A method for isolation and fluorescent labeling of rat neutrophils for intravital microvascular studies.

A method for the isolation, fluorescent labeling, and reinfusion of rat neutrophils for in vivo investigation of white blood cell function in the microcirculation is described. The cell surface morphology and function of the labeled and unlabeled neutrophils were compared in vitro and in vivo. The morphology of the labeled and unlabeled cells was visually assessed by differential interference contrast microscopy before and after exposure to the chemotactic peptide FMLP. Labeled and unlabeled cells were morphologically similar under normal and stimulated conditions. The chemotactic responsiveness of the labeled and unlabeled cells was evaluated in vitro by quantifying the movement of cells across a porous membrane in response to FMLP. No significant difference in chemotactic responsiveness was found. The in vivo behavior of the labeled neutrophils was quantitatively studied by examining their interactions with the venular endothelium in the rat cremaster muscle. Rolling velocity for labeled neutrophils and unlabeled white blood cells was measured at different flow velocities and wall shear rates. No difference was found between the labeled and unlabeled cells. It is concluded that this isolation and labeling procedure yields cells possessing normal morphology and function. These labeled neutrophils may be useful for in vivo study of their behavior in the microcirculation.

Noradrenergic and peptidergic innervation of the extrinsic vessels and microcirculation of the rat cremaster muscle.

The noradrenergic and peptidergic innervation of the extrinsic vessels and microcirculation of the rat cremaster muscle was examined. Catecholamine-containing nerves were identified histochemically by glyoxylic acid-induced fluorescence and tyrosine hydroxylase immunoreactivity (TH-IR). The extrinsic pudic-epigastric artery and vein as well as the entire intramuscular arteriolar network was innervated by noradrenergic axons. The capillaries and intramuscular venules of the cremaster muscle were devoid of a noradrenergic innervation. Immunohistochemical double-labeling demonstrated that most, if not all, of the TH-IR axons also possessed neuropeptide Y immunoreactivity (NPY-IR), implying colocalization of the norepinephrine and NPY in the perivascular nerves. No vasoactive intestinal peptide immunoreactivity (VIP-IR) was found, except for occasional VIP-IR axons associated with the pudic-epigastric artery. Substance P immunoreactive (SP-IR) axons formed a sparse plexus around the arteries and larger arterioles. Calcitonin gene-related peptide immunoreactivity (CGRP-IR) had a similar distribution to the SP-IR axons. CGRP-IR was also observed in axons alongside some smaller arterioles and capillaries. The extrinsic vessels and intramuscular arteriolar network of the rat cremaster muscle are innervated by noradrenergic axons which contain NPY and by presumed sensory nerves containing SP and/or CGRP. Both types of nerves may contribute to regulation of microvascular function.

Response of the arteriolar network in rat cremaster muscle to intraarterial infusion of nicotine.

The response of the arteriolar network in rat cremaster muscle to continuous intraarterial infusion of nicotine was studied. Measurements were made of mean femoral arterial pressure, inside vessel diameter, red blood cell velocity and volumetric flow rate in each of four series-coupled arteriolar segments. Nicotine was continuously infused in a cumulative fashion in doses of 12.5, 25, 50 and 100 micrograms/kg/min. After a 10 min infusion of each dose, measurements were made again in each arteriole and compared with the values obtained prior to infusion of nicotine. Arterial pressure increased in a graded fashion with increasing dose of nicotine up to 50 micrograms/kg/min. The smaller arterioles demonstrated a dose dependent vasoconstriction and reduction in flow rate which were maximal at a dose of 50 micrograms/kg/min. In another series of experiments, the microvascular responses to nicotine infusion were obtained in the acutely denervated microvasculatured. The nicotine-induced flow reduction was significantly diminished by denervation. In a separate series of experiments nicotine was infused at doses of 25 and 50 micrograms/kg/min, and plasma catecholamine concentrations were determined. Plasma norepinephrine and epinephrine were significantly elevated at only the higher dose. Responses in denervated tissues suggest that plasma catecholamine concentrations were approximately threshold for arteriolar responses. It is concluded that the nicotine-induced flow reduction in rat skeletal muscle is due primarily to enhanced release of norepinephrine from vasomotor nerves with little or no influence from circulating catecholamines.

Innervation of the microcirculation.

Neurogenic control of the peripheral circulation is accomplished by alterations in nerve discharge to the pre- and postcapillary vascular network in the various organs. The postganglionic sympathetic adrenergic nerves constitute the most important efferent pathway for neural control. The physiologic response of the microvasculature to neural influences depends on a number of factors: the pattern of distribution of the innervation to the microvessels is one of the more important determinants. In addition to its influence on the contractile state of vascular smooth muscle, the adrenergic nerves also have a trophic influence on the smooth muscle cells. Following surgical denervation of a vascular bed, the adrenergic nerve terminals degenerate, and subsequently reinnervate, the vasculature. During the period following denervation, a number of functional and morphologic changes occur in the smooth muscle. This review emphasizes those aspects of the structure and function of adrenergic nerves that may have particular relevance for microsurgery.

Effects of chronic smoke exposure on the cardiovascular responses to acute nicotine infusion in the rat.

Rats were exposed daily to cigarette smoke for 17-22 weeks in order to characterize mean arterial pressure and regional hemodynamic effects of chronic smoke exposure and to determine if cardiovascular reactivity to acute nicotine infusions is altered by chronic smoke exposure. Urethane-anesthetized animals were instrumented with miniaturized pulsed-Doppler flow probes on the iliac and mesenteric vascular beds. Under resting conditions sham-smoked and smoke-exposed animals had similar levels of mean arterial pressure and mesenteric blood flow; however, resting heart rate was lower in the smoke-exposed group, while iliac blood flow was elevated in the smoke-exposed group. Acute nicotine infusion (6.25, 12.5 and 25 micrograms/kg per min) produced equivalent, dose-dependent pressor effects as well as increases in iliac and mesenteric resistance in sham and smoke-exposed groups. Thus, chronic cigarette smoke-exposure in rats may exert significant cardiovascular effects other than on arterial pressure such as lowered heart rate and elevated blood flow to skeletal muscle beds, while cardiovascular responses to nicotine are not altered by chronic smoke-exposure.

Response of the microcirculation in rat cremaster muscle to peripheral and central sympathetic stimulation.

Microvascular diameter and flow responses to peripheral and central sympathetic stimulation were measured in different segments of the arteriolar network in rat cremaster muscle. For peripheral stimulation, a bipolar electrode was placed on the internodal segment of the lumbar sympathetic chain between ganglia L1 and L2. For central stimulation, a bipolar electrode was stereotaxically implanted in the posterior hypothalamus. Inside vessel diameter, red blood cell velocity, and volumetric flow rate were recorded in response to electrical stimulations of varying magnitude in four series-coupled segments of the arteriolar network: 1A, 2A, 3A, and 4A. Systemic arterial pressure was also monitored. The vasoconstriction and flow reduction produced by stimulation of the lumbar chain was graded with the frequency of stimulation over the range of 0.5-16.0 Hz in all arteriolar segments. Examination of the relation between stimulation frequency and vasoconstrictor response measured as percent of control diameter indicated a sequence of responsiveness to peripheral stimulation where 4A = 3A greater than 2A = 1A. No changes in diameter were recorded in the venous microcirculation at any level of stimulation. Stimulation of the posterior hypothalamus with currents of 38-300 microA for 60 seconds produced graded vasoconstriction in only 3A and 4A vessels. Fluorescence histochemistry for biogenic amines was used to examine the distribution of innervation to the microvasculature. All segments of the arteriolar network from 1A to 4A possessed an adrenergic innervation; no vessels of the venous network were found to be innervated. The results indicate that the pattern of response of the arteriolar network in rat cremaster muscle to peripheral and central sympathetic stimulation is segmentally differentiated and consistent with the distribution of the vasomotor innervation.

Measurement of erythrocyte velocity by use of a periodic differential detector.

An optical velocimeter employing a linear array of photodiodes has been developed and utilized for measuring erythrocyte velocities in the microcirculation. A magnified image of a microvessel is projected and aligned on a one-dimensional array of photodiodes. Photocurrent from odd-ordered diodes is summed, photocurrent from even-ordered diodes is summed, and a signal proportional to the difference between these two currents is produced by a differential amplifier. The center frequency of the output signal of the differential amplifier is proportional to the erythrocyte velocity. After lowpass filtering the output of the differential amplifier, a signal proportional to its frequency and therefore velocity is produced by a frequency-voltage converter. In vitro calibration with a moving dried smear of erythrocytes illustrated a linear relation between the output of the frequency-voltage converter and erythrocyte velocity for a wide range of velocities and magnifications. The system produces a stable zero output at zero velocity and had an estimated frequency response of greater than 40 Hz in vivo. Volumetric flow rates computed from velocity and diameter measurements at arteriolar bifurcations in the rat cremaster muscle were consistent with mass conservation.

Adrenergic innervation in the microcirculation of the bat wing.

The distribution of adrenergic nerves to the microcirculation of the wing web of the bat was studied in whole-mount preparations employing glyoxylic acid fluorescence histochemistry. A dense perivascular adrenergic nerve plexus consisting of various-sized nerve bundles surrounded the arteries. The accompanying veins were sparsely innervated by small nerve bundles or single axons. Arterioles also possessed a dense perivascular plexus comprised of a loose network of small axon bundles. Muscular venules accompanying most arterioles were supplied by a small number of varicose axons which surrounded these vessels in a loose network. The majority of the terminal arterioles were devoid of any direct innervation. The proximal segment of some terminal arterioles was innervated by one or two axons which were observed to diverge from the vessels and terminate in the tissue. The true capillaries, postcapillary venules, and lymphatics possessed no direct adrenergic innervation. Surgical sectioning of the appropriate nerve trunks or administration of 6-hydroxydopamine caused complete disappearance of all fluorescent adrenergic axons in the wing web. Faint acetylcholinesterase-positive staining was observed in a perivascular plexus surrounding the arteries and arterioles. A much diminished acetylcholinesterase activity was observed after treatment with 6-hydroxydopamine which implies that this enzymatic activity resides in the adrenergic nerves.

A mathematical representation of the microvascular network in the bat's wing.

We previously have published a description of a mathematical representation for general vascular networks. In this paper we will illustrate the application of this operator representation to the microvascular network found in the wing web of the bat. We will show that this method follows the rules of vector algebra, it can be used for determination of Orthogonality of two vectors, and can be used in calculating resistance through the microvascular network. The method can also be used for representation of vessel networks in three dimensions.

Correlation of fluid exchange in the microcirculation and macrocirculation.

Accurate, quantitative information relating to hydraulic conductivity (Lp) of the capillary wall in any organ of tissue is essential as the first step toward understanding fluid homeostasis. Any perturbation which permanently alters fluid balance will ultimately lead to death and destruction of tissues in the living organism. This report summarizes some recent information relating to Lp measurements in single capillary and whole organ experiments. It is shown that the single capillary and whole organ methods do not yield similar quantitative information and some possible reasons for this are discussed. It is hypothesized that capillary membrane permeability is governed by dynamic processes which may change in response to both the metabolic requirements of the organ tissue cells and overall metabolic homeostasis of the organism. It follows from this hypothesis that fluid balance in tissues is a passive process determined solely by the Starling forces, the functional significance of which is to provide an aqueous medium to aid the diffusion of nutrient substrate from blood to tissue cells.

Pre- and postcapillary vascular responses to sympathetic nerve stimulation in DOCA-hypertensive dogs.

Measurements of precapillary resistance (Ra),postcapillary resistance (Rv), and mean capillary hydrostatic pressure (Pci) were made during sympathetic nerve stimulation (SNS) under constant-flow perfusion in isolated hindlimbs from deoxycorticosterone acetate (DOCA)-hypertensive dogs. We found that both pre- and postcapillary vascular responses to SNS were greater in the DOCA-hypertensive group when compared to the control group. Intraarterial injections of norepinephrine produced a dose-response curve for precapillary vessels in the hypertensive group that was asymmetrically shifted to the left (increased slope) and exhibited a significant decrease in vasoconstrictor threshold. these results, coupled with our earlier observations, suggest that the hyperresponsiveness of the precapillary segment in DOCA-hypertensive dogs could be attributed to both structural and intrinsic alterations of the resistance vessels. We present evidence that suggests, however, that the increased postcapillary resistance with SNS may be explained by a structural alteration causing a decrease in the diameter of existing postcapillary vessels, or may be due to a decrease in the actual number of postcapillary vessels, or both. It is concluded that in this model of hypertension, postcapillary vascular changes also contribute to the overall increase in total peripheral resistance.

Are acetylcholine-induced increases in 42K efflux mediated by intracellular cyclic GMP in turtle cardiac pace-maker tissue?

1. 42K efflux has been measured from small strips of turtle sinus venosus tissue in order: (a) to characterize further the pharmacology of the acetylcholine response and (b) to test whether cyclic guanosine 3':5'-monophosphoric acid (cyclic GMP) is the intracellular mediator of this response. 2. The 42K wash-out curves show that the fractional escape rate (FER) of 42K efflux is nearly constant after 60-80 min, indicating that after this time period 42K FER is controlled by barrier-limited diffusion from a single intracellular compartment. 3. The threshold of the dose-response relationship for the acetylcholine-induced increase in 42K FER is about 10(-8) M and the Km is 2.75 x 10(-7) in non-eserinized preparations. 4. This acetylcholine response is completely blocked by atropine; but nicotinic blockers produce no detectable reduction of it. 5. Exogenous application of lipid-soluble analogues of cyclic GMP (dibutyryl or 8-bromo-cyclic GMP applied at 2-3 mM for 30 min) failed to mimic the acetylcholine-induced augmentation of 42K FER. 6. Experiments in which sodium nitroprusside (5 x 10(-4) M for 30 min) was applied in order to stimulate the guanylate cyclase and hence produce a large, maintained increase in intracellular cyclic GMP failed to show a significant increase in 42K FER. 7. When acetylcholine (10(-6)M) was applied in the presence of O[Ca2+]0 (in an attempt to inhibit the guanylate cyclase) there was no significant reduction in the acetylcholine-induced increases in 42K FER. 8. Hence, these three indirect tests indicate that the muscarinic acetylcholine-induced increase in 42K FER in cardiac pace-maker tissue is unlikely to be mediated entirely by changes in the levels of intracellular cyclic GMP.

Changes in 42K efflux produced by alterations in transmembrane calcium movements in turtle cardiac pace-maker tissue.

1. 42K efflux has been measured from small strips of turtle sinus venosus which were electrically paced. Three different procedures for altering transmembrane calcium influx have been utilized to test whether changes in 42K efflux may be modulated by changes in intracellular calcium levels. 2. No significant changes in the 42K fractional escape rate (FER) were observed when external calcium was reduced to O mM or increased to 4 x normal (10 mM). In these experiments extracellular divalent cation concentration was held constant by adding or removing magnesium ions. 3. Application of 10 mM-Ba2+ also failed to alter 42K FER consistently. In red blood cells and snail neurones addition of barium ions has been shown to reduce significantly the calcium-mediated potassium current. 4. A tenfold increase in pacing rate (0.5-5 Hz) resulted in an augmented 42K FER, but repetition of this rate change in O mM-Ca2+ indicated that this increase in 42K FER was not strongly dependent on the amount of calcium entry. 5. Attempts to load the pace-maker cells with calcium by using the ionophore A23187 (10 micrograms ml . -1 of 2.0 x 10(-5) M) consistently resulted in very large increases in 42K FER. However, this effect (i) was blocked by atropine and (ii) was markedly reduced by pretreating the tissues with hemicholinium, indicating that A23187-induced release of acetylcholine from the endogenous nerve terminals was responsible for the observed increase in 42K FER. 6. In summary, three different experimental tests indicate that the majority of the 42K efflux is not tightly linked to transmembrane calcium movement in sinus venosus pace-maker tissue.

Femur-bending properties as influenced by gravity: I. Ultimate load and moment for 3-G rats.

Fresh experimental bones can withstand greater bending forces and moments after 1.0 to 2.5 weeks of 3-G exposure. This appears more attributable to a 50% greater strength of bone material than to effects upon size or shape, and is most measurable for animals of 5 to 8 weeks of age. Experimental bone material seems to grow to its mature level at a younger age rather than there being so marked an effect upon the mature level itself. We simulated 3.1 G by chronic centrifugation of 66 albino rats and compared them to 63 1-G controls. Extrapolation of the simplest mathematical description of the present results to weaker, zero-G bones could be tested by a total of 60 space-based control and experimental animals. A flight of only 15 animals would be necessary for comparison to ground-based control animals. This is consistent with reports of bone demineralization during space-flight. In light of the differences in bone histology, however, extrapolation of these results to humans would be premature and, if at all applicable, are most likely to be so for children rather than adults.