Monitoring transport in the rabbit ear chamber

MICROVASCULAR

RESEARCH

l&t,

204-209 (1982)

BRIEF COMMUNICATIONS Monitoring

Transport LAWRENCE

Depurtment

in the Rabbit Ear Chamber’,*

J. NUGENT AND RAKESH K.

of Chemical Engineering, Carnegie-Mellon Pittsburgh, Pennsylvania 15213 Received January

JAIN University,

13, 1982

Wayland and co-workers have studied extravascular transport of macromolecules in the mesenteries of the cat and rat (Nakumura and Wayland, 1975; Fox and Wayland, 1979). In these investigations, macromolecular species, tagged with the fluorescent label fluorescein isothiocyanate (FITC) were injected directly into the circulatory system, and movement of molecules from individual microvessels into and through the interstitial space was monitored spatially and temporally using a computer-based image digitizing, storage, and processing system. Using a one-dimensional diffusion model with a time-dependent boundary condition located outside the capillary wall, these data were analyzed to obtain “apparent” interstitial diffusion coefficients for the test molecules studied. In their investigations, they reported that the time-dependent concentration behavior within the plasma could not be evaluated. A simple technique has been developed, as a goal in the present work, to observe and quantify intra- and extravascular transport of fluorescent-labeled molecules in mature granulation tissue grown in a rabbit ear chamber following a pulse iv injection. Three unique features characterize our approach: (1) Elaborate image digitizing, data storage, and computerized image processing systems are not required (our method employs a photometric analyzer used in many laboratories for RBC velocity measurements); (2) in addition to “apparent” interstitial diffusion coefficients, this method facilitates monitoring of plasma pharmacokinetics in individual microvessels; (3) the rabbit ear chamber preparation, unlike the mesenteric preparation, allows transport studies to be conducted in the same animal tissue repeatedly without inducing trauma, inherently restricts transport to a thin tissue plane,3 and can be used to study transport in tumors or other explants (Williams, 19.54).The molecular species studied in this ’ Presented in part at the Annual Meeting of the American Institute of Chemical Engineers, New Orleans, Louisiana, November 1981. ’ Research support granted by the American Cancer Society, National Science Foundation; a National Institutes of Health Doctoral Fellowship to L.J.N. and a National Institutes of Health Research Career Development Award to R.K.J. 3 The upper and lower chamber plates restrict transport to a thin tissue, whereas Fox and Wayland (1979) physically contacted the mesentery with an oil layer to ensure this condition. 204 0026-2862/82/050204-06$02.00/0 Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.

BRIEF COMMUNICATIONS

205

work are sodium fluorescein (Na-F, MW =376) and FITC-conjugated bovine serum albumin (FITC-BSA, MW = 67,000). MATERIALS

AND METHODS

A modified Sandison-Clark transparent chamber (One of a Kind, Ltd., Lincoln Park, N.J.) is surgically implanted in the ear of a white New Zealand rabbit, weighing between 2 and 3 kg (Three Springs Kennel Co., Zelienople, Penn.), following the procedure described in Zawicki et al. (1981). Once the granulation tissue has matured, about 4 to 6 weeks postimplant, the animal is brought to a quiescent state with I cc/kg of nembutal 60% ip. The ear containing the transparent chamber is extended to the specimen plane of a Zeiss universal microscope (Morgan Instruments Inc., Cincinnati, Ohio) which has been adapted for intravital fluorescence television microscopy. The tissue is transilluminated with the tissue-specimen plane at the front principal focus of a long working distance Neofluar objective (20 x /0.57) via a 100-W mercury vapor lamp (MI-38-00-184060) driven by a low-ripple DC power supply (MI-91-01-04). A long working distance (13 mm) substage condenser (MI-46-55-50) gathers the disperse incident beam to a point coincident with the tissue plane. Focused images are formed at the end of a phototube and impinge on the face plate of an attached SIT television camera (Model 4410 SIT, Cohu Inc., San Diego, Calif.). Sufficient stimulating radiation with spectral characteristics appropriate for FITC excitation is provided by the mercury light source in conjunction with FITC-exciter (MI91-00-31) and red suppressor (BG 23) filters (MI-46-78-00). A barrier filter (MI46-78-73) positioned in front of the SIT video camera renders a minimal background light level, which serves as a baseline voltage corresponding to zero concentration in the absence of fluorochrome in the video field. The video image was continuously displayed on an image shearing monitor (Model 907, Instrumentation for Physiology and Medicine Inc., San Diego, Calif.) and recorded on videotape (Model AV-3650, Sony, Inc.) with U.S. standard framing rates (30 fps). Automatic gain controls on both the SIT camera and video recorder were set to a manual mode to ensure a linear voltage response between light intensity and fluorochrome concentration.4 After the video gains are adjusted in the optical system to ensure a linear response and a suitable black-level setup is attained, preinjection control images are recorded for 15 min to identify the structural and functional state of the vascular region of interest. Next, the molecular species Na-F and FITC-BSA (Sigma Chemical CO., St. Louis, MO.) are injected iv as a pulse into the auricular vein of the contralateral ear at a dosage of 100 mgikg body wt as a 2.5% solution in physiological saline. Movement of molecules is then observed and video taped. The transport process is quantified off-line temporally at discrete positions with subsequent video photometric analysis. ’ Intensity of fluorescence as a function of concentration by the SIT camera was found to be linear over three orders (0.1-10 mg/ml) covered the range found in Go.

as measured in small-bore glass tubes of magnitude. The concentrations studied

206

BRIEF

COMMUNICATIONS

DATA ANALYSIS The videotapes represent an analog voltage record of the transient spatial distributions in light intensity resulting from the transport of the fluorescent test molecules within the microvasculature. Continuous playback of the video record of each experiment permitted intensity-time profiles to be generated at selected regions in the vascular bed. As part of this process the video signals are fed to a video photometric analyzer (Model 204, Instrumentation for Physiology and Medicine Inc.) where a voltage-sensing window of specified position and size is activated and delineated within the televized scene. The analog signal forming the intensity level at this locus is readily sampled in real time and transcribed into digital form using a digital voltmeter recorder (Model D9, Acurex Corp., Mountain View, Calif.). The relative distance between successive window positions can be precisely determined using an image shearing monitor. Frame-by-frame gray-level digitization can be unwieldy and data reduction time consuming. Since the temporal component of the fluorescent intensity response is extracted from the video record at a known position within the vascular bed, digitization and data reduction are accomplished in one step. Furthermore, this approach is more amenable to making dynamic measurements of transport processes. RESULTS AND DISCUSSION To illustrate the feasibility of our technique we will present our results on plasma pharmacokinetics and extravascular diffusion. Plasma pharmacokinetics. Initially we measured the fluorescent light intensity-time response at the center line of several vessels (I.D., IO-30 pm) in the same animal. However, the responses obtained at different axial positions within a given vessel or those obtained simultaneously from different vessels within a given tissue showed a high degree of variability. This perceived difference in blood pharmacokinetics was thought to be a result of the strong light absorption characteristics of hemoglobin, the concentration of which depends upon the hematocrit. To minimize this absorption effect, we confined our fluorescent measurements to the erythocyte-free plasma layer of the vessel. As anticipated, for a given test molecule, we obtained reproducible pharmacokinetic data in all measured vessels of the ear chamber.5 The concentration-time profiles obtained from individual vessels (Fig. 1) were found to be a composite of two exponential decays suggesting that the tracer was cleared from the blood compartment at two main rates, expressed by: C(t) = Aeear + Be-@.

Average half-lives 7l = In 2/c~ and r2 = In 2/p determined for Na-F and FITCBSA are shown in Table 1 for one animal. These half-lives are in general agreement with values of Grotte (1956). ’ Since the fluorescent intensity-time profiles, when measured in the erythrocyte-free plasma layer, were found to be independent of the diameter, hemoglobin absorption was believed to be minimal.

207

BRIEF COMMUNICATIONS

TIME

( SEC.)

FIG. I. Normalized concentration-time profiles for Na-F and FITC-BSA measured from within the erythrocyte-free plasma layer in individual vessels of the rabbit ear chamber; points (0) represent experimental data, solid lines (--) represent the biexponential fit to the data from which half-lives are determined.

Extravascular rransport. In order to estimate the apparent diffusion coefficient of Na-F and FITC-BSA, fluorescence was measured as a function of time at various positions perpendicular to the vessel wall. Since the tissue is only 40 pm in thickness and the chamber plates introduce a no-flux condition above and below the tissue plane, the data obtained were modeled as one-dimensional diffusion: 6c/& C(x, 0) C(O, 2) C(=, t)

= = = =

D(6’C/6x2), 0, fU>, 0,

where C(x, I) is the test molecule concentration at distance x from a specified origin at time t, and D is the apparent diffusion coefficient, assumed to be constant. Since the tracer is not present in the extravascular space prior to

TABLE 1 ESTIMATED

PARAMETERS FOR PLASMA PHARMACOKINETICS

FITC-BSA

IN THE RABBIT

AND EXTRAVASCULAR

Plasma pharmakocinetics” (set) 72

71 Na-F FITC-BSA

7.5 2267

k 0.8 -c 133

” Mean 2 SD; n = 4 in the same animal. ’ Mean 2 SD; n = 3 in the same animal.

32.9 3574

DIFFUSION OF Na-F

AND

EAR CHAMBER

k 8.2 _f 498

Extravascular diffusion ’ D x

10'

(cm’isec)

16.3 t 4.0 0.59 _c 0.05

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208

TIME (SEC.) FIG. 2. Concentration-time profiles for Na-F measured within the tissue of the rabbit ear chamber. The upper curve is the concentration function at the origin f(t), measured just outside the capillary wall; points (0) represent experimental data, solid line (-4 represents the fourth-order polynomial fit to the data. The lower curve is the concentration response at a distance X, = 20 pm; points (0) represent experimental data. solid line (-) represents the prediction using the onedimensional diffusion model with a best fit value of DNa.Fof 1.1 x 10e6 cm’isec.

injection, C(x, 0) = 0. If only short-term data (t G 10 set) are used, we can neglect the interaction of nearby vessels, C(a, t) = 0, and seek a solution applicable to small penetration distances.6 In addition, we can choose the origin (X = 0) extravascularly at a position near the capillary wall, and measure a timedependent concentration function, C(0, f) = f(t). The solution to this system for constant D is readily obtained (Carslaw and Jaeger, 1959): z C(x, t) = 2n-“2

i

h f[t

- x2/(4Dp2)]e-“’

dp.,

where A = x(4Dt)-“’ and p is the integration variable. Nakumura and Wayland solved this system constraining f(t) to a linear function, since the fluorescent intensity was observed to increase in this manner for small times. However, we observed a nonlinear rise in concentration at the origin, and, therefore, we used a fourth-order polynomial representation for f(t). Mode1 predictions (Fig. 2) showed excellent agreement with the data obtained for small times yielding best fit (Ralston and Rabinowitz, 1978) values of D for the test molecules studied (Table 1). The capability of monitoring concentration within the erythrocyte-free plasma layer in conjunction with similar measurements within the tissue offers the prospect of quantifying permeabilities at the microvascular level. REFERENCES H. S., AND JAEGER, J. C. (1959). “Conduction of Heat in Solids,” 2nd Ed., pp. 62-63. Oxford Univ. Press (Clarendon), London/New York. Fox, J. R., AND WAYLAND, H. (1979). Interstitial diffusion of macromolecules in the rat mesentery.

CARSLAW,

Microvasc.

Res. 18, 255-276.

6 The one-dimensional diffusion model was not able to fit our data at large penetration distances from the capillary (X 5 60 pm) or at large times (I 2 10 set) presumably due to the transport interaction of adjacent capillaries.

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G. (1956). Passage of dextran molecules across the blood-lymph barrier. Acta Chir. Stand. (Suppl.) 211, I-84. NAKUMURA, Y., AND WAYLAND, H. (1975). Macromolecular transport in the cat mesentery. Microvasc. Res. 5, l-21. RALSTON, A., AND RABINOWITZ, P. (1978). “A First Course in Numerical Analysis,” pp. 332-409. McGraw-Hill, New York. WILLIAMS, R. G. (1954). Microscopic studies in living mammals with transparent chamber methods. In “International Review of Cytology” (G. H. Bourne and J. F. Danielli, eds.), Vol. III, pp. 359-398. Academic Press, New York. ZAWICKI, D. F., JAIN, R. K., SCHMID-SCHOENBEIN, G. W., AND CHIEN, S. (1981). Dynamics of neovascularization in normal tissues. Microvasc. Res. 21. 27-47.

GROTTE,