The skin blood flow response in wound healing.

Although vasodilation is conventionally held to be the predominant microvascular response to a wound, there has been no previous attempt to actually quantitate skin blood flow within and in the neighborhood of wounds. In particular, there has been no differentiation between sites with primarily nutritive (NUTR) blood flow and those with considerable arteriovenous (AV) perfusion. We used our previously described model of cutaneous blood flow in the rat to study the blood flow response to wounding. We measured skin blood flow at the centers and at the undisturbed perimeters of wounds placed at the back, a NUTR site, and at the paw, an AV site, in 11 Wistar Kyoto rats. Measurements were performed at baseline, and then at 3 hr, 24 hr, 72 hr, and 7 days postwounding. At 3 hr, flow at the center of the back wound had increased to 11.3 +/- 1.4 ml/min/100 g from a baseline of 2.1 +/- 0.1 ml/min/100 g and remained elevated at 7 days (8.3 ml/min/100 g). Flow at the perimeter of the back wound rose as well, but not as high as at wound center, to twice the baseline level (4.1 ml/min/ 100 g at Day 7). Flow values at control sites on the back did not increase from baseline. Flow at the center of the paw wound rose from 7.2 +/- 0.5 ml/min/100 g at baseline to 15.6 +/- 4.3 ml/min/100 g at Day 3 but then fell back to 6.9 +/- 0.9 ml/min/100 g at Day 7. There was only a very small increase in the basal temperature wound response at the paw perimeter. Blood flow at all wound sites showed a response to heat. At the back, heating to 44 degrees stimulated an 80% increase in blood flow at baseline. This degree of increase was maintained at both the center and the perimeter of the back wound. In contrast, although there was also a thermal response at the paw wound center, it was of much lower magnitude than the nonwounded baseline response. As a result, the heat-stimulated flow value actually fell over the 7 days to approximately half of the baseline level. At the paw wound periphery, there was an initial fall in the heat stimulated response, but it then recovered to the baseline level and remained stable over the 7 days. Thus, the skin blood flow response seen at the paw wound challenges the conventional concept of vasodilation as the expected wound blood flow response. The mechanisms of blood flow response in the healing wound may be more complex than the simple inflammatory vasodilation conventionally postulated.

Skin blood flow in the Wistar-Kyoto rat and the spontaneously hypertensive rat.

1. Using laser Doppler techniques in man, we have previously demonstrated differences in skin blood flow properties at sites with primarily nutritive (NUTR) perfusion, such as the elbow or knee, as compared to sites such as the finger pulp, with predominantly arteriovenous anastomotic (AVA) perfusion. 2. Basal and heat stimulated flow is greater at AVA sites. In man, blood pressure changes are reflected primarily by changes at AVA rather than NUTR sites. 3. These blood pressure induced changes affect the red blood cell velocity (VEL) component at AVA sites more than microvascular volume (VOL). 4. Given these findings in man, we decided to compare skin blood flow properties in a suitable animal model. 5. We chose the Wistar-Kyoto (WKY) and Spontaneously Hypertensive Rat (SHR) strains, in view of the marked difference in systemic blood pressure in these two related strains. 6. Skin blood flow varied considerably at different skin sites in the rats. Skin sites with hair covering, on the back and at the base of the tail, showed low basal and heat stimulated blood flow. 7. In contrast, the plantar surface of the paw behaved similarly to the finger or toe pulps in man, with 3-4-fold higher basal flow than the hair covered areas and a 7-8-fold rise with local heating to 44 degrees C. 8. Furthermore, there was a 25% greater blood flow at the plantar paw surface in the SHR rats as compared to the WKY rats, corresponding to the 25% higher systemic blood pressure in these animals. 9. The heat induced increase in flow at the plantar surface of the paw was primarily a result of a marked increase in VEL rather than VOL. 10. The higher flow at this site in SHR as compared to WKY rats was likewise ascribable to an increase in VEL, VOL being equivalent in the two strains.

The effect of increasing temperature on skin blood flow and red cell deformability.

Using laser Doppler techniques in nine healthy volunteers, we contrasted the effect of increasing local skin temperature at the elbow, a skin site with nutritive microvasculature, and the finger pulp, with predominantly arteriovenous anastomic (AVA) perfusion). We also assessed flow at the finger dorsum, with contributions of both types of microvasculature. In parallel with the laser Doppler studies, we determined the effect of increasing temperature on the red cell deformability of our subjects, using the new technique of Cell Transit Time Analysis (CTTA). Thermal stimulation produced very large increases in skin blood flow at all three sites tested. However, the magnitude and the pattern of increase were different at the three sites. At the finger pulp, there was a linear approximately threefold increase in flow as temperature increased from the basal level to 44 degrees C. At the elbow, basal flow was considerably lower than at the finger pulp and increased very slowly until skin temperature reached 38 degrees C. From that point, flow increased sharply, reaching tenfold the basal level at 44 degrees C. The thermally induced increase at the finger dorsum was intermediate between the other two sites, with a pattern resembling the elbow more than the finger pulp. These differences among the sites were attributable to substantially different patterns of change in the two components of flow, microvascular volume and velocity. At the finger pulp, there was very little increase in microvascular volume with increasing temperature. The curve was practically flat from basal temperature to 44 degrees C. In contrast, there was a linear increase in red blood cell velocity of about 300%. At the elbow, both microvascular volume and red blood cell velocity exhibited a parallel curvilinear pattern of equivalent increase, on the order of 300% for each. There was only a small increase in both parameters until the temperature reached 38 degrees, at which point there was a sharp increase in both. At the finger dorsum, the situation was intermediate, again resembling the elbow more than the finger pulp. Cell Transit Time Analysis revealed a progressive decrease in red cell transit time (TT), from 3.28 ms at 28 degrees C to 2.48 m at 44 degrees C, an overall change of 24%. The decrease in TT was accompanied by an increase in transit frequency, measured as counts s-1 (C s-1), from 3.1 to 5.3, an overall change of 71%. The changes in both TT and C/S were essentially linear.(ABSTRACT TRUNCATED AT 400 WORDS)

Competitive binding kinetics in ligand-receptor-competitor systems. Rate parameters for unlabeled ligands for the formyl peptide receptor.

We describe the real-time kinetics of the competition of different ligands for the same receptor and use the computer routine SAAM to simulate this competition. Based on the simulation, we have developed two experimental approaches whereby the parameters of the interaction of nonlabeled ligands with their receptor can be detected; briefly, the analysis of the nonlabeled ligands depends on the perturbation of the kinetics of interaction of labeled ligands with the receptor with which they are in competition. The approach relies primarily upon an analysis of the kinetics of the competition between fluorescent and nonfluorescent ligands using a real-time, homogeneous binding assay in the fluorescence flow cytometer. A secondary approach depends upon an examination of the kinetic impact of antagonists on the responses of cells stimulated by agonists at the same receptor. Experimental verification of these approaches has been obtained using the N-formyl peptides (and their antagonists) which bind to receptors on human neutrophils and produce rapid cell stimulation. We find that agonistic N-formyl peptides have residence times of minutes while nonstimulatory antagonists have residence periods of, at most, a few seconds at these receptors. The limitations and general range of applicability of these procedures are discussed. The main advantage of these approaches is that they permit the evaluation of kinetic parameters of unlabeled ligands, even those which bind weakly or which have brief residence times--properties which make analyses by conventional methods difficult.

A convenient on-line device for reagent addition, sample mixing, and temperature control of cell suspensions in flow cytometry.

We describe a simple and inexpensive device which permits the addition of up to three different solutions into a cell suspension which is on-line in a flow cytometer. The mixing chamber houses a disposable plastic cuvette stirred with a magnetic stirrer. The sample chamber is attached to a circulating water bath, hence accurate temperature control is achieved. Because the system is prepressurized and the sample line is very short, the delay time-between the point of sample modification and the point of analysis is reduced to a few seconds. Thus reagents may be added rapidly, and kinetic measurements of high temporal resolution are possible. Because the temperature of the sample chamber is regulated, binding can be observed over longer time periods than was previously possible. We demonstrate the usefulness of this device in determining the binding of fluoresceinated hexapeptide to human neutrophils under conditions where the stimulus is infused into the cell suspension while on-line in the cytometer.

Neutrophil degranulation detected by right angle light scattering: spectroscopic methods suitable for simultaneous analyses of degranulation or shape change, elastase release, and cell aggregation.

We have used simultaneous spectrometric analysis of right angle scattering and elastase release from human neutrophils to demonstrate the similarity of these two measures of degranulation. Both responses depend on the presence of cytochalasin B, and are similar in kinetics, dose-response, and dependence on receptor occupancy at the formyl peptide receptor. This scattering response is shown to be largely independent of cell aggregation. In the absence of cytochalasin B, a rapid and transient right angle scatter response of a different character, probably associated with cell ruffling, is detected. Either right angle response can be detected by flow cytometry.

The dynamics of ligand-receptor interactions. Real-time analyses of association, dissociation, and internalization of an N-formyl peptide and its receptors on the human neutrophil.

Parallel cytometric and fluorimetric analyses of the interaction of a fluoresceinated N-formyl hexapeptide (N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, Nle = norleucine) with its receptors on human neutrophils are presented. The cytometric analyses take advantage of the ability of the fluorescence flow cytometer to discriminate free and receptor-bound ligand in a homogeneous real-time assay. The spectrofluorometric analysis relies on a high affinity antibody to fluorescein to discriminate free and bound ligand. We find that the number of receptors for formyl peptides on the surface of a resting cell is 53,000 +/- 13,000 (Kd approximately 0.6 +/- 0.2 nM). We use commercially available cytometric standards to calibrate the cytometer and we obtain similar values for the number of receptors. The temperature dependence of the kinetics of ligand-receptor interactions have been examined. The association rate constant varies from approximately 3 X 10(8) M-1 min-1 at 4 degrees C to approximately 10(9) M-1 min-1 at 37 degrees C (delta H approximately 8 kcal/mol). While ligand internalization is blocked at 4 degrees C, at 37 degrees C internalization proceeds at an initial rate of approximately 24% of the occupied receptors/min following a latency period of approximately 20-30 s. Intermediate rates and longer latency periods are found at 15 and 25 degrees C. Dissociation of the ligand is heterogeneous and depends both on the length of time of association and the temperature. After short periods of association, the ligand dissociates with t1/2 approximately 1-5 min. After longer periods (30 min at 15 degrees C or 100 min at 4 degrees C), but while the ligand-receptor complex remains on the cell surface, t1/2 increases to greater than 30 min. It appears that the ligand-receptor complex undergoes an alteration in affinity, with a time course at elevated temperatures, which parallels or lags behind the time course of the participation of the occupied receptors in cell activation.

Ligand/receptor internalization: a kinetic, flow cytometric analysis of the internalization of N-formyl peptides by human neutrophils.

Fluorescence flow cytometry was used to measure the internalization of the fluorescent ligand N-formyl-nle-leu-phe-nle-tyr-lys-fluorescein by human neutrophils. The internalization process was monitored by the accessibility of the receptor-bound fluorescent ligand to quenching following a change in the pH of the extracellular medium from 7.4 to 3.0. In such a pH change, extracellular ligand or fluorescein are quenched immediately (excitation 488 nm). In contrast, intracellular fluorescein (derived from fluorescein diacetate) or intracellular ligand are quenched with half-times of approximately 20 or approximately 40 sec, respectively, at 37 degrees C. The fraction of internalized ligand is calculated by resolving the fast and slow components of the quenching process. Temporal resolution of the internalization process in this system depends upon two factors. We have previously shown that it is possible to examine essentially continuously the kinetics of ligand binding in the nM concentration range without removing the free ligand (Sklar LA, Finney DA, Cytometry 3:161, 1982). We have now modified a Becton Dickinson FACS IV sample head assembly to permit direct addition of reagents into the cell suspension while on-line. This enables us to change the suspension pH and evaluate internalization with a time resolution of a few seconds. We observe that internalized ligand can be detected within 1 min and that the rate is proportional to the number of receptors occupied. The rate is essentially linear over the first few minutes and approximately 60% of the receptor-bound ligand is internalized after 3 min.

Purification of cybrids by fluorescence-activated cell sorting.

A general method to isolate and purify substantial numbers of viable cybrids from cultured mammalian cells immediately following cytoplast-cell fusion is described. This method uses cytoplasts whose mitochondria are selectively stained in vivo by the cationic fluorescent rhodamine dye, rhodamine 123. Large numbers of highly purified, rhodamine-stained cytoplasts are fused to appropriate recipient cell lines and then the fusion mixture is sorted based on forward angle scatter and fluorescence parameters. Plating the positively sorted population in culture for as short as 12 h eliminates contaminating cytoplasts which, lacking a nucleus, are unable to adhere or survive. The resultant population, based on an analysis of genetic markers, is 75-100% cybrids, an enrichment of 1000- to 10,000-fold over the initial fusion mixture. Cybrids purified by cell sorting may be useful for detailed molecular studies of mitochondrial DNA gene expression and in the specific induction of new mitochondrial DNA mutants.

Analysis of ligand-receptor interactions with the fluorescence activated cell sorter.

The binding of a fluorescent peptide to human neutrophils is analyzed with a fluorescence activated cell sorter. We examine steady-state and kinetic features of the ligand-receptor interaction (in the presence of unbound ligand) and we show tht the number of receptors may be estimated without resorting to any external references for calibration. These methods are applicable to other fluorescent ligands with affinities greater than 10(8) M-1.