| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Guest Access | Sign In via User Name/Password
|
|
|
|||||||
Guest Access | Sign In via User Name/Password |
|||||||||
* From the Beckman Laser Institute (Mr. Mahmood, Ms. Hanna, Mr. Han, Mr. Jung, and Dr. Chen) and the Pulmonary and Critical Care Division (Dr. Brenner), University of California Irvine Medical Center, Orange, CA; and the US Army Institute of Surgical Research (Mr. Jordan and Drs. Yershov and Walton), Fort Sam Houston, San Antonio, TX.
Correspondence to: Zhonping Chen, MD, Beckman Laser Institute, UC Irvine Medical Center, Building 53, Room 119, 101 City Dr South, Orange, CA 92868; e-mail: zchen{at}laser.bli.uci.edu
Abstract
Background: Optical coherence tomography (OCT) is an evolving technology that is capable of delivering real-time, high-resolution images of tissues. The purpose of this study was to evaluate the feasibility of using OCT for detecting airway pathology in a septic animal model.
Methods: The tracheas of New Zealand white rabbits were inoculated endobronchially with various concentrations of live Streptococcus pneumoniae bacteria. After the development of pneumonia/sepsis, the animals were killed. OCT tracheal images and corresponding histologic specimens from these experimental animals were compared to control rabbit tracheas for morphologic features and quantitative tracheal mucosal thickness measurements.
Results: The results revealed significant airway mucosal thickening in the experimental group that was consistent with tracheal edema. Morphologic changes, including epithelial denuding and mucosal sloughing, were evident in regions of the experimental tracheas.
Conclusion: This study suggests that OCT is a potentially valuable imaging modality that is capable of evaluating superficial airway pathology with high-resolution in vivo images. Numerous applications of OCT can be envisioned in the realm of pulmonary medicine and thoracic surgery that may substantially increase the precision and accuracy of current bronchoscopic diagnostic and surgical techniques.
Key Words: bioengineering • pathology • trachea • tracheal injury • tracheal surgery
Optical coherence tomography (OCT) is a novel imaging modality that may help to improve the medical diagnostic modalities of pulmonary and airway diseases. Analogous to ultrasound, OCT uses the reflection of light waves to construct high-resolution structural representations of complex tissues.1 Because OCT is based on light reflection rather than sound, it can be performed "noninvasively"; without requiring direct physical contact with the tissues being imaged. This property is of potential benefit in imaging structures such as the eyes or large airways. Second, because light has a substantially shorter wavelength than sound, OCT can produce images of considerably higher spatial resolution, in some cases up to one to two orders of magnitude higher, than current ultrasound capabilities.2 Real-time images of tissues with resolutions of 1 to 15 µm can be obtained.345 Since light is scattered and absorbed within most biological tissues, OCT suffers from the disadvantage that it has a limited depth of penetration (approximately 2 to 3 mm with current technology). However, OCT can be performed with relatively inexpensive flexible fiberoptic imaging probes, enabling many clinically relevant tissue and organ surface and subsurface structures to be imaged.6
Applications of OCT have been investigated in a range of medical disciplines. However, translational work to pulmonary applications of OCT has been relatively lacking. Although research conducted within the past few years has described some aspects of normal airway structures as observed by OCT,78 no studies have analyzed the ability to detect airway pathology. Because of the high tissue resolution capabilities and flexible fiberoptic bronchoscopic applicability of OCT, it has the potential to detect changes that are associated with various diseases that present in the superficial layers of the respiratory tract. It has even been suggested that OCT may eventually replace the need for biopsy, and become a method of "optical biopsy," though this is obviously highly speculative at this time.9
The purpose of this study was to investigate the potential utility of OCT in detecting airway pathology. In order to demonstrate this concept, an animal model of airway disease was employed in which the airways of live rabbits were inoculated with live Streptococcus pneumoniae bacteria. This bacterial lung infection and Pneumococcus induce an inflammatory process within the airways with resultant airway edema.10 This study was designed to demonstrate the feasibility of using OCT imaging of tracheas of S pneumoniae-infected rabbits to detect pathologic changes indicative of airway edema and inflammation compared to controls.
Materials and Methods
Description of Technology
A schematic representation of the OCT interferometry principles and design used in these studies is illustrated in Figure 1
. Short-coherence-length broadband laser light is emitted from a light source toward a partially reflecting mirror that acts as an optical beam splitter.1 One of the resulting light beams is directed toward a precisely controlled reference mirror at a specific distance from the beam splitter. The other beam is aimed toward the biological tissue to be examined. Light directed toward the reference mirror reflects back with a time delay (proportional to the distance traveled), while the reflected light beam from a biological tissue consists of multiple reflected "echoes" that are determined by the optical reflectivity of structures within the sample. These two reflected light beams (the returning reference and sample arms) are then recombined at the partially reflecting mirror creating an interference signal that is directed toward a photodetector for reading. Varying the position of the reference mirror allows investigation of different tissue depths. The pattern of interference created by the two recombined light beams is decoded and plotted on a logarithmic two-dimensional scale to reconstruct an image of the structures within the biological sample. Many successive axial measurements are obtained along a longitudinal surface, and the images are combined to create extremely high-resolution cross-sectional images of the tissues.
|
The normal control group (group 1) consisted of four rabbits from which 10 tracheal specimens were harvested. As part of an unrelated study to evaluate acute hemorrhagic shock hemodynamics, successive amounts of blood were removed and replaced with saline solution within a 3-h period. At the completion of these experiments, the rabbits were killed, and their tracheas were removed, maintained in isotonic saline solution, and imaged on site at the Beckman Laser Institute on the campus of the University of California at Irvine.
In the saline solution stored control group (group 2), the tracheas were harvested in the manner mentioned above. The tracheas were imaged immediately after resection, then stored in saline solution. Serial OCT images were obtained daily up to 4 to 5 days postresection to note the effects of saline solution on the mucosa and submucosa of the tracheas.
At the Brook Army Hospital Institute of Research in San Antonio, TX, two additional groups of New Zealand White rabbits (groups 3 and 4) were anesthetized, intubated with a 3.0-mm cuffed endotracheal tube, and mechanically ventilated for a period of several hours. The intubated control group (group 3; consisting of three animals from which six tracheal specimens were obtained) was allowed to recover immediately following the above-mentioned procedure. These animals subsequently underwent a panel of diagnostics tests that was similar to that which the experimental group of rabbits underwent (described in the next paragraph).
The experimental group (group IV; consisting of 12 animals from which 29 tracheal specimens were attained) was first inoculated with various quantities of S pneumoniae using a sterile pediatric suction catheter. This infected group of animals was monitored at the time of exposure and at 24, 48, 72, and 96 h postexposure via blood work, pulmonary function tests, vital sign data, CT scans, and flow cytometry; cultures of BAL fluid were used to confirm diagnosis of pneumonia. On the fourth day following inoculation, the surviving rabbits were killed, and their tracheas were excised and placed in isotonic saline solution packed in ice. These tissues were then sent via overnight delivery and were imaged at the University of California at Irvine.
Previous experience with OCT imaging demonstrated that the tracheal microstructure remained intact without evident change for a period of up to 4 days in cold isotonic saline solution. Nonetheless, an effort was made to image all specimens as soon as possible, usually within 2 days of excision. Furthermore, throughout their manipulation, care was taken to ensure that the architecture of the tracheas was not damaged.
Specimen Imaging
The specimens from all four groups of animals underwent the same OCT imaging procedures. Tracheas were cut open longitudinally along their musculofibrous membranes and were divided into sections that were approximately 1 x 2 cm. Each tracheal specimen yielded two samples, representing the upper and lower trachea. Triangular notches were cut into opposite ends of each specimen to delineate the intended line of image acquisition, perpendicular to the cartilage rings. The tracheas were secured to pieces of cork using metal pins placed along their perimeter and covered liberally by a layer of water-based lubricant (K-Y Jelly; McNeil PPC; Fort Washington, PA) in order to prevent desiccation during imaging (Fig 2
). The tracheas were then placed on a moveable sample platform and a visible-light guiding beam was used to match the line of image acquisition with the triangle notches already present. Images of various size and resolution were obtained using the prototype 1,310-nm broadband (
= 80 nm), superluminescent diode laser-based OCT device that was constructed in our laboratory. The images constructed were displayed using a logarithmic intensity scale with the most backscattering areas represented in white and the least backscattering areas represented in black.
|
OCT/Histology Matching and Statistical Analysis
The OCT images and corresponding histologic preparations of the control group (group 1), the saline solution stored control group (group 2), the intubated control group (group 3), and the infected tracheal specimens group (group 4) were compared on the basis of the morphology of the structures present. The tracheal mucosal thickness was measured as the average distance between all the apices of the cartilage rings and the tracheal lumen attained from each OCT image. This was performed for every image. Measurements were made using digital imaging software and image editing software (Photoshop; Adobe; San Jose, CA). The values obtained from the animal groups were then compared using analysis of variance statistical analysis (SYSTAT, version 10.0; Systat Software Inc; Richmond, CA) comparing the combined control groups (groups 1 to 3) against the infected group (group 4) [after it was shown that the control subgroups were statistically similar to each other].
Results
High-resolution images of the normal control group (group 1), the saline solution stored control group (group 2), the intubated control group (group 3), and the experimental infected tracheal specimens group (group 4) were obtained using OCT and revealed the ability to detect tissue structures that had been seen previously in other studies.78 These included the epithelium, lamina propria, submucosa with glandular tissue, and various layers of tracheal cartilage and trachealis muscle (Fig 3, 4 ).
|
|
The mean (± SD) tracheal mucosal thickness above the cartilage rings for the combined control groups (groups 1 to 3) was 150 ± 2.5 µm, whereas that for the pneumonia-infected group (group 4) was 228 ± 1.7 µm. There is a significant difference (p < 0.005) [Fig 5 ] between the infected mucosal thicknesses and the thicknesses from the other two control groups. There were no differences among the values obtained from the three control subgroups (p = 0.50). There were no significant differences between upper vs lower tracheal specimens, and storing the tracheal sample in saline solution for up to 4 days had no impact on image appearance or the thickness of the mucosal and submucosal layers.
|
Other commonly used imaging modalities, such as CT scanning, MRI, and ultrasound, have the ability to image subsurface architectures, yet none offer the soft-tissue resolution of 1 to 15 µm that OCT does. Hence, those other modalities cannot be used to reliably depict mucosal changes in the superficial airways. Novel endoscopic confocal imaging techniques, on the other hand, have remarkable soft-tissue resolution (< 10 µm); however, they suffer from very shallow depth of penetration (generally, < 800 µm).11 Of the current imaging modalities, only OCT offers a unique combination of high resolution (1 to 15 µm) and a depth of penetration (2 to 3 mm) that is adequate for imaging superficial airway anatomy and pathology. Moreover, using modern fiberoptic technology, OCT can and has been used with endoscopic devices, allowing for real-time, in vivo imaging.12
Given the real-time tissue-resolution capabilities of OCT, its flexible fiberoptic bronchoscope compatibility, and its relatively inexpensive optical components, OCT has the potential to some day become a useful tool in pulmonary diagnostic medicine.1314 This study was designed to demonstrate the feasibility of high-resolution OCT in detecting disease-induced airway changes and some of the capabilities of OCT imaging of complex airway tissue. A variety of possible pulmonary applications for this technology can be envisioned, including bronchogenic carcinoma detection. OCT may be studied to screen for early neoplastic changes and to assess the submucosal spread or degree of dysplasia in endobronchial masses. OCT might also have a role in the treatment of airway malignancy by increasing the efficacy of tumor resection and by evaluating the effects of therapy.
More investigation needs to be conducted before OCT can be used in routine thoracic and pulmonary clinical settings. Research needs to be carried out to evaluate the ability of OCT to recognize alterations in models of respiratory pathologies, such as bronchogenic carcinoma and inhalation injury. Imaging of human airway specimens displaying a range of pathologic processes will be an important step toward clinical application validation. Parallel efforts need to be made toward the further development of OCT technology. Faster image acquisition rates are needed before OCT can consistently make high-resolution, larger area images from a moving respiratory tract. Better resolution and contrast will also be needed to increase the sensitivity and specificity with which OCT can detect airway pathology.15 Specific probes and delivery methods need to be optimized for bronchoscopic approaches.
There are a number of specific limitations to this study. All specimens were imaged in vitro. Even though care was taken to maintain the original tissue architecture throughout the preparation of the sample, some changes inevitably occur during preparation. These limitations may be overcome once in vivo measurements can be readily performed. There are limitations in quantitatively describing some of the differences between images obtained from the four animal groups. Our method of comparing mucosal thickness measurements and qualitatively describing morphologic changes overcomes some of the limitations in objectively describing image comparisons but cannot fully define the changes seen.
The current "first-generation" OCT prototype device for pulmonary applications has limitations as well. The current resolution falls short of the "gold standard" of tissue biopsy preparation and hematoxylin-eosin staining. This gap will improve as OCT technology advances in the future. Some of the discrepancies between OCT imaging and light microscopy are due to the principle that the two methodologies examine different tissue properties and optical reflectivity in OCT vs those in dye absorption in hematoxylin-eosin staining. One of the advantages of OCT is its ability to avoid the changes in tissue that occur as a result of histologic processing, which alters the tissue due to the excision, fixation, and microtoming processes.16
OCT represents a promising new imaging technology for pulmonary diagnostics. In addition to structural imaging, future OCT technology will also be capable of providing functional information such as localized blood flow and tissue birefringence. Improved resolution, acquisition time, and probes will enable real-time fiberoptic airway imaging at near histologic levels of resolution.
Acknowledgements
Thanks to Teri Kennedy and Tanya Burney for their help in conducting these studies and coordinating the collaborations, and for their technical assistance.
Footnotes
Abbreviation: OCT = optical coherence tomography
The authors have no conflicts of interest to disclose.
This research was supported by Department of Defense grant FA 9550-04-1-0101, Philip Morris grant USA-32598, National Science Foundation grant BES-86924, and National Institutes of Health grants EB-00293, NCI-91717, and RR-01192. Support from the Beckman Laser Institute Endowment is also gratefully acknowledged.
Received for publication October 19, 2004. Accepted for publication March 2, 2006.
References
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
