Bruce Klitzman

Microvascular hematocrit and red cell flow in resting and contracting striated muscle

Microvascular hematocrit and its possible relation to oxygen supply were systematically examined. We studied the red cell volume fraction (hematocrit) in arterial blood and in capillaries under a variety of circumstances. Control capillary hematocrit averaged 10.4 +/- 2.0% (SE) and arteriolar (14.2 micrometer ID) hematocrit averaged 13.9 +/- 1.2% in cremaster muscles of pentobarbital-anesthetized hamsters. Carotid artery hematocrit was 53.2 +/- 0.6%. The low microvessel hematocrit could not be entirely explained by a high red cell flux through arteriovenous channels other than capillaries (shunting). Hematocrit was not only low at rest, but varied with physiological stimuli. A 1-Hz muscle contraction increased capillary hematocrit to 18.5 +/- 2.4%, and maximal vasodilation induced a rise to 39.3 +/- 9.5%. The quantitative relations between capillary red cell flux, arterial hematocrit, and total blood flow could be explained by a two-element model of microvascular blood flow that incorporated a relatively slow-moving plasma layer (1.2 micrometer). Such a model would generate a low microvessel hematocrit and might reduce the diffusion capacity of individual capillaries, but would not reduce time-averaged red cell flux or alter steady-state vascular oxygen supply.

Engineering the tissue which encapsulates subcutaneous implants. I. Diffusion properties

This report uses normal rat subcutis as a reference point to provide a quantitative analysis of small analyte transport through the tissue which encapsulates implants. Polyvinyl alcohol (PVA) with 60- and 350-micron mean pore size (PVA-60, PVA-350), nonporous PVA (PVA-skin), and stainless-steel cage (SS) specimens were implanted in the subcutis of Sprague-Dawley rats for 4 weeks to elicit a range of capsular wound-healing tissues. Histologic examination showed that the capsular tissue which formed around PVA-skin and SS specimens was densely fibrous and avascular. That forming around PVA-60 and PVA-350 was less densely fibrous and more vascular. The fibrous content of capsular tissue and subcutis was determined from eosin-stained histologic sections. Dual-chamber diffusion measurements of sodium fluorescein (Mw 376 g/mol) through capsular tissue and normal rat subcutis were used to quantitatively compare the effective diffusion coefficients of small analytes on the order of glucose. The two most fibrous capsular tissues exhibited diffusion coefficients that were statistically (p < 0.05) less than that determined for rat subcutis by 50 and 25% for PVA-skin and SS, respectively. The diffusion coefficients of the less dense capsular tissue which formed around the porous implants were not statistically different from subcutis. The experimentally measured diffusion coefficients of the two most fibrous capsular tissues were closely predicted by a simple two-component diffusion model consisting of an aqueous interstitium with an array of impenetrable bodies equal in volume fraction to the fibrous content of the tissue. This model overestimates the diffusion coefficients measured for the least fibrous tissues. Using the diffusion coefficient measured for the PVA-skin capsular tissue, a finite difference model predicts that a 200-microns-thick capsular layer would increase from 5 to 20 min the time required for subcutaneously implanted sensor to detect 95% of the blood analyte concentration. This study suggests that the fibrous capsule forming around a subcutaneously implanted smooth-surface sensor imposes a significant diffusion barrier to small analytes such as glucose, thus increasing the lag time of the sensor by as much as threefold. A corollary observation is that a sensor with a porous surface which allows tissue ingrowth may be more responsive to blood analyte fluctuations as a result of its a more vascular and less fibrous encapsulation tissue.

Quantification of longitudinal tissue pO2 gradients in window chamber tumours : impact on tumour hypoxia

SummaryWe previously reported that the arteriolar input in window chamber tumours is limited in number and is constrained to enter the tumour from one surface, and that the pO2 of tumour arterioles is lower than in comparable arterioles of normal tissues. On average, the vascular pO2 in vessels of the upper surface of these tumours is lower than the pO2 of vessels on the fascial side, suggesting that there may be steep vascular longitudinal gradients (defined as the decline in vascular pO2 along the afferent path of blood flow) that contribute to vascular hypoxia on the upper surface of the tumours. However, we have not previously measured tissue pO2 on both surfaces of these chambers in the same tumour. In this report, we investigated the hypothesis that the anatomical constraint of arteriolar supply from one side of the tumour results in longitudinal gradients in pO2 sufficient in magnitude to create vascular hypoxia in tumours grown in dorsal flap window chambers. Fischer-344 rats had dorsal flap window chambers implanted in the skin fold with simultaneous transplantation of the R3230AC tumour. Tumours were studied at 9–11 days after transplantation, at a diameter of 3–4 mm; the tissue thickness was 200 μm. For magnetic resonance microscopic imaging, gadolinium DTPA bovine serum albumin (BSA-DTPA-Gd) complex was injected i.v., followed by fixation in 10% formalin and removal from the animal. The sample was imaged at 9.4 T, yielding voxel sizes of 40 μm. Intravital microscopy was used to visualize the position and number of arterioles entering window chamber tumour preparations. Phosphorescence life time imaging (PLI) was used to measure vascular pO2. Blue and green light excitations of the upper and lower surfaces of window chambers were made (penetration depth of light ~50 vs >200 μm respectively). Arteriolar input into window chamber tumours was limited to 1 or 2 vessels, and appeared to be constrained to the fascial surface upon which the tumour grows. PLI of the tumour surface indicated greater hypoxia with blue compared with green light excitation (P < 0.03 for 10th and 25th percentiles and for per cent pixels < 10 mmHg). In contrast, illumination of the fascial surface with blue light indicated less hypoxia compared with illumination of the tumour surface (P < 0.05 for 10th and 25th percentiles and for per cent pixels < 10 mmHg). There was no significant difference in pO2 distributions for blue and green light excitation from the fascial surface nor for green light excitation when viewed from either surface. The PLI data demonstrates that the upper surface of the tumour is more hypoxic because blue light excitation yields lower pO2 values than green light excitation. This is further verified in the subset of chambers in which blue light excitation of the fascial surface showed higher pO2 distributions compared with the tumour surface. These results suggest that there are steep longitudinal gradients in vascular pO2 in this tumour model that are created by the limited number and orientation of the arterioles. This contributes to tumour hypoxia. Arteriolar supply is often limited in other tumours as well, suggesting that this may represent another cause for tumour hypoxia. This report is the first direct demonstration that longitudinal oxygen gradients actually lead to hypoxia in tumours.

Engineering the tissue which encapsulates subcutaneous implants. II. Plasma-tissue exchange properties.

This study assesses the plasma-tissue exchange characteristics of the capsular tissue that forms around implants and how they are affected by implant porosity. The number of vessels and their permeability to rhodamine were measured by intravascular injection of the fluorophore tracer into Sprague-Dawley rats that hosted for 3-4 months polyvinyl alcohol (PVA) and polytetrafluoroethylene (PTFE) subcutaneous implants. Rats were implanted with four pore sizes of PVA--a nonporous PVA (PVA-skin), and 5, 60, and 700 micron mean pore sizes (PVA-5, PVA-60, and PVA-700, respectively)--and two pore sizes of PTFE: 0.50 (PTFE-0.5) and 5.0 (PTFE-5) mean micron pore sizes. Photodensitometric image analysis was used to quantify the local tracer extravasation and, hence the permeability coefficients of isolated vessels around the implants. The number of functional vessels within 100 microm of the implants highlighted by the lissamine-rhodamine tracer were counted with fluorescence microscopy and with H&E stained sections using brightfield microscopy. The permeability of vessels did not vary substantially with implant pore size but generally were lower than those measured for surrounding subcutis. Pore size, however, had a dramatic effect on the vascular density of tissue-encapsulating implants: the number of microvessels (under 10 microm in radius) within the tissue surrounding the porous implants was higher than the number around nonporous implants. Pore sizes on the order of cellular dimensions incited optimal neovascularization; the vascular density around PVA-60 implants was six times higher (p < .001) and three times higher (p < .001) than those around PVA-0 implants in the fluorescent images and in brightfield, respectively. Moreover, brightfield microscopy showed the number of vessels around PVA-60 implants was almost double those in normal subcutis. The results suggest that optimal vascular density around long-term implants, such as sensors, biofluid cell constructs, and immunoisolated cell systems, may be engineered with pore size.

Analysis of oxygen transport to tumor tissue by microvascular networks

We present theoretical simulations of oxygen delivery to tumor tissues by networks of microvessels, based on in vivo observations of vascular geometry and blood flow in the tumor microcirculation. The aim of these studies is to investigate the impact of vascular geometry on the occurrence of tissue hypoxia. The observations were made in the tissue (thickness 200 microns) contained between two glass plates in a dorsal skin flap preparation in the rat. Mammary adenocarcinomas (R3230 AC) were introduced and allowed to grow, and networks of microvessels in the tumors were mapped, providing data on length, geometric orientation, diameter and blood velocity in each segment. Based on these data, simulations were made of a 1 mm x 1 mm region containing five unbranched vascular segments and a 0.25 mm x 0.35 mm region containing 22 segments. Generally, vessels were assumed to lie in the plane midway between the glass plates, at 100 microns depth. Flow rates in the vessels were based on measured velocities and diameters. The assumed rate of oxygen consumption in the tissue was varied over a range of values. Using a Greens function method, partial pressure of oxygen (PO2) was computed at each point in the tissue region. As oxygen consumption is increased, tissue PO2 falls, with hypoxia first appearing at points relatively distant from the nearest blood vessel. The width of the well-oxygenated region is comparable to that predicted by simpler analyses. Cumulative frequency distributions of tissue PO2 were compared with predictions of a Krogh-type model with the same vascular densities, and it was found that the latter approach, which assumes a uniform spacing of vessels, may underestimate the extent of the hypoxic tissue. Our estimates of the maximum consumption rate that can be sustained without tissue hypoxia were substantially lower than those obtained from the Krogh-type model. We conclude that the heterogeneous structure of tumor microcirculation can have a substantial effect on the occurrence of hypoxic micro-regions.

Direct demonstration of instabilities in oxygen concentrations within the extravascular compartment of an experimental tumor

To test the hypothesis that temporal variations in microvessel red cell flux cause unstable oxygen levels in tumor interstitium, extravascular oxygenation of R3230Ac mammary tumors grown in skin-fold window chambers was measured using recessed tip polarographic microelectrodes. Red cell fluxes in microvessels surrounding pO2 measurement locations were measured using fluorescently labeled red cells. Temporal pO2 instability was observed in all experiments. Median pO2 was inversely related to radial distance from microvessels. Transient fluctuations above and below 10 mm Hg were consistently seen, except in one experiment near the oxygen diffusion distance limit (140 microm) where pO2 fluctuations were <2 mm Hg and median pO2 was <5 mm Hg. Vascular stasis was not seen in these experiments. These results show that fluctuations in red cell flux, as opposed to vascular stasis, can cause temporal variations in pO2 that extend from perivascular regions to the maximum oxygen diffusion distance.

Decreased analyte transport through implanted membranes: differentiation of biofouling from tissue effects.

Membrane biofouling and tissue changes in the foreign body response are known to cause detrimental reductions of analyte transport into implanted biosensors. The relative contribution of each phenomenon is unknown. Hollow fiber microdialysis probes were employed to assess the effect of subcutaneous implantation on glucose flux through polymeric membranes in rats over 8 days and to differentiate the transport effects of biofouling versus tissue changes. Three commercially available membranes were examined: poly(ether sulfone) (PES), polyacrylonitrile (PAN), and polycarbonate (PC). As measured by glucose recovery (the ratio of microdialysis glucose to blood glucose concentrations), transport through PES membranes was significantly less on day 2 than day 0 (39% decrease, p < 0.05) whereas PAN and PC showed no significant decreases in flux until day 8 (42 and 43%, respectively). Application of a transport model to glucose recovery data obtained before implantation in vivo and after explantation indicated that mass transport resistances originating from biofouling and tissue compartments increased between days 0 and 8. However, on average the biofouling layer adherent to the probe created substantially less resistance to glucose transport (12-24% of total) than did the tissue that surrounded the probe. These results suggested that future material developments for biosensors should be directed at understanding and modifying transport properties of tissues at the implant site.

Engineering the tissue which encapsulates subcutaneous implants. III. Effective tissue response times

The results of two previous studies have shown that implant porosity can be used to increase both the measured diffusion coefficients and the vascularity within the tissue encapsulating long-term subcutaneous implants. This study investigates the hypothesis that the analyte concentrations within the tissue surrounding porous implants will respond more quickly to changes in plasma levels than does the densely packed, avascular fibrous capsule surrounding nonporous implants. The average concentration of lissamine-rhodamine was measured in tissue within 100 microm of the following implants at four different times following injection of the tracer: PVA-skin, PVA-5, PVA-60, PVA-700 (polyvinyl alcohol nonporous, 5 microm, 60 microm, and 700 microm mean pore sizes, respectively) and PTFE-0.5 and PTFE-5 (polytetrafluoroethylene 0.5 microm and 5 microm mean pore sizes, respectively). The results were compared to those of unimplanted subcutaneous tissue (SQ). In addition, the data were analyzed with a simple two-compartment model in which a tissue response time constant (taup) was extracted. As in the case of vascular density, the cellular dimension of the PVA-60 pore sizes produced surrounding tissue with the optimum response times to changes in plasma concentrations. The concentrations of rhodamine within the tissue surrounding the PVA-60 implant were the highest at all time points and responded to the change in plasma rhodamine concentration approximately three times more quickly (taup = 764 s) than the fibrous tissue encapsulating the nonporous PVA-skin (taup = 2058 s) and more than twice as quickly as SQ (taup = 1627 s). The overall mass transfer rate between plasma and the tissue surrounding the different implants calculated from the permeability and density of vessels from the previous study correlated very well (r2 = 0.7, p < .02, slope of 0.98) with the reciprocal of the tissue response time constant (taup).

Perivascular Oxygen Tensions in a Transplantable Mammary Tumor Growing in a Dorsal Flap Window Chamber

Fischer 344 rats with R3230 Ac mammary carcinomas implanted in dorsal flap window chambers served as a model to obtain measurements of perivascular and stromal oxygen tension in normal and tumor tissues using Whalen recessed-tip microelectrodes (3- to 6-microns tip). Perivascular measurements were made adjacent to vessels with continuous blood flow. Thus the measurements and models provided are reflective of conditions leading to chronic hypoxia. Perivascular oxygen tensions averaged 72 +/- 13 mmHg in normal tissue vessels adjacent to tumor, 26 +/- 5 mmHg in tumor periphery, and 12 +/- 3 mmHg in tumor central vessels. There was a significant trend toward lower perivascular oxygen tensions in the tumor center (Kruskal-Wallis test, P = 0.002). A similar tendency was seen with a limited number of stromal measurements. Krogh cylinder models, which incorporate these data for perivascular oxygen tension, along with morphometric data obtained from the same tumor model suggest that hypoxic regions will exist between tumor vessels in the tumor center unless O2 consumption rates are well below 0.6 ml/100 g/min. The low perivascular measurements observed near the tumor center combined with the theoretical considerations suggest, for this model at least, that tissue oxygenation may best be improved by increasing red cell velocity and input pO2 and reducing oxygen consumption. The low perivascular oxygen tensions observed near the center also suggest that conditions conducive to increased red cell rigidity exist, that drugs which can decrease red cell rigidity could improve tumor blood flow and oxygenation, and that the endothelium of those vessels may be susceptible to hypoxia-reoxygenation injury.

Augmented tissue oxygen supply during striated muscle contraction in the hamster. Relative contributions of capillary recruitment, functional dilation, and reduced tissue PO2.

To investigate the relative contributions of alterations in blood flow, capillary density, and tissue PO2 to elevated oxygen delivery in working muscle, we conducted experiments on the suffused hamster cremaster muscle, using in vivo microscopic techniques. Muscle PO2 was measured during striated muscle twitch contraction at 1 Hz. Tissue oxygenation was changed by using suffusion solutions equilibrated with 0%, 5%, 10%, 21%, or 50% oxygen. Contraction caused an increase in capillary density (capillary recruitment), whose magnitude was related to the equilibration gas and, thus, to the suffusate PO2. Capillary recruitment first increased as the oxygen content was raised, peaked with 10% oxygen, and then diminished with higher oxygen content. Arteriolar functional dilation was also observed; when oxygen was raised above 21%, dilation was decreased. The data suggest that oxygen supply is increased primarily by arteriolar conductance changes with low suffusion solution oxygen (0% to 5%), and by capillary recruitment and increased PO2 gradients above 10% oxygen. When vasomotor tone was increased by addition of norepinephrine to the suffusion medium, the changes observed were similar to those observed when oxygen was increased. Therefore, we propose that the altered microvascular responses during vasoconstriction are a function of vascular tone rather than the levels of tissue PO2. A model is proposed which may partially explain the relations among vascular tone, functional dilation, and capillary recruitment. Our data also suggest that tissue PO2 may not be precisely regulated about a narrowly defined set point in this striated muscle but that, instead, tissue PO2 is a dependent variable controlled by the integrated effects of capillary recruitment, functional vasodilation, and altered metabolism.