Joanne Lofthouse

In vivo diffusion of immunoglobulin G in muscle: effects of binding, solute exclusion, and lymphatic removal

Previously, we demonstrated that immunoglobulin G (IgG), dissolved in an isotonic solution in the peritoneal cavity, transported rapidly into the abdominal wall when the intraperitoneal (ip) pressure was >2 cmH2O. We hypothesized that this was chiefly caused by convection and that diffusion of IgG was negligible. To investigate the role of diffusion, we dialyzed rats with no pressure gradient across the abdominal wall muscle for 2 or 6 h with an ip isotonic solution containing125I-labeled IgG. At the end of the experiment, the animal was euthanized and frozen and abdominal wall tissue was processed to produce cross-sectional autoradiograms. Quantitative densitometric analysis resulted in IgG concentration profiles with far lower magnitude than profiles from experiments in which convection dominated. In other in vivo experiments, we determined the lymph flow rate to be 0.8 × 10-4ml ⋅ min-1 ⋅ g-1and the fraction of extravascular tissue (θs) available to the IgG to be 0.041 ± 0.001. An in vitro binding assay was used to determine the time-dependent, nonsaturable binding constant: 0.0065 min-1 × duration of exposure. A non-steady-state diffusion model that included effects of θs, time-dependent binding, and lymph flow was fitted to the diffusion profile data, and the IgG diffusivity within the tissue void was estimated to be 2 × 10-7cm2/s, a value much higher than that published by other groups. We also demonstrate from our previous data that convection of IgG through tissue dominates over diffusion at ip pressures >2 cmH2O, but diffusion may not be negligible. Furthermore, nonsaturable binding must be accounted for in the interpretation of tissue protein concentration profiles.Previously, we demonstrated that immunoglobulin G (IgG), dissolved in an isotonic solution in the peritoneal cavity, transported rapidly into the abdominal wall when the intraperitoneal (ip) pressure was > 2 cmH2O. We hypothesized that this was chiefly caused by convection and that diffusion of IgG was negligible. To investigate the role of diffusion, we dialyzed rats with no pressure gradient across the abdominal wall muscle for 2 or 6 h with an ip isotonic solution containing 125I-labeled IgG. At the end of the experiment, the animal was euthanized and frozen and abdominal wall tissue was processed to produce cross-sectional autoradiograms. Quantitative densitometric analysis resulted in IgG concentration profiles with far lower magnitude than profiles from experiments in which convection dominated. In other in vivo experiments, we determined the lymph flow rate to be 0.8 x 10(-4) ml.min-1.g-1 and the fraction of extravascular tissue (theta s) available to the IgG to be 0.041 +/- 0.001. An in vitro binding assay was used to determine the time-dependent, nonsaturable binding constant: 0.0065 min-1 x duration of exposure. A non-steady-state diffusion model that included effects of theta s, time-dependent binding, and lymph flow was fitted to the diffusion profile data, and the IgG diffusivity within the tissue void was estimated to be 2 x 10(-7) cm2/s, a value much higher than that published by other groups. We also demonstrate from our previous data that convection of IgG through tissue dominates over diffusion at ip pressures > 2 cmH2O, but diffusion may not be negligible. Furthermore, nonsaturable binding must be accounted for in the interpretation of tissue protein concentration profiles.

In vivo effects of hydrostatic pressure on interstitium of abdominal wall muscle

Fluid loss from the peritoneal cavity to surrounding tissue varies directly with intraperitoneal hydrostatic pressure (Pip). According to Darcys law [Q = -KA(dPif/dx)], fluid flux (Q) across a cross-sectional area (A) of tissue will increase with an increase in either hydraulic conductivity (K) or the interstitial fluid hydrostatic pressure gradient (dPif/dx, where x is distance). Previously, we demonstrated that in the anterior abdominal muscle (AAM) of rats, dPif/dx increases by only 40%, whereas K rises fivefold between Pip of 1.5 and 8 mmHg. Because K is a function of interstitial volume (thetaif), we hypothesized that perturbations of Pip would change Pif and expand the interstitium, increasing thetaif. To test this hypothesis, we used dual-label quantitative autoradiography (QAR) to measure extracellular fluid volume (thetaec) and intravascular volume (thetaiv) in the AAM of rats within the Pip range from -2.8 to +8 mmHg. thetaif was obtained by subtraction (thetaec - thetaiv). dPif/dx was measured with a micropipette and a servo-null system. Local thetaiv did not vary with Pip and averaged 0.010 +/- 0.002 ml/g, and thetaif averaged 0. 19 +/- 0.01 ml/g at Pif </=1.2 mmHg. However, thetaif doubled between Pif of 1.2 and 4.2 mmHg (from 0.20 +/- 0.00 to 0.39 +/- 0.01 ml/g, respectively) but did not increase with further increases in Pif. This nonlinear pressure-volume relationship does not explain the fivefold increase in K with Pip. Because the interstitial matrix contributes to the interstitial resistance to fluid flow, and because hyaluronan (HA) is the only component of the matrix that is not anchored to the tissue, we hypothesized that the loss of interstitial HA was responsible for the continued decrease in interstitial resistance to fluid flow. We determined HA concentration in the rat AAM and adjacent subcutaneous tissue (SC) at Pip = 0 mmHg and after 2 h of dialysis at constant Pip = 6 mmHg. The HA content (normalized to dry weight) in the AAM was reduced from 487 +/- 16 to 360 +/- 27 micrograms/g dry tissue (n = 4, P < 0.05) and increased from 528 +/- 72 to 1,050 +/- 136 mg/g dry tissue (n = 4, P > 0.001) in the SC. We conclude that the mechanisms responsible for the increase in K with Pip include expansion of the interstitium, dilution of interstitial macromolecules, and washout from the AAM to SC of interstitial macromolecules responsible for resistance to fluid flow.

In vivo hydraulic conductivity of muscle: effects of hydrostatic pressure

We and others have shown that the loss of fluid and macromolecules from the peritoneal cavity is directly dependent on intraperitoneal hydrostatic pressure (Pip). Measurements of the interstitial pressure gradient in the abdominal wall demonstrated minimal change when Pip was increased from 0 to 8 mmHg. Because flow through tissue is governed by both interstitial pressure gradient and hydraulic conductivity ( K), we hypothesized that K of these tissues varies with Pip. To test this hypothesis, we dialyzed rats with Krebs-Ringer solution at constant Pip of 0.7, 1.5, 2.2, 3, 4.4, 6, or 8 mmHg. Tracer amounts of125I-labeled immunoglobulin G were added to the dialysis fluid as a marker of fluid movement into the abdominal wall. Tracer deposition was corrected for adsorption to the tissue surface and for local loss into lymphatics. The hydrostatic pressure gradient in the wall was measured using a micropipette and a servo-null system. The technique requires immobilization of the tissue by a porous Plexiglas plate, and therefore a portion of the tissue is supported. In agreement with previous results, fluid flux into the unrestrained abdominal wall was directly related to the overall hydrostatic pressure difference across the abdominal wall (Pip = 0), but the interstitial pressure gradient near the peritoneum increased only ∼40% over the range of Pip = 1.5-8 mmHg (20-28 mmHg/cm). K of the abdominal wall varied from 0.90 ± 0.1 × 10-5cm2 ⋅ min-1 ⋅ mmHg-1at Pip = 1.5 mmHg to 4.7 ± 0.43 ×10-5cm2 ⋅ min-1 ⋅ mmHg-1on elevation of Pip to 8 mmHg. In contrast, for the same change in Pip, abdominal muscle supported on the skin side had a significantly lower range of fluid flux (0.89-1.7 × 10-4vs. 1.9-10.1 × 10-4ml ⋅ min-1 ⋅ cm-2in unsupported tissue). The differences between supported and unsupported tissues are likely explained in part by a reduced pressure gradient across the supported tissue. In conclusion, the in vivo hydraulic conductivity of the unsupported abdominal wall muscle in anesthetized rats varies with the superimposed hydrostatic pressure within the peritoneal cavity.