A method of examining arteriolar function in situ using the in vitro-perfused hydronephrotic rat kidney is described. This approach facilitates direct visualization of arteriolar contractile responses in a well-controlled experimental environment while avoiding the consequences of traumatic microdissection and the accompanying exposure to ischemia, hypothermia, or hypoxia. The preparation has a remarkably well-preserved myogenic reactivity, exhibiting precisely graded vasoconstriction over the range in perfusion pressure subtending normal renal autoregulatory responses (i.e., 80-180 mm Hg). Using this preparation, the inhibitory effects of hypoxia on arteriolar myogenic reactivity have been demonstrated. The range over which reduced pO2 affected arteriolar reactivity in this model corresponded closely to that reported to alter vascular tone in vivo. A technique of adapting the model to incorporate simultaneous monitoring of arteriolar fluorescence measurements and contractile responses is also described. This approach has been used to examine the relationship between arteriolar contractility and NADH autofluorescence during the hypoxia-induced activation of ATP-sensitive K channels. Future applications may include the use of intravital fluorescent dyes to examine, for example, microvascular endothelial calcium signaling in an intact, functioning arteriole.
It is possible to estimate acoustic parameters that describe the microscopic anatomy of live tissues. The same rf echo signals used to form diagnostic ultrasound images undergo additional signal processing to compute D, the average scatterer size; SNR, a measure of the number density of scatterers; and IBC, a measure of tissue echogenicity. With these three parameters it is possible to identify the sources of ultrasonic scattering in kidneys, observe functional changes in live organs, and produce images of each parameter. Although this chapter has emphasized the more basic research elements of possible applications, similar methods have been used to study changes in tissue morphometry during the progress of disease and during the course of therapy in several organ systems. Advances in diagnostic ultrasound are providing safe and low-cost method for studying soft tissues in vivo.
The information presented in this chapter clearly shows the usefulness of SEM for analyzing the components of the wall of microvessels. The literature contains several methodologies that allow one to successfully expose single smooth muscle cells that line microvessels. It appears that chemical and enzymatic digestion steps will continue to be the principal elements of any methodology for exposing these cells. The combination of microdissection, prior to processing the specimen for SEM, with digestion has allowed better preservation of the tissues by reducing the amount of material obscuring the tissue of interest as well as reducing the length of time the tissue is exposed to chemicals. The next important improvement was the mobilization step of selected vessel segments so that they could be mounted "end on" for circumferential viewing and photographing. Finally, at least three different mathematical approaches have been developed and tested for the quantitation of single cell length and width determination.
A method of examining arteriolar function in situ using the in vitro-perfused hydronephrotic rat kidney is described. This approach facilitates direct visualization of arteriolar contractile responses in a well-controlled experimental environment while avoiding the consequences of traumatic microdissection and the accompanying exposure to ischemia, hypothermia, or hypoxia. The preparation has a remarkably well-preserved myogenic reactivity, exhibiting precisely graded vasoconstriction over the range in perfusion pressure subtending normal renal autoregulatory responses (i.e., 80-180 mm Hg). Using this preparation, the inhibitory effects of hypoxia on arteriolar myogenic reactivity have been demonstrated. The range over which reduced pO2 affected arteriolar reactivity in this model corresponded closely to that reported to alter vascular tone in vivo. A technique of adapting the model to incorporate simultaneous monitoring of arteriolar fluorescence measurements and contractile responses is also described. This approach has been used to examine the relationship between arteriolar contractility and NADH autofluorescence during the hypoxia-induced activation of ATP-sensitive K channels. Future applications may include the use of intravital fluorescent dyes to examine, for example, microvascular endothelial calcium signaling in an intact, functioning arteriole.
Cell volume changes and regulation are thought to be important in the physiology and pathology of neurons. Osmotic challenges alter a number of physiologic parameters including electrical properties, chemosensitivity, and Na pump activity. Thus, a good method for correlating images of the living cell's surface with electrophysiologic measures is needed, and we are exploring the use of confocal light microscopy for imaging neurons in culture.Isolated neurons from ganglia of Aplvsia California were plated on polylysine coated coverslips in L15 media with 20% hemolymph at room temperature. Cells stained with “Dil” were imaged with a Bio—Rad MRC—600 and an Olympus BH—2. Images were displayed using Bio—Rad's maximum projection procedure, or Vital Images’ Voxel View software run on an IBM RISC 6000 Powerstation 320. Electrophysiologic properties were assessed after imaging.
In mice, rats, hamsters, dogs, and monkeys, doses of > or = 1.1 microgram/kg/day of r-metHuG-CSF stimulated neutrophil production in bone marrow. There was a resulting rapid release of mature neutrophils from the marrow storage pool into circulation and a distinct neutrophilia maintained for the dosing period in each study. R-metHuG-CSF corrected cyclic hemopoiesis in dogs, established sustained endogenous recovery after lethal bone marrow suppression, and elicited differention and maturation induction and enhanced functional activity of mature neutrophils. Reversible side effects due to an exaggerated pharmacological activity of the growth factor were observed at doses of > or = 115 micrograms/kg/day, with the exception of a rodent-specific osteopathy, which occurred in some animals after 3 months at > or = 5.7 micrograms/kg/day. The only irreversible adverse effect was seen in monkeys at doses of 1150 micrograms/kg/day (equivalent to 200 times the recommended human dose) when leukocyte counts exceeded 100 x 10(9)/liter. It consisted of hyperleukocytosis, leukostasis in terminal capillaries of the brain, followed by tissue hypoxia and intracerebral hemorrhage. Local tolerance of r-metHuG-CSF was good and no hypersensitivity reactions were observed. Antibody formation was minimal in monkeys but occasionally marked in dogs, which is not suprising due to the species difference in the amino acid sequence of the factor. The use of recombinant canine G-CSF, however, did not elicit formation of neutralizing antibodies in dogs. Moreover, relatively few untoward effects were seen in reproductive toxicology except when maternal toxicity or weight loss of dams occurred. In addition, negative results were obtained in all genotoxicity studies performed.
