The objectives of this study are to define the distribution of collagen and hyaluronic acid in the interstitium of the mesentery and, with the same data, evaluate sensitivity of UV-IR absorption for estimating protein and fluid. Because two molecules cannot occupy the identical space, collagen and hyaluronic acid exclude protein and fluid in the interstitium. This exclusion modulates the volume of free fluid in the interstitium and thus edema formation. The experimental procedure estimates protein mass and fluid volume by using differential absorption at 280 and 320nm (corrected for collagen scattering) and 1000 and 1500nm, respectively, in accordance with the Lambert-Beer relationship. Sampling a TV image provides an array of 512 x 512 bytes of data. Using a 20X objective in our TV-microscope system yields a microregion sample of about 2um2. TV images are grabbed, digitized, integrated and stored within 5 seconds for each wavelength. The experimental protocol involves obtaining 3 sets of control data, then superfusing with either collagenase or hyaluronidase or a combination. TV sampling will be done every 15 minutes for I hour, followed by 30 minute intervals until enzyme action is completed. Enzyme application causes degeneration of the interstitial constituents thereby reducing the extent of exclusion and permitting a more uniform distribution of protein and fluid to occur. Comparing the distribution of protein and fluid before and after the enzyme should reveal the distribution of the interstitial constituents. Microregions which exhibit increases in fluid and protein following collagenase treatment will represent regions of high collagen content. Similarly, microregions which exhibit disproportionate increases in protein following hyaluronidase treatment will reflect regions which had high hyaluronic acid content. The ability of the UV-IR absorption procedure to detect these changes of distribution will reveal the sensitivity and specificity of the method. This study may serve to identify the true nature of the perivascular protein concentration critical to the evaluation of the mechanisms of transvascular protein and fluid transport and possibly identify the extent to which alterations of the distribution and integrity of the interstitial matrix may contribute to edema formation.
