B) Higher-magnification view of denuded basement membrane in same tracheal mucosal wound. The epithelium is completely removed, and the basement membrane and underlying connective tissue are not significantly damaged. (C) Higher-magnification view of edge of tracheal mucosal wound shown in (A). Here the edge of the original wound is marked by the thin arrow, and the extent of reepithelialization is marked by the thick arrow. In this image, the height of the normal epithelium is 22.0 μm and that of the new epithelial cells is4.3 μm. e, epithelium; c, cartilage; sm, smooth muscle; bm, basement membrane.

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4. Notes1. We have found that there is a learning curve to this procedure. The investigator

should plan several practice sessions using animals that are not vital to the overall experimental design (e.g., not precious transgenic animals on the firstattempt).

2. There are several important complications to the intubation and tracheal injury that may lead to death of the animal or may preclude the use of the animal in the planned experiment. Among these are the following:a. Puncture of the hypopharynx with the otoscope speculum or with the stylette.b. Placement of the stylette into the esophagus. This can lead to puncture with a

resulting infection of the retrosternal and left pleural spaces.c. Puncture of the trachea, which can lead to a pneumomediastinum and

infection.d. Tracheitis owing to the mucosal injury. The incidence is low if proper clean

procedures are employed in creating the injury.e. Bleeding owing to the mucosal injury or owing to the intubation. The incidence

is very low with proper technique.3. The procedure may be scaled to larger animals (e.g., rabbits) with relative ease.

Instead of an otoscope, one may employ a pediatric laryngoscope with a very short, curved (“Mac”) blade, such as those used in the care of human neonates. The stylette to be used may be of the same size as for guinea pigs (1.5 mm in diameter) or may be larger if a larger injury is required. The procedure is otherwise the same.

Fig. 5. Grid arrangement on a hemacytometer. The outside corner boxes are subdivided into smaller boxes. The outside corner box has a length of 1 mm, and an inside smaller box has a length of 0.25 mm. The corresponding length in pixels on a digitized image can be used to calibrate dimensions in other images taken with the same imaging system at the same magnification.

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4. The use of this procedure in rats and mice is more problematic. The problem is the physical size of the pharynx, which many times precludes direct visualization of the vocal cords using the technique we have described—particularly in the mouse. We have had some success in doing blind intubations with a very thin (0.7-mm-diameter) stylette in mice. This stylette has a very small bend at its tip instead of a proper beveled edge. In the anesthetized mouse, one can insert the stylette gently into the mouth and guide it by feel to the hypopharynx. With a finger on the cervical neck over the trachea, one can feel when the stylette has entered the trachea. The stylette then can be torqued and withdrawn. The investigator should be prepared for a substantially higher mortality rate with this procedure. We found that the method of Jou et al. (10) can be adapted to perform a blind intubation of a mouse using a 1-mL syringe barrel; tracheal injury then can be done using a very thin metal wand as we describe.

5. Other intubation procedures, such as those suggested in refs. 8–11, can be combined with the use of mechanical injury to the trachea as we describe. In adapting other techniques, direct visualization of the glottis and vocal cords is necessary to avoid injuring these structures with the metal wand.

6. Other methods of tracheal injury can be adapted to the method we present. Instead of a metal wand, a catheter of appropriate diameter through which a selected agent (e.g., naphthalene) can be instilled. Alternately, a narrow brush (such as those used for human bronchoscopy) can be employed to create an abrasive injury.

7. The types of digital cameras, microscopes, and computers that may be used in these basic measurements is diverse. Key considerations in building such a system are based on the end points of the work: What are the goals of the investigator, and what other potential uses would a digital photomicrography system have? Microscopes from any good vendor that have a port and an adapter for attaching a digital camera may be used. In considering a digital camera, the limits of the camera’s resolution should be ascertained. In our experience, a typical 2 megapixel digital camera photographing a tracheal ring will have a limit of approx 0.1 μm using a ×4 objective and a ×10 camera adapter, and approximately 0.02 μm using a ×20 objective and a ×10 camera adapter. For the types of measurements we describe in this chapter, these limits are more than acceptable. Other issues, including color fidelity, reliability, and other features of the camera should be considered according to the investigator’s needs. Finally, we utilized NIH Image since it can make the measurements necessary for this method and is distributed without cost. Other suitable image analysis programs are available for either Windows- or Macintosh-compatible computers.

8. There are other issues that should be considered in morphometric analysis of airway epithelium and of airways in general. A tracheal ring must be oriented in the proper plane on section; deviations from this can lead to progressive error in measurements. A comprehensive analysis of these limitations has been published (12).

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AcknowledgmentsThis work was supported by grants HL-60531 and HL-63300 from the

National Heart, Lung and Blood Institute.I thank John Kim, M.D., and Valerie McKinnis, M.D., for their assistance

in developing the tracheal injury technique; Delbert Dorscheid, M.D., Ph.D., for his helpful comments in morphometry analysis; and Karen Dirr for original artwork.

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