The purpose of this study was to evaluate the laser-tissue interactions of engineered human skin and in-vivo pig skin following exposure to a single 3.8 micron laser light pulse. The goal of the study was to determine if these tissues shared common histologic features following laser exposure that might prove useful in developing in-vitro and in-vivo experimental models to predict the bioeffects of human laser exposure. The minimum exposure required to produce gross morphologic changes following a four microsecond, pulsed skin exposure for both models was determined. Histology was used to compare the cellular responses of the experimental models following laser exposure. Eighteen engineered skin equivalents (in-vitro model), were exposed to 3.8 micron laser light and the tissue responses compared to equivalent exposures made on five Yorkshire pigs (in-vivo model). Representative biopsies of pig skin were taken for histologic evaluation from various body locations immediately, one hour, and 24 hours following exposure. The pattern of epithelial changes seen following in-vitro laser exposure of the engineered human skin and in-vivo exposure of pig skin indicated a common histologic response for this particular combination of laser parameters.
Two-dimensional electrophoresis and histomorphometry were used to determine if equivalent protein changes occurred within native rabbit corneas and engineered corneal tissue models following in vitro exposure to single pulse, 1540 nm laser light operating at a pulse width of 0.8 milliseconds. Frozen sections of exposed tissues were processed to detect laser-induced protein changes. Isoelectric points, molecular weights and relative densities were used to characterize corneal proteins of interest that were then identified using MALDI-MS peptide fragment analysis. Increasing radiant exposures of corneal tissues were associated with progressively more severe necrosis of the epithelium and stroma in both the native and engineered tissues.
Recent advances in the field of tissue engineering have led to the development of complex three-dimensional tissue constructs. It has become clear, however, that the traditional tools used for studying standard cell cultures are not always adequate for diagnostically studying thick, highly-scattering cultured tissues. Furthermore, many techniques used for studying three-dimensional constructs are invasive or require exogenous fluorophores, which damage the tissue and prevent time-course studies of tissue development. An integrated optical coherence tomography (OCT) and multi-photon microscope (MPM) has been constructed for visualizing 3-D engineered tissues. OCT was used for imaging structure and cell organization, while MPM was used for assessing functional properties of cells. We demonstrate technical developments involved in the construction of this instrument and its use in the non-destructive investigation of cell movement and tissue organization in engineered tissues. Cells labeled with GFP and exogenous fluorescent probes have also been imaged with OCT and confocal microscopy. Studies indicate that an integrated microscope has the potential to be an enabling diagnostic tool for future studies in the growth and organization of engineering tissues and in cell-cell and cell-matrix interactions.
In vitro exposures of explant rabbit corneas to single pulse 1540 nm infrared laser light operating at a pulse width of 0.8 milliseconds resulted in coagulative necrosis of both the corneal epithelium and stroma. Histomorphometric data correlated with increasing tissue radiant exposures. Histologic alterations in the corneal stroma were typical of matrix remodeling within the beam path and reactive to antibodies against matrix metalloproteinase-2. A two-dimensional electrophoretic analysis, using a mini-gel format, was developed to determine if specific corneal protein changes within tissue sections could be detected. Frozen sections taken through the center of the laser lesion were evaluated for proteomic data using tissue isoelectric focusing in the first dimension and polyacrylamide gel electrophoresis in the second dimension. Histomorphometric data describing the extent of the laser lesions were compared to the isoelectric points, molecular weights and relative densities of individual corneal proteins. Increasing radiant exposures of corneal tissues were associated with characteristic histomorphometric and proteomic changes.
Significant damage to rabbit corneal tissue was produced by a single pulse, in vitro exposure of 1540 m infrared laser light operating in either millisecond or nanosecond pulse widths. Millisecond pulse widths of infrared laser light produced a marked coagulative necrosis of both the corneal epithelium and stroma. We also noted histologic alterations in the stromal matrix within the beam path that we interpreted as matrix remodeling. To test this interpretation, we used an indirect immunohistochemical procedure to detect Matrix Metalloproteinase-2 (MMP-2) activity. Immunohistochemistry revealed that the MMP-2 reaction was mostly limited to the margins of the beam path. In addition, the MMP-2 reaction was less intense than expected given the significant tissue changes observed in the histologic sections. Exposure of rabbit corneal tissue to the nanosecond pulse widths produced a less severe coagulative necrosis of the tissue when compared to the millisecond exposures. However, a markedly stronger immunohistochemical pattern than would have been predicted from the histologic sections was observed, with approximately half of the beam path filled with MMP-2 reaction product. These data suggest an association between infrared laser pulse width and the degree of extracellular matrix remodeling in rabbit corneal tissue.
Single pulse, 1540 nm laser light with a pulse width of 1 microsecond altered the morphologic appearance of explant rabbit and pig corneas following ex vivo exposure. Using digital images of the post-exposure corneas projected onto a measuring grid, we could accurately locate the relative position of the circular laser lesion in the embedded tissue. This allowed us to section through the lesion with micrometer precision and accurately resolve the inside edge, middle and outside edge of the laser lesion. All tissue sections used for morphometric analysis were taken through the middle of the lesion. Several features of the response to laser exposure may reflect species-specific tissue differences. The rabbit corneal epithelium showed a homogeneous coagulative necrosis with a distinct demarcation between necrotic and normal epithelium. The pig epithelium also showed a distinct demarcation between necrotic and normal epithelium, however, there were several remarkable differences in the tissue response between the two species including coagulative necrosis pattern and nuclear morphology. These changes suggested a different and less severe response of the pig epithelium to the laser light when compared to the rabbit epithelium.
Dutch Belted rabbit corneas and corneal equivalent (CE) tissue were exposed to 0.8 millisecond pulses of 1540 nm laser light. We report the single pulse ED50 for Dutch belted rabbits and for in-vitro corneal equivalent tissues. A histological comparison between the two tissues is presented. Remarkable similarities between the two models in both location and extent of damage are noted. We postulate which cellular energy absorption mechanisms are significant at 1540 nm and how this relates to the histopathology presented.
Mechanisms of tissue damage are investigated for skin and cornea exposures from 1540 nm ('eye safe') laser single pulses of 0.8 milli-seconds. New skin model data point out the advantages of using the Yucatan mini-pig versus the Yorkshire pig for in-vivo skin laser exposures. Major advantages found include similarities in thickness and melanin content when compared with human skin. Histology from Yucatan mini-pig skin exposures and the calculation of an initial ED50 threshold indicate that the main photon tissue interaction may not be solely due to water absorption. In-vitro corneal equivalents compared well with in-vivo rabbit cornea exposure under similar laser conditions. In-vivo and in-vitro histology show that initial energy deposition leading to damage occurs intrastromally, while epithelial cells show no direct injury due to laser light absorption.
We investigate the use of in-vitro corneal equivalents as a replacement model for in-vivo rabbit corneas used in laser damage threshold studies. In-vitro corneal equivalents (CE) were exposed to 0.8 millisecond (ms), 1540 nanometer (nm) single laser light pulses ranging from 124 J/cm2 to 58 J/cm2. After exposure, CE's were evaluated opthalmoscopcopically, imaged using confocal microscopy and examined histologically to investigate the mechanisms of laser induced damage. Preliminary results indicate that the 50% damage threshold for CE's, ED50, is approximately 70 J/cm2 with a 0.5 mm diameter spot size. Additional data points are required to determine a statistically significant ED50. Preliminary exposures of in-vivo rabbit corneas using the same laser parameters as the CE's are also reported. Comparisons between the in-vivo and in-vitro models are presented. Histopathological images from both models show remarkable similarities in the location and extent of damage throughout the full thickness of each tissue.
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