THE REGULATION OF BRANCHIAL BLOOD-FLOW IN THE BLACKTIP REEF SHARK, CARCHARHINUS-MELANOPTERUS (CARCHARHINIDAE, ELASMOBRANCHII)

The innervation of the branchial vasculature of the blacktip reef shark, Carcharhinus melanopterus, appears similar to that described for other elasmobranchs, Isolated perfused gill arch preparations were used in a study of vascular responses to branchial nerve stimulation. The pre-addition of 10(-5) M concentrations of pancuronium to the perfusate prevented skeletal muscle contractions and significantly reduced (P < 0.001) changes in perfusion pressure in response to nerve stimulation, The adductor muscles received an extensive innervation by nerve fibres ramifying from the gill arch, In pharmacological studies, pancuronium completely antagonized the normal vasoconstrictive response to acetylcholine, At low concentrations, noradrenaline produced a vasoconstriction, but this changed to a vasodilatation at concentrations above 10(-5) M, The use of antagonists demonstrated the presence of both alpha-, and beta-adrenoceptors in the branchial vasculature, Anatomical studies failed to show adrenergic nerves in the gills, suggesting al lack of a spinal autonomic ('sympathetic') innervation, Adrenergic regulation of gill blood flow may involve the release of catecholamines into the blood stream from chromaffin tissue stores

Estimating cell diffusivity and cell proliferation rate by interpreting IncuCyte ZOOM™ assay data using the Fisher-Kolmogorov model.

BACKGROUND: Standard methods for quantifying IncuCyte ZOOM(™) assays involve measurements that quantify how rapidly the initially-vacant area becomes re-colonised with cells as a function of time. Unfortunately, these measurements give no insight into the details of the cellular-level mechanisms acting to close the initially-vacant area. We provide an alternative method enabling us to quantify the role of cell motility and cell proliferation separately. To achieve this we calibrate standard data available from IncuCyte ZOOM(™) images to the solution of the Fisher-Kolmogorov model. RESULTS: The Fisher-Kolmogorov model is a reaction-diffusion equation that has been used to describe collective cell spreading driven by cell migration, characterised by a cell diffusivity, D, and carrying capacity limited proliferation with proliferation rate, λ, and carrying capacity density, K. By analysing temporal changes in cell density in several subregions located well-behind the initial position of the leading edge we estimate λ and K. Given these estimates, we then apply automatic leading edge detection algorithms to the images produced by the IncuCyte ZOOM(™) assay and match this data with a numerical solution of the Fisher-Kolmogorov equation to provide an estimate of D. We demonstrate this method by applying it to interpret a suite of IncuCyte ZOOM(™) assays using PC-3 prostate cancer cells and obtain estimates of D, λ and K. Comparing estimates of D, λ and K for a control assay with estimates of D, λ and K for assays where epidermal growth factor (EGF) is applied in varying concentrations confirms that EGF enhances the rate of scratch closure and that this stimulation is driven by an increase in D and λ, whereas K is relatively unaffected by EGF. CONCLUSIONS: Our approach for estimating D, λ and K from an IncuCyte ZOOM(™) assay provides more detail about cellular-level behaviour than standard methods for analysing these assays. In particular, our approach can be used to quantify the balance of cell migration and cell proliferation and, as we demonstrate, allow us to quantify how the addition of growth factors affects these processes individually