Blood Flow In Experimental Animals Research Articles

Natriuretic peptides relax human intrarenal arteries through natriuretic peptide receptor type-A recapitulated by soluble guanylyl cyclase agonists.

Natriuretic peptides, BNP and ANP increase renal blood flow in experimental animals. The signalling pathway in human kidney vasculature is unknown. It was hypothesized that BNP and ANP cause endothelium-independent relaxation of human intrarenal arteries by vascular natriuretic peptide receptor-A, but not -B and -C, which is mimicked by agonists of soluble guanylyl cyclase sGC. Human (n=54, diameter: 665±29µm 95% CI) and control murine intrarenal arteries (n=83, diameter 300±6µm 95% CI) were dissected and used for force recording by four-channel wire myography. Arterial segments were pre-contracted, then subjected to increasing concentrations of BNP, ANP, phosphodiesterase 5-inhibitor sildenafil, sGC-activator BAY 60-2770 and -stimulator BAY 41-2272. Endothelial nitric oxide synthase (eNOS) dependence was examined by use of L-NAME and eNOS knockout respectively. Molecular targets (NPR A-C, sGC, phosphodiesterase-5 and neprilysin) were mapped by PCR, immunohistochemistry and RNAscope. BNP, ANP, sildenafil, sGC-activation and -stimulation caused concentration-dependent relaxation of human and murine intrarenal arteries. BNP responses were independent of eNOS and were not potentiated by low concentration of phosphodiesterase-5-inhibitor, sGC-stimulator or NPR-C blocker. PCR showed NPR-A and C, phosphodiesterase-5, neprilysin and sGC mRNA in renal arteries. NPR-A mRNA and protein was observed in vascular smooth muscle and endothelial cells in arteries, podocytes, Bowmans capsule and vasa recta. NPR-C was observed in tubules, glomeruli and vasculature. Activation of transmembrane NPR-A and soluble guanylyl cyclase relax human preglomerular arteries similarly to phosphodiestase-5 inhibition. The human renal arterial bed relaxes in response to cGMP pathway.

Use of calcium channel blockers for glaucoma

Calcium channel blockers (CCBs), which alter the intracellular calcium concentration by modifying calcium flux across cell membranes and affect various intracellular signaling processes, have been long and widely used to treat essential hypertension and certain types of cardiac diseases such as angina pectoris. Among five subtypes of calcium channels, only specific agents for L-type calcium channels have been used as therapeutics. Animal experiments have indicated that topical application of CCBs, especially verapamil, caused significant intraocular pressure (IOP) reductions, while ocular hypotensive effects in humans were not substantial. Although the results obtained for nifedipine and nimodipine were not always consistent, CCBs generally dilate isolated ocular vessels and increase ocular blood flow in experimental animals, normal humans, and patients with open-angle glaucoma (OAG). Several single-centered, hospital-based, prospective studies have suggested that nimodipine, brovincamine, and nilvadipine had beneficial effects on visual function not only in normal humans but also in patients with OAG, while the results of population-based and case-controlled studies were not always consistent with those obtained in hospital-based studies. In vitro studies showed that CCBs exerted neuroprotective effects on neurons undergoing apoptosis and necrosis. Although the neuroprotective effects of CCBs have been well documented in experimental cerebral ischemia models, no controlled studies have shown the clinical efficacy of CCBs in stroke or cerebral ischemia. Neuroprotective effects also were documented in retinal ganglion cells and photoreceptors in experimental animals. Some ophthalmic beta-adrenoceptor antagonists, especially betaxolol, interact with L-type calcium channels and show calcium channel-blocking activity, which may be partly responsible for the neuroprotective effects of these drugs reported in experimental animals. Based on the reported findings of CCBs and that the results of clinical studies in acute cerebral ischemia may not be directly applicable to a chronic neurodegenerative ocular disorder, such as OAG, CCBs deserve future study to investigate strategies that are additive or synergetic to ocular hypotensive therapy for OAG, especially in patients with lower IOP.

神経細胞死研究の成果と臨床応用

Molecular mechanisms underlying ischemic neuronal death have been extensively investigated these twenty years. While several drugs such as glutamate receptor antagonists and free radical scavengers attracted much attention as potential neuroprotive agents, randomized clinical trials have failed to show any beneficial effect of those drugs for acute ischemic stroke patients. However, recent advance in molecular biology of NMDA receptor may explain why NMDA receptor antagonists were toxic rather than beneficial in ischemic stroke patients. NMDA receptors consisted of two subtypes, synaptic and extrasynaptic receptors. It is likely that activation of synaptic receptors results in survival while that of extrasynatpic ones causes cell death in cultured neuron. Antagonists specific to extrasynaptic receptor could be promising for neuroprotection. Hibernation and hypothermia both show robust protection against decreased levels of cerebral blood flow in experimental animals. Ischemic tolerance is well known phenomenon in that sublethal ischemic stress induces resistance of neurons against subsequent severe ischemic insult. Recent studies using cDNA microarray revealed that gene expression of cell metabolism and ion channel was down-regulated in tolerant brain. Metabolic downregulation would underlie the protective action of hibernation, hypothermia and ischemic tolerance. Therapeutic application of metabolic downregulation could be further investigated in next few years.

The effects of chronic phosphodiesterase-5 inhibitor use on different organ systems

Phosphodiesterase-5 (PDE-5) inhibitors selectively inhibit PDE-5 enzymes that are present in various tissues like penile tissue, platelets, vascular, and smooth muscle tissue. The drug's actions on these tissues have lead to the successful therapeutic use in patients suffering from conditions such as erectile dysfunction (ED) and pulmonary hypertension. PDE-5 inhibitors (PDE-5i) act on the erectile tissue causing penile smooth muscle relaxation and vasodilatation leading to penile erection. In addition, in particular when used in conjunction with prostaglandin inhibitors, PDE-5i cause vasodilatation in pulmonary vasculature hence decreasing both the pulmonary arterial pressure and resistance. PDE-5i have also shown to mildly decrease blood pressure, increase cardiac index, and increase coronary blood flow in experimental animals as well as in human studies. The Food and Drug Administration (FDA) has approved three PDE-5i for the treatment of ED: sildenafil (Viagra), vardenafil (Levitra), and tadalafil (Cialis) and one for pulmonary hypertension: sildenafil (Revatio). These agents are highly selective for PDE-5 enzymes as compared to other subclasses of PDE enzymes and have the almost identical pharmacological action but slightly different pharmacokinetics. Only little data exist about long-term use of PDE-5i and their effects on different organ system. This paper reviews the current information available on chronic PDE-5 inhibitor use.

Modification of the radioactive microsphere technique for assessment of regional blood flow to the craniofacial skeleton in the New Zealand rabbit

Background The radioactive microsphere technique has been used extensively for the accurate measurement of tissue blood flow in experimental animals. However, the need for carotid artery cannulation for injection of microspheres may significantly impair blood flow to the head and neck region. Objective This study assessed an alternative technique of injecting microspheres directly into the left ventricle to ensure a symmetric distribution of microspheres to the head. Methods Twenty-five New Zealand rabbits (seven to nine weeks of age) underwent blood flow measurement to the orbitozygomatic complex (OZC) regions bilaterally. Under general anesthesia, a sternotomy was performed and a cannula was inserted into the left ventricle. Radioactive microspheres were injected after a stabilization period of 15 min. Blood pressure and cardiac output were measured during the procedure and the animals were sacrificed at the end of the experiment. Tissue samples were harvested bilaterally from the OZC, hemimandibles, masseter muscles and overlying skin for measurement of blood flow. Results There was no significant difference in blood flow between the right and left side specimens. Animals were hemodynamically stable during the procedure. Conclusion Assessment of blood flow to the craniofacial region in experimental animals using radioactive microspheres may be facilitated by sternotomy and direct intracardiac injection.

The effects of sandostatin (Octreotide, SMS 201-995) infusion on splanchnic and hepatic blood flow in an experimental model of hepatic metastases.

Manipulation of hepatic blood flow may improve drug delivery to hepatic tumour. Somatostatin and its long acting analogues are known to elicit effects upon hepatic and splanchnic blood flow in experimental animals and patients with portal hypertension. This study investigates the effects of SMS 201-995 (sandostatin) infusion on hepatic, splanchnic and tumour blood flow in an experimental model of liver metastases. Hepatic tumour was induced by the intraportal inoculation of 10(6) HSN sarcoma cells and blood flow measured using the dual reference microsphere method before and after infusion of SMS 201-995. There was a significant decrease in hepatic arterial flow and a significant increase in the tumour:liver blood flow ratio associated with a marked reduction in blood flow to normal hepatic parenchyma. Portal venous inflow and tumour blood flow were not significantly affected. SMS 201-995 infusion may lead to preferential delivery of concomitantly injected cytotoxic drugs to hepatic tumour. In addition, the reduction in growth of hepatic tumour may be due to a reduction in nutritive, arterial blood flow to hepatic tumour.

Measurement of optic nerve blood flow with iodoantipyrine: Limitations caused by diffusion from the choroid

Radioactively labelled iodoantipyrine has been used to measure optic nerve blood flow in experimental animals. The reliability of blood flow measurements with this technique may be limited by diffusion of tracer from the nearby choroid. The activity of tracer in the peripapillary choroid and anterior optic nerve was measured in a series of in vivo optic nerve blood flow experiments in cats. The diffusion of iodoantipyrine into cat optic nerve segments was also measured in vitro, and a model for diffusion of tracer from the peripapillary choroid into the anterior optic nerve was developed. The activity profiles established by simple diffusion experiments were similar to the activity profiles in the anterior optic nerve established by blood flow experiments. Apparent blood flow measurements in the anterior optic nerve made with iodoantipyrine may be largely influenced by tracer diffusion from the nearby choroid. For measurements of tracer activity which are statistically similar between control and experimental optic nerve samples, and with a total diffusion time of 60 sec (a well-done experiment), real blood flow in the experimental sample may differ by as much as 60% from the control at a point 50 μm from the choroid. At 300 μm from the choroid, the maximum undetectable difference because of diffusion declines to approx. 12%. These estimates assume an autoradiographic technique which can reliably detect tissue tracer activity differences of ± 10%. Measurements of optic nerve blood flow made with diffusible tracers are affected by diffusion from the choroid and should be reported with estimated limits of reliability.

Delineation of impaired regional myocardial perfusion by positron emission tomography with H2(15)O.

Positron emission tomography with 15O-labeled water (H2(15)O) can be used to delineate abnormal regional myocardial blood flow in experimental animals. To determine the feasibility of this method in humans, we studied 33 subjects (9 normal volunteers and 24 patients with angiographically documented coronary artery disease) at rest and after myocardial hyperemia induced with intravenous infusion of dipyridamole. At rest, the myocardial region demonstrating the lowest relative H2(15)O activity exhibited 71 +/- 8% of activity in the region with peak activity in control subjects and 62 +/- 17% in patients (p = NS). After the dipyridamole infusion, differences between the two groups were accentuated. In control subjects, activity in the region with lowest relative radioactivity averaged 77 +/- 5% of that in the region with peak activity. In patients, it averaged 55 +/- 22% of activity in the region with peak activity (p less than 0.01). Results in patients with ischemia with or without a history of remote myocardial infarction were not significantly different. In 22 of the 24 patients, the region with lowest relative perfusion corresponded anatomically to the region of myocardium distal to a stenosis. Thus, delineation of regional myocardial perfusion in patients with coronary artery disease is possible with positron emission tomography and H2(15)O. Further studies will be necessary to prospectively determine sensitivity and specificity.

Microsphere estimates of blood flow: methodological considerations.

The microsphere technique is a standard method for measuring blood flow in experimental animals. Sporadic reports have appeared outlining the limitations of this method. In this study we have systematically assessed the effect of blood withdrawals for reference sampling, microsphere numbers, and anesthesia on blood flow estimates using radioactive microspheres in dogs. Experiments were performed on 18 conscious and 12 anesthetized dogs. Four blood flow estimates were performed over 120 min using 1 X 10(6) microspheres (15 microns) each time. The effects of excessive numbers of microspheres (13 million), pentobarbital sodium anesthesia (30 mg/kg), and replacement of volume loss for reference samples with dextran 70 were assessed. In both conscious and anesthetized dogs a progressive decrease in gastric mucosal blood flow and cardiac output was observed over 120 min. This was also observed in the pancreas in conscious dogs. The major factor responsible for these changes was the volume loss due to reference sample withdrawals. Replacement of the withdrawn blood with dextran 70 led to stable blood flows to all organs. The injection of excessive numbers of microspheres did not modify hemodynamics to a greater extent than did the injection of 4 million microspheres. Anesthesia exerted no influence on blood flow other than raising coronary flow. We conclude that although blood flow to the gastric mucosa and the pancreas is sensitive to the minor hemodynamic changes associated with the microsphere technique, replacement of volume loss for reference samples ensures stable blood flow to all organs over a 120-min period.

Measurement of renal blood flow by cine computed tomography

Renal blood flow can be a major determinant of solute and water excretion. Several methods have been developed during the last 20 to 30 years to measure renal blood flow in experimental animals and in man [1–4]. The distribution of renal blood flow to the cortex and medulla may also influence sodium excretion [5]. Regional flow rates have therefore also been measured [1, 5–7]. However, doubts have been expressed as to the accuracy of these measurements, partly due to theoretical problems and partly because of disagreement in results between techniques [1, 6]. The recent availability of cine–computed tomography (CT) [8, 9] offers a promising new approach to measuring renal blood flow in man and experimental animals. With short scanning time and a high repetition rate, this technique can record indicator dilution curves in major vessels as well as in different layers of tissue using contrast material as a soluble indicator. CT images give a cross sectional view of the anatomy, eliminating the superimposition problem commonly encountered with other techniques. In addition, selective cannulation of renal vessels can be avoided due to the superb contrast resolution of CT scanners, which allows for opacifying vessels and tissues with only small amounts of contrast medium. The goal of the present study was to explore the capability of high speed CT scanning for measuring regional, renal blood flow in the dog.

Effects of (-)-cis-2,3-dihydro-3-(4-methylpiperazinylmethyl)-2-phenyl- 1,5-benzothiaze pin-4-(5H)-one hydrochloride (BTM-1086) on ulceration, gastric secretion and mucosal blood flow in experimental animals.

Anti-ulcerous and anti-secretory effects of (-)-cis-2, 3-dihydro-3-(4-methylpiperazinylmethyl)-2-phenyl-1, 5-benzothiazepin-4-(5H)-one hydrochloride (BTM-1086), a new peptic-ulcer therapeutic agent, were studied in rats. BTM-1086 effectively prevented the ulceration of pylorus-ligated rats. A remarkable inhibition of gastric secretion was found at a dose of 0.1 mg/kg s.c. within a short period (6 h) after pylorus ligation. In the stomach-perfused rats, BTM-1086 (0.02 to 0.04 mg/kg i.v.) distinctly inhibited the teragastrin- and carbachol-induced gastric acid secretion, but only weakly inhibited the histamine-induced secretion. BTM-1086 also had a depressive effect on the gastric secretion stimulated by such secretagogues as tetragastrin, carbachol and histamine in rats with acute gastric fistulae. BTM-1086 increased the gastric mucosal blood flow in normal and indomethacin-induced ischemic rats. It also increased the gastric mucosal blood flow in water-immersion stress mcie at a dose equivalent to that used to prevent the development of ulcers.These results suggest that the antiulcer effects of BTM-1086 are mainly due to the inhibition of gastric acid secretion and to the increase of gastric mucosal blood flow.

Evaluation of changes in epicardial blood flow in experimental animals by cardiothermography.

When exposed to ambient air, the heart loses heat and its epicardial temperature becomes mainly perfusion dependent. Cardiothermography was used in this study to identify the extent and degree of epicardial cooling following reduction of coronary blood flow in 21 open chested dogs. The cold-ischemic area was easily identified by the dark color, reflecting lower epicardial temperature. Differences in temperature could be accurately identified (± 0.2 °C) by isotherm analysis. Six unipolar ECG electrodes were sutured to the epicardium 2–3 mm apart, between the normal and the ischemic zone. Following complete occlusion of the left anterior descending coronary artery (LAD), a cold area was detected within 11.7 ± 2.3 s. Within 60 s the ischemic area reached its maximum size. Myocardial cooling was 2.0 ± 0.4 °C from control state and reached its lowest value within 2 min. The zone of myocardial cooling corresponded precisely in all dogs with the ischemic area detected by ECG. ST elevation at the border of the ischemic zone was 4.5 ± 2.0 m V while toward the ischemic zone it was 14.1 ± 4.6 m V. Cooling of the epicardium was seen when coronary blood flow was reduced beyond 40–50%; this coincided with the ECG ST elevation. Further decrease of coronary blood flow increased the size of the ischemic area. Between 70–100% of coronary blood flow reduction, the size of the ischemic zone did not change although there was a slight further drop in the temperature at complete occlusion. The capability of cardiothermography to detect changes in the size of the ischemic area was tested under two interventions. Hypotension, induced by bleeding, caused a marked increase in the size of the cold-ischemic area in four dogs after LAD occlusion, while elevation of blood pressure after LAD occlusion induced by methoxamine caused a decrease in the size ofischemic area in six out of eight dogs studied. It is concluded that changes in epicardial temperature are useful indicators of myocardial ischemia, and that cardiothermography can assess the degree and extent of myocardial ischemia in experimental animals.

Measurement of cerebral blood flow with microspheres.

Labeled microspheres have been used recently to measure cerebral blood flow in experimental animals. The technique allows repeated measurements of regional, as well as total, blood flow to the brain. The microsphere technique has been used to study physiological regulation of cerebral blood flow, and can be used to examine regional cerebral blood flow in experimental models of disease states.

The determination of hepatic blood flow in the rat using xenon- I33

The measurement of hepatic blood flow in rats undergoing surgical trauma using different anaesthetic techniques has been carried out using the 133Xe clearance method. Injection into the hepatic artery is followed by monitoring of the liver radioactivity. The complex clearance curves have been analysed using a computer. Details of the surgical technique and the care of the animal while undergoing examination are given. The physiological basis for the clearance curves observed is analysed and a survey of the various parameters which may produce changes in clearance rates is made. Comparison with other investigations shows that rat liver blood flows obtained using the 133Xe clearance method are consistent with others using non-radioactive techniques; because of its simplicity and ease of repetition, the Xenon method is recommended as a way of evaluating drug effects on hepatic blood flow in experimental animals.

The measurement of the Ostwald solubility coefficient of krypton in blood and ocular tissue (relevant to blood flow measurement)

The clearance of radioactive krypton from ocular tissue has been used to measure choroidal blood flow in experimental animals. A demonstration of the validity of this method by the author (1975), by studying the diffusion of krypton in ocular tissue, required knowledge of the solubility and diffusion coefficients of krypton in the different ocular tissues but no measurements of these coefficients have been reported in the literature. The results of measurements of the solubility of krypton using scintillation counting, are presented.

Gastric mucosal blood flow measurement

1. 1. Pertechnetate clearance ( C Tc) by the stomach before and after betazole stimulation was compared to regional measurements of gastric blood flow utilizing nuclide (Chromium-51 and Cerium-141) labeled microspheres in five piglets. 2. 2. Pertechnetate clearance closely correlated (correlation coefficient 0.926) with mucosal blood flow in the gastric corpus measured by the microsphere technique. Betazole increased blood flow in the corpus region by 100% but did not alter this relationship. Except in one experiment, microsphere blood flow valves in the antrum and fundus were unchanged by betazole and did not significantly correlate with pertechnetate clearance. 3. 3. Pertechnetate clearance appears to be a reliable method of determining gastric mucosal blood flow in experimental animals and may be considered as a noninvasive method for measuring such flow in humans.

Construction of a regional cerebral blood flow probe using a peltier device

A commercially available Peltier device has been used to construct a probe for the determination of regional cerebral blood flow in experimental animals. The Peltier device provides a hot and a cold brass surface for application to the exposed cortex. Blood flowing through the tissue affects the temperature of the hot and cold plates in different directions whereas a change in ambient temperature affects the two plates equally. A thermocouple attached to each plate and connected in series monitors the differential temperature. The output of the flow probe is stable and very sensitive to changes in cerebral blood flow, but is not linear.

An Optically Recording Tambour Flowmeter and a Compensator for Large Volume Blood Flow

An optically recording flowmeter for measuring rate and volume of blood flow in experimental animals by the phlebotomy method is described. The advantages of the flowmeter are its great accuracy, sensitivity, linear calibration, minimal time lag, and above all its simplicity when used with optically recording manometers. In conjunction with a simple ``compensating switch'' for the outflowing blood, it permits measurement of very large volume flows without reduction of the arterial blood pressure.