Curiosity Killed the Dog
Appendix 1: A case study of basic research involving the cardiovascular system
Evaluation of Animal Research at Leeds Medical School
Prepared by John J. Pippin, MD, FACC
Research Consultant, PCR
The effect of stimulation of the atrial receptors on plasma renin activity in the dog. Journal of Physiology 1988;398:411-21.
Left atrial balloon distension to stimulate atrial receptors produced reflex decreased plasma renin level and increased urine output. This effect was blocked by cervical vagosympathetic nerve trunk cooling, which the research team concluded meant that the afferent limb of this reflex involves myelinated vagal fibers.
The effect of stimulation of left atrial receptors on coronary blood flow inanaesthetized dogs. Journal of Physiology 1989;408:45-56.
Left atrial balloon distension, while simultaneously blocking normal reflex tachycardia, results in decreased coronary blood flow. This response was abolished by cooling the cervical vagosympathetic trunk or giving bretylium. The research team concluded that the afferent limb of this reflex response involves myelinated vagal fibers, and the efferent limb involves cardiac sympathetic pathways.
The effect of distension of the urinary bladder on activity in efferent vagal fibers in anaesthetized dogs. Quarterly Journal of Experimental Physiology 1989;74:493-501.
Bladder distension was produced by volume infusion. In 8 dogs, 11 efferent vagus fibers were identified which responded to changes in carotid artery pressure. These fibers also responded to bladder distension by causing reflex decrease in vagus nerve activity. It was also found that the bladder distension-mediated decrease in vagus activity was affected by the level of carotid sinus pressure.
Efferent limb of the atrial receptor reflex decrease in plasma renin activity in anaesthetized dogs. Quarterly Journal of Experimental Physiology 1989;74:503-9.
Left atrial balloon distension produced decreased plasma renin activity and increased urine output. Tachycardia and natriuresis were blocked. The reflex plasma renin decrease was abolished by renal denervation. The team concluded that the efferent limb of this reflex involves the renal nerves.
The effect of distension of the urinary bladder on activity in efferent renal fibres in anaesthetized dogs. Journal of Physiology 1989;409:357-69.
Bladder distension was produced by volume infusion. In 11 dogs, 26 efferent vagal fibers were identified which responded to changes in carotid artery pressure. These fibers also responded to bladder distension by increasing renal nerve activity In 6 other dogs, 13 efferent fibers showed a graded response to bladder distension, which was proportional to intravesical pressure and inversely proportional to carotid artery pressure.
The effect of stimulation of the atrial receptors on plasma cortisol level in the dog. Journal of Physiology 1989;413:299-313.
Left atrial balloon distension produced reflex decrease in plasma cortisol levels, and increased urine output. Cooling of the cervical vagosympathetic trunk abolished these responses, suggesting that the afferent limb of this reflex involves the vagal nerves. Hypophysectomy abolished the plasma cortisol decrease, but not the diuretic response, suggesting that the efferent limb of this reflex requires the pituitary gland.
The effects of ventricular end-diastolic and systolic pressures on action potential and duration in anaesthetized dogs. Journal of Physiology 1992;457:75-91.
Performed on open-chest dogs with blockade of autonomic influences on the heart. Elevation of left ventricular peak systolic pressure, while keeping end-diastolic pressure constant, shortened the monophasic cardiac action potential. Elevation of end-diastolic pressure, while keeping peak systolic pressure constant, lengthened the monophasic cardiac action potential. Changes in myocardial segment lengths were compared to ventricular pressures, and suggested that the changes in action potential were related to contraction-excitation feedback.
Reflex responses to stimulation of mechanoreceptors in the left ventricle and coronary arteries in anaesthetized dogs. Journal of Physiology 1993;472:769-83.
This study was done to identify the mechanism for reflex vasodilation in response to increased aortic root pressure in dogs (demonstrated in other dog research). Cannulations, variable perfusions, and pressure and flow measurements involved the aortic root, left ventricular apex, left atrium, carotid sinuses, and hindlimb arteries. A series of experiments using variable aortic, ventricular and combined pressures suggested that the reflex increased vasodilation due to increased aortic root pressure is from stimulation of coronary artery baroreceptors.
Afferent discharges from coronary arterial and ventricular receptors in anaesthetized dogs. Journal of Physiology 1993;472:785-99.
This study came directly from the previous study, and was designed to determine if there was a contribution from left ventricular receptors to aortic root pressure-mediated reflex vasodilation. Simultaneous aortic root, left ventricular, coronary arterial and vagus nerve instrumentation, measurements, and changes in pressure were used to assess differential effects and vagal responses. Vagal afferent fibers were classified as myelinated (10) or non-myelinated (11), based upon conduction velocities. Non-myelinated fibers showed variable responses to aortic and ventricular pressure changes, and to drug infusions. Myelinated fibers showed a more consistent and graded response to pressure changes, and were more sensitive to coronary arterial than to left ventricular pressure changes. The team concluded that reflex vasodilation was likely mediated by myelinated vagal afferent fibers, which were likely attached to coronary arterial mechanoreceptors.
Reflex vascular responses from aortic arch, carotid sinus and coronary baroreceptors in the anaesthetized dog. Experimental Physiology 1996;81:397-408.
Reflex vascular responses to independently altered pressures in aortic, coronary and carotid baroreceptors were measured. Increased pressures produced vasodilation in all cases, more prominently in coronary and carotid baroreceptors. The time for post-vasodilation recovery of vascular resistance was longest for the coronary artery. The reason(s) for this difference was unknown.
Delayed sympathetic efferent responses to coronary baroreceptor unloading in anaesthetized dogs. Journal of Physiology 1996;497(part 1):261-9.
This study was to determine whether distension of subdiaphragmatic veins produces reflex vasoconstriction and interacts with the carotid baroreflex. An extensive perfusion circuit was devised to control carotid and thoracic aortic pressures, splanchnic and limb blood flows, and cardiopulmonary blood flows. Elevated splanchnic pressures produced increased carotid baroreflex responses and vasoconstriction in splanchnic and limb vascular territories. These responses were not affected by cutting vagal or phrenic nerves, but were prevented by cutting peripheral nerves supplying the affected regions.
Reflex vascular responses to abdominal venous distension in the anesthetized dog. American Journal of Physiology 1996;271(3 part 2):H1049-56.
This study came directly from study #10, and was designed to identify the location of the reflex determinant which results in slower post-vasodilation recovery of vascular resistance after coronary artery pressure changes, compared to carotid artery pressure changes. An extensive perfusion circuit was devised to control aortic root, aortic arch, coronary artery and carotid artery perfusion and pressure. Rapid coronary artery afferent responses to increased and decreased pressures excluded prolonged activation of these afferents as the mechanism. Increased coronary artery pressure produced a reflex decrease in either renal or lumbar sympathetic nerve activity, which recovered slowly after coronary pressure decreased. The team concluded that the delayed recovery of coronary arterial resistance post-vasodilation was due to delayed recovery of the efferent limb of the reflex, mediated by either renal or lumbar sympathetic nerves.
Vascular responses to stimulation of carotid, aortic and coronary artery baroreceptors with pulsatile and non-pulsatile pressures in anaesthetized dogs. Experimental Physiology 1996;81: 969-81.
Deriving from several previous studies, this was designed to investigate the ranges of pressures required to elicit various baroreceptor reflex vascular responses, and the influences of pulsatile v. non-pulsatile flow. An extensive perfusion circuit was devised to control perfusion and pressure for the 3 baroreceptor regions investigated. Cardiopulmonary bypass and induced ventricular fibrillation were used to prevent contamination of coronary artery baroreceptor pressure. Data analysis was limited to those dogs which demonstrated specific minimum pressure responses. Reflex responses were obtained at lower coronary perfusion pressures than carotid artery or aortic arch perfusion pressures. Carotid responses, but not coronary or aortic responses, were influenced by pulsatile v. non-pulsatile flow.
Mechanisms responsible for changes in abdominal vascular volume during sympathetic nerve stimulation in anaesthetized dogs. Experimental Physiology 1997;82:925-34.
This study was designed to determine whether sympathetic-mediated decreased abdominal blood flow is due to decreased blood volume or vasoconstriction. Abdominal perfusion was controlled for either volume or pressure, and was drained at constant pressure via the IVC. Various experiments demonstrated predominantly vasoconstrictive influence, which was inversely related to the magnitude of sympathetic stimulation. The passive component increased after splenic pedicle ligation, which was interpreted to show that passive volume changes were important after splenic pedicle ligation (!?).
Reflex vascular responses to alterations in abdominal arterial pressure and flow in anaesthetized dogs. Experimental Physiology 1997;82:995-1005.
This study was designed to determine if there are abdominal vascular baroreceptors in the dog. Abdominal circulation was isolated, maintained by aortic perfusion, and drained from the IVC to a reservoir. Perfusion pressure responses were larger at higher carotid sinus pressures. Responses were unaffected by cutting vagus, phrenic and splanchnic nerves, but were abolished by cutting the spinal cord at T12. The team concluded that there is evidence for abdominal baroreceptors, with the afferent pathway involving the spinal cord.
Structure and in vitro function of human subcutaneous small arteries in mild heart failure. American Journal of Physiology 1998;274(5 part 1):C1298-305.
This study compared morphology tension generation and response to ramipril for small, subcutaneous arteries among patients with mild heart failure and healthy controls. Compared to normal controls and placebo-treated mild heart failure patients, the ramipril-treated patients' arterial segments showed enhanced vasoconstrictor responses to both norepinephrine and angiotensin II. The team concluded that this enhanced response may identify upregulation of receptor-mediated events.
Blood mobilization from the liver of the anaesthetized dog. Experimental Physiology 1998;83:513-22.
This study was designed to evaluate the contribution of the liver to overall abdominal vascular capacitance and compliance in the dog. The liver was vascularly isolated, perfused through the hepatic artery and portal vein, and drained at constant pressure from the hepatic veins. Results indicated that the dog's liver (like the spleen in other experiments) has a major capacitance role and is highly compliant. The team suggested that if this is also true for humans, the liver would be the most important controllable blood reservoir in the body.
Reflex vascular responses in the anaesthetized dog to large rapid changes in carotid sinus pressure. American Journal of Physiology 1998;275(4 part 2):H1169-77.
This study examined reflex vascular responses to large rapid increases and decreases in carotid sinus pressure. An extensive perfusion circuit was devised to control perfusion and pressure in the carotid sinus, thoracic aorta, abdominal circulation and a hindlimb. Results showed that reflex-mediated vasodilation occurred more rapidly and more completely than vasoconstriction. The team concluded that if similar findings occurred in humans in response to alternating gravitational forces, there may be a predisposition to syncope.
Reflex control of splanchnic blood volume in anaesthetized dogs. Journal of Physiology 1998;513(part 1):263-72.
Various vascular isolation techniques were used to control perfusion and pressure within splenic, hepatic and caudal circulations. These regions were drained through the IVC, portal and femoral veins. Variation of carotid sinus pressure showed greater volume changes during constant pressure perfusion than during constant volume perfusion. The liver's capacitance role greatly exceeded that of the splanchnic circulation, but large passive changes in splanchnic flow occurred in response to flow changes.
Absence of early resetting of coronary baroreceptors in anaesthetized dogs. Journal of Physiology 1998;513(part 2):543-9.
This study was designed to determine whether coronary baroreceptors reset differently from carotid and aortic arch baroreceptors, after sustained distension at low and high pressures. An extensive perfusion circuit was devised to control perfusion and pressure in the carotid, aortic arch and coronary artery baroreceptors. Results indicated rapid resetting of carotid baroreceptors, but delayed resetting of coronary baroreceptors.
Reflex responses from the main pulmonary artery and bifurcation in anaesthetized dogs. Experimental Physiology 2000;85:411-20.
Open-chest dogs with cardiopulmonary bypass and blockade of secondary modulation of responses from other reflexes. Proximal aspects of the pulmonary circulation were vascularly isolated, pefused through the left PA, and drained through the right PA to maintain desired PA pressure. Results showed that pulmonary baroreceptor stimulation produces proportional pressor and hyperventilatory responses. Greater vascular responses were obtained at lower PA pressures when negative thoracic pressure was introduced, indicating that the response would likely be greater in a closed-chest animal.
Absence of reflex vascular responses from the intrapulmonary circulation in anaesthetized dogs. Experimental Physiology 2000;85:421-30.
Open-chest dogs with cardiopulmonary bypass and blockade of reflexogenic mechanisms not being measured in the study. Left lung vascular territory was isolated, and was perfused through the left PA and drained through the left pulmonary veins. Pressures were controlled to the carotid, aortic and coronary baroreceptors, and to the heart chambers. Results indicated that the lung was innervated, but changes in pulmonary arterial or venous pressure did not produce changes in systemic or limb vascular resistances. Different dogs showed different results in responses to pulmonary pressure changes and effects upon phrenic nerve activity.
Reflex effects of independent stimulation of coronary and left ventricular mechanoreceptors in anaesthetized dogs. Journal of Physiology 2000;528:349-58.
This study was designed to examine reflex responses to independently controlled stimulation of ventricular and coronary artery mechanoreceptors. Manipulations performed included mechanical ventilation, cardiopulmonary bypass, balloon occlusion of LV outflow to prevent coronary perfusion, LV apical cannulaton to regulate LV volume, carotid and aortic root cannulation to regulate pressures. Results indicated that coronary baroreceptors were important for cardiovascular hemodynamic control, with minimal if any contribution from ventricular mechanoreceptors. The authors note that this is contrary to previous dog studies which showed significant reflex responses attributed to ventricular mechanoreceptors.
Reflex vascular responses to independent changes in left ventricular end-diastolic and peak systolic pressures and inotropic state in anaesthetized dogs. Journal of Physiology 2001;532 (part 2):549-61.
This study was designed to examine the nature and physiological role of ventricular mechanoreceptor-mediated reflex vasodilation in the dog. Manipulations included mechanical ventilation, cardiopulmonary bypass, isolation of ventricular pressures from the coronary arteries and aorta by a LV outflow tract balloon, and cannulation of the LV apex, carotid and coronary arteries, aortic root and abdominal aorta. Increased carotid or coronary pressure produced vasodilation, which was unaffected by inotropic stimulation or changes in ventricular peak systolic pressure, and was influenced in a small but proportional manner by increases in end-diastolic pressure. The team concluded that the only effective ventricular determinant of reflex responses was increased filling volume, and that the influence was so small as to be unimportant under normal conditions.
Responses to stimulation of coronary and carotid baroreceptors and the coronary chemoreflex at different ventricular distending pressures in anaesthetized dogs. Experimental Physiology 2001;86:381-90.
This study was done because most previous work involving the effects of ventricular mechanoreceptors was flawed due to inadequate localization of stimuli to specific reflexogenic areas (authors' words). Manipulations included mechanical ventilation, cardiopulmonary bypass, aortic valve balloon obstruction, and cannulation of the LV apex, carotid and coronary arteries, and aorta. Results showed that ventricular mechanoreceptor stimulation, either alone or in conjunction with changes in other reflexogenic areas, has minimal effect on reflex responses.
Influence of dietary salt intake on the response of isolated perfused mesenteric veins of the dog to vasoactive agents. American Journal of Hypertension 2003;16:6-10.
This study was designed to examine the influence of dietary salt intake upon the vasoconstrictor response of capacitance vessels in the dog. Lengths of mesenteric vein were studied ex vivo, after feeding dogs low, intermediate and high salt diets. Vascular responses to cumulative doses of norepinephrine and acetylcholine were determined. Results demonstrated greater vasoconstrictive response to both drugs in dogs fed high salt diets, and it was suggested that this may explain the hypertensive effects of dietary salt in humans.
Phasic negative intrathoracic pressures enhance the vascular responses to stimulation of pulmonary arterial baroreceptors in closed-chest anaesthetized dogs. Journal of Physiology 2004;555(part 3):815-24.
This study came from the study (#21) which showed greater responses to pulmonary baroreceptor reflexes in the presence of negative intrathoracic pressure, suggesting that a closed-chest model may be more accurate. Manipulations included mechanical ventilation, cardiopulmonary bypass, isolation of pulmonary perfusion, and cannulation of the carotid and coronary arteries, aortic arch and abdominal aorta. Using apparently arbitrary definitions of threshold pressures, set points and rates of change, the effects of phasic intrathoracic pressures upon the stimulus-response curve of PA baroreceptors was examined. Results showed displacement of the stimulus-response curve to lower pressures during phasic (presumably physiological) intrathoracic pressures.
Pulmonary arterial distension and vagal afferent nerve activity in anaesthetized dogs. Journal of Physiology 2004;555(part 3):805-14.
This study was a variation of the above study (# 27), designed to examine afferent activity from pulmonary baroreceptors under conditions of simulated physiological changes in intrathoracic pressure. Manipulations included mechanical ventilation, cardiopulmonary bypass, isolation of pulmonary perfusion, pulsatile pulmonary perfusion to simulate physiological blood flow, monitoring of vagus nerve impulses, and use of open and closed-chest measurements. Results showed that pulmonary baroreceptor activity was triggered at lower perfusion pressures in closed-chest dogs with phasic negative intrathoracic pressure.
Critique of the research work
By way of overview, this team's research involves a single area of physiological expertise and a single animal preparation. It has successfully mined those attributes to carry out largely repetitive and unproductive animal studies, using their own and others' previous findings (often incorrect, as noted by the team in the justifications for some studies) to carry on with minor variations upon very few central themes. By doing so, they have published scientific articles for over 16 years, without apparent correlation with, or influence upon, similar areas of human physiology or medicine. This body of work amounts, in my view, to a startling example of the pursuit of disconnected scientific knowledge with no clear human benefits, and to the detriment of dogs.
Twenty-seven of 28 published papers reported studies in anaesthetized dogs, and other than variations in the extent of instrumentation and manipulation, this was a single animal preparation. I am not aware of other such long basic science research projects using such a narrow focus.
The team mined the research area related to atrial and ventricular receptors and responses for several years, then switched to evaluation of vascular baroreceptors for several more years, then switched to evaluation of pulmonary baroreceptors for the most recent several years. The nature of their research was very similar in all three closely related areas, enabling them to do the same basic research with small variations for a very long time, and without conspicuous human benefit.
Very evident in this collection of papers is the characteristic use of one study to justify the next. In many cases, unanswered (usually unforeseen) questions arising from one study produced the rationale for a later study In several instances, the team invoke conflicting or erroneous results from previous studies (sometimes their own) to justify another study.
In many of the reports, the team draw conclusions from study results in the absence of any apparent study hypothesis. This suggests that they may have developed their hypotheses from the results of these studies.
In many cases, the team do not suggest how study results may relate to human physiology or disease.
In several studies, the team report variable physiological responses among animals subjected to the same experimental conditions, or appear to select animals with specific responses for data analysis. Are they excluding animals who don't produce predetermined responses, or who have uninterpretable results? In either case, this likely invalidates the study results and conclusions.
I do not see evidence that the team correlate their study results with similar investigations in humans. Even when they suggest how specific findings may be important for humans (such as with gravitational changes, blood volume regulation or salt loading), the team do not correlate with relevant findings from human studies or provide data to suggest similar mechanisms.
Virtually all of the team's studies could be classified as irrelevant to human disease and medicine, even if they weren't biologically and mechanistically unsupportable. This is because the neural and biomechanical mechanisms they examined in dogs have been known for many years in humans.
There is an extreme degree of manipulation of the team's dog model to create non-physiological conditions of convenience favoring their investigations. Their basic dog model is anaesthetized, mechanically ventilated, extensively cannulated, and rendered useless by artificial isolation of the circulations to be studied. Aside from the well-documented effects of such stresses upon research animals' physiological and biochemical responses, such manipulations completely remove the animal model from the influences of an intact and interactive biological system. When a major argument for such animal research is the need for `an intact biological system', the extensive modifications of anatomy physiology, perfusion, respiration, intrathoracic pressure, sympathetic innervation and many other characteristics of that intact system make these study results non-physiological and lacking relevance... even for dogs, let alone humans. Just one example is the team's own recognition that the open-chest dog model used in many of their studies produces different baroreceptor responses than a closed-chest model, and is thus not valid.
The extent of manipulation of this dog model is truly ingenious and technically challenging. Such manipulations include not only all those mentioned above (#10), but also variable and non-physiological patterns of perfusion to isolated vascular territories, artificial and contrived patterns of vascular drainage to maintain pressures within a vascular territory, the use of external reservoirs to recirculate blood, cutting of the spinal cord and various nerves (vagus, renal, phrenic, splanchnic and peripheral), simulated pulsatile flow and respiratory intrathoracic pressure changes, ex vivo measurements of vascular responses, balloon occlusions of pulmonary veins and LV outflow tract, LV apical cannulation, blockade of reflexogenic mechanisms which may influence those being studied, and sustained ventricular fibrillation to eliminate influence on coronary artery baroreceptors.
In one study (#13) designed to evaluate various baroreceptor responses, the animal model was a VF dog maintained on life support for the purpose of the study. Even under these conditions, data analysis was limited to dogs with specific desired responses. Under what circumstances could this animal model, or any of the extensively compromised animals used in these studies, produce results worthy of consideration? How can we imagine there may be some association with human responses, when the responses are not even applicable to the species being studied?
This work provides an exceptional example of a common practice: the manipulation of animal models for convenience and usefulness, regardless of the effects upon the validity of results obtained. This is not uncommon among those researchers who propose and perform studies to satisfy their scientific curiosity and sustain their careers, without sufficient regard for potential applications to humans.