Hemodynamic Alterations in Hypertensive Obese Rabbits

Animal Preparation

The experimental protocols for this study were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center and were carried out according to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and the guidelines of the Animal Welfare Act. Sixteen female New Zealand White rabbits were used in this experiment. Before the experimental day rabbits were housed in cages in the animal care facility in a humidity- and temperature-controlled room with a 12-hour light/dark cycle. After at least 2 weeks of acclimation they were randomly assigned to one of two groups: (1) a group that received a maintenance diet²¹ of 100 to 120 g/d standard rabbit chow (Laboratory Rabbit Chow 5321, Purina Mills) or (2) a group that was fed ad libitum with standard rabbit chow to which 15% extra fat (corn oil and animal fat mixture) was added.

Experiments were conducted after an 8- to 12-week period of dietary manipulation. General anesthesia was induced in rabbits with 4% to 5% isoflurane with an oxygen flow of 1 L/min and was administered with a face mask. Anesthesia was maintained with 2% to 3% isoflurane. The right femoral artery was cannulated, and a catheter (PE-90) was advanced into the abdominal aorta for measurement of arterial blood pressure and withdrawal of a reference blood sample during the microsphere procedure. The right carotid artery was isolated and another catheter (PE-90) was advanced into the left ventricle for microsphere injection. The placement of this catheter tip in the ventricle was verified by observation of the ventricular pulse pressure. Bupivacaine (0.25%) was administered locally around both incision sites so that there would be no discomfort from the surgical incisions during the remainder of the experiment.

Protocol

Rabbits were allowed to recover from the anesthesia for 1 to 1.5 hours while positioned in a Lucite rabbit restrainer (Plas Labs). During this time the femoral arterial catheter was connected to a pressure transducer to allow continuous recording of arterial pressure and heart rate. Signals from the transducer were sent to an analog-to-digital convertor and analyzed with a personal computer and customized software that has already been tested and implemented in this laboratory.²² Heart rate and pressures were recorded for 30 minutes after the recovery period; the averaged values over the 30 minutes were used for statistical analyses.

After blood pressure measurements cardiac output was determined with the use of radioactive microspheres (⁴⁶Sc, 15±3 μm diameter, New England Nuclear) as described by Stanek et al.²³ Briefly, dry microspheres were suspended in a 1.3–specific gravity dextrose solution with one drop of 0.05% Tween 80, and a precoiled 70 cm length of PE-90 tubing was filled with 0.4 mL of the microsphere suspension. The microsphere injection coil was then interposed between the left ventricular catheter and an infusion pump (Gilford 105-S), and the femoral catheter was connected to a withdrawal pump. Reference sampling of arterial blood (2 mL/min) for cardiac output determination was started 10 seconds before the start of the microsphere infusion and ended 55 to 60 seconds after the infusion had been completed; sampling time for the withdrawal of the 3 mL of reference blood was recorded. Approximately 2×10⁶ microspheres were injected over a period of approximately 20 seconds; the injection was followed by flushing of the infusion catheter with saline. The residual activity in the infusion coils and connectors was determined and subtracted from the preinjection activity for determination of the amount of activity injected.

After injection of the microspheres the rabbit was euthanized with an overdose of pentobarbital sodium. The heart ventricles, kidneys, lungs, diaphragm, liver, spleen, stomach, brain, ovaries, adrenals, large and small intestines, and selected fat depot samples were removed, separated immediately from extraneous fat and fibrous tissue, and weighed on a precision balance. Both the tissue samples and reference blood sample were then placed in glass vials and counted on a gamma counter (Searle 1185) for radioactivity determination. Radioactivity for the entire organ was directly determined for the right and left ventricles, kidneys, spleen, ovaries, and adrenals. For the lungs, liver, stomach, brain, and intestines, total organ weights were recorded, and then only a sample of the organ was counted for radioactivity. Total organ blood flows (milliliters per minute) and organ blood flows indexed for organ weight (milliliters per minute per gram) were calculated (see “Calculations” as follows).

Calculations

Cardiac output was calculated as

where

Both absolute (milliliters per minute) and standardized (milliliters per minute per kilogram) cardiac output values were calculated.

For tissue blood flow calculations the number of microspheres in the tissue was calculated as the ratio of the number of microspheres in the sample to the sample weight times the total organ weight. Tissue blood flow per gram of tissue was calculated as the ratio of the number of microspheres in the selected organ to the total number of injected microspheres times total cardiac output and then divided by organ weight. Peripheral resistances were calculated by dividing mean arterial blood pressure (millimeters of mercury) by respective flows (milliliters per minute); resistances normalized for tissue weight were calculated by dividing mean arterial blood pressure by normalized tissue flow (milliliters per minute per gram).

Statistical Analyses

Right- and left-side organ weights and normalized tissue flows were compared in the lungs, kidneys, adrenals, and ovaries using paired t tests. Since there were no significant differences in the variables measured (P>.05), right and left sides were combined in further calculations and analyses. Analyses of group differences between obese and lean rabbits were then performed using unpaired t tests. All data are expressed as mean±SEM; statistical significance was accepted at a value of P≤.05.

Body Weight, Resting Heart Rate, and Arterial Pressures

Before the dietary manipulation period there was no significant difference between groups in body weight (P>.05). However, 8 to 12 weeks of a high-fat diet produced obesity and hypertension in rabbits. Body weight was higher by 43% (P≤.05) and mean arterial pressure higher by 19% (P≤.05) in the obese rabbits (Table 1⇓). Elevations in both systolic and diastolic pressures contributed to the rise in mean pressure. Resting heart rate was also higher by 23% (P≤.05). Blood pressure values recorded for both lean and obese rabbits in the present experiment were slightly higher than those recorded previously in this laboratory,²⁴ probably because of the occlusion of the right carotid artery for placement of the ventricular catheter.

Cardiac Output

Obesity-associated hypertension in the rabbits was accompanied by a 37% increase in absolute resting cardiac output (812±59 and 593±47 mL/min, respectively; P≤.05). When indexed for body weight, however, cardiac output did not differ between groups, averaging 156±11 mL/min per kilogram for the obese rabbits and 162±13 mL/min per kilogram for the lean rabbits. The increase in pressure in the obese rabbits was proportionally less than the increase in cardiac output, so that total peripheral resistance was 18% lower for the obese rabbits (P≤.05, Table 2⇓). Although resistance indexed for body weight tended to be higher (+21%) in the obese rabbits, this failed to reach statistical significance (P=.06, Table 2⇓).

Regional Blood Flow

Increased body weight in the obese rabbits was accompanied by increased weights of many of the body organs (Table 3⇓). Both the right and left ventricles were approximately 30% heavier in the obese rabbits, and kidneys were approximately 24% heavier (P≤.05). Other organs demonstrating significant weight increase in the obese rabbits included the liver (40%), ovaries (40%), adrenals (26%), diaphragm (15%), and spleen (47%) (all P≤.05). The increases in organ weights were accompanied by higher absolute blood flows in some organs. Right and left ventricular flows were higher in the obese rabbits by 40% and 77%, respectively (P≤.05); combined flow to the kidneys was increased by 40% (P≤.05). In addition, absolute flow was higher in the lungs (+124%) and ovaries (+50%) (P≤.05).

After indexing for tissue weight, blood flow to the large intestine was 68% higher in the obese rabbits compared with the lean rabbits (P≤.05) (Table 3⇑). Although normalized blood flow tended to be higher in the obese rabbits in the left ventricle (+35%, P=.06), kidneys (+12%, P=.09), and lungs (+126%, P=.08), these values did not reach statistical significance.

Blood flows in four selected fat depots are listed in Table 4⇓. In all sites sampled the blood flow per gram of fat tissue was significantly lower in the obese than lean rabbits (all P≤.05), with differences ranging from 55% to 73%. Consequently, calculated tissue resistance was increased significantly in three of the four fat depots, with increases ranging from 128% to 276%. Absolute resistances in other tissues (Table 2⇑) did not differ between lean and obese rabbits, except in the left ventricle, kidneys, and large intestine, where resistances were 39%, 14%, and 27% lower, respectively, in the obese rabbits. When indexed for organ weight the differences in the left ventricle and large intestine were no longer significant. Although normalized kidney resistance tended to be higher in the obese rabbits (+12%, P=.09), this value did not reach statistical significance.

Overall and Regional Blood Flows and Resistances

Previous studies have shown that both weight gain and hypertension are independently associated with alterations in overall and regional blood flow. With obesity, in the presence or absence of concomitant hypertension, absolute values of cardiac output are significantly increased both in humans and in animal models^{4 7 8 9 10 11 12 19 25} and are usually associated with an increase in plasma volume. However, in accord with the present findings, most research indicates that cardiac output does not differ between lean and obese individuals when it is indexed for body weight or body surface area.^{4 7 10 11 13 19 25 26} In contrast, in the ventromedial hypothalamic lesioned rat, obesity is not associated with alterations in central hemodynamics despite significant increases in blood pressure.^{27 28} This disparity with the present findings may represent a species difference or may be unique to the ventromedial hypothalamic method of producing obesity. Alternatively, the differences may result from the comparatively smaller increases in body weight and blood pressure produced in these studies.

It has been stated that obese individuals have an increase in cardiac output out of proportion to the increase in fat mass, thus suggesting that blood flow to nonadipose tissue is increased.^{4 13 20} This has been verified in obese rats, in which increases in flow to the kidneys, liver, and testes have been documented,¹⁹ and in obese hypertensive dogs, in which renal blood flow has been shown to increase.¹⁰ A preliminary report also documented increases in flow to the gastrointestinal tract, heart, and brain in the obese dog model.¹⁴ In contrast, the ventromedial hypothalamic rat model demonstrated a decrease in the proportion of flow to the kidney in obesity-associated hypertension.²⁷ The present study demonstrated that nonadipose flow in obesity-associated hypertension was significantly increased in the ventricles, kidneys, and large intestine. Even indexed for the increase in organ size, flow tended to be higher in the left ventricle and kidneys. The increase in flow to the left ventricle may be related in part to the greater metabolic work associated with the higher heart rate in the obese rabbits.

In the present study the increase in heart rate appeared to be responsible for most of the increase in cardiac output because the calculated stroke volume did not differ between lean and obese rabbits (2.7±0.6 and 3.1±0.7 mL, respectively). This is in accord with data from studies of obesity-associated hypertension in dogs,¹⁰ which demonstrated a 57% increase in heart rate concomitantly with a 56% increase in cardiac output. Data from obese adolescents⁸ and adults²⁹ also suggest that elevations in heart rate, rather than in stroke volume, are associated with obesity-related increases in cardiac output. However, the literature is not entirely in agreement on the contributions of heart rate and stroke volume to the increased cardiac output, as there are assertions that the elevated cardiac output in obesity is maintained by an elevated stroke volume and a normal or somewhat low heart rate.^{11 30}

Overall and regional resistances in different forms of hypertension in nonobese animals are generally increased.^{4 5 6 26} On the other hand, normotensive obese individuals usually demonstrate a decrease in overall resistance, as has been demonstrated in studies of obese adolescents⁸ and adults.^{3 30} The effect of obesity-associated hypertension on peripheral resistance varies depending on the predominant abnormality, with some subjects demonstrating decreased overall and regional peripheral resistances^{3 4 14 31} and others demonstrating increased overall resistance.^{3 4 25}

The present study demonstrated that obesity-associated hypertension was associated with an 18% decrease in overall resistance. The magnitude of the decrease in absolute overall resistance is similar to that noted in the dog model of obesity-associated hypertension.^{10 13 32} When resistance is normalized for body weight or body surface area, however, both the present study and a previous study of obesity-associated hypertension in dogs¹⁰ noted a 20% increase in resistance, whereas obese hypertensive humans have shown an increase of 37%.²⁶ However, the data on regional resistances in obesity are not clear. Obesity-associated hypertension in dogs was not associated with a change in regional vascular resistance,¹⁴ whereas in obese adolescents a decrease in forearm vascular resistance has been noted.⁸ On an absolute basis we noted decreases in renal, left ventricular, and intestinal resistances. When resistance values were normalized for organ weight, there was a tendency toward an increase in renal resistance, but this value did not reach statistical significance. Normalized left ventricular and intestinal resistances were approximately 20% lower in obese rabbits, but this was not significant.

Increases in regional flow associated with obesity-associated hypertension might not be related to any hemodynamic abnormality but could be related to increases in organ size. Indeed, many of the increases in absolute organ flow seen in the present study were reduced or eliminated when flow was indexed to account for the increase in organ weight. In contrast to the present study Rocchini¹³ did not find an increase in organ weight in obese compared with lean dogs despite a significant increase in body weight and suggested³³ that weight gain was therefore associated with regional hemodynamic abnormalities. This conclusion may be the result of (1) the short 6-week experimental period, which may be insufficient for producing significant increases in organ weight, (2) the relatively small weight gain reported (2.5 kg), or (3) the large inherent variability among animal subjects in the dog model. Using an 8- to 12-week period of obesity in the rabbit model, we were able to demonstrate increased organ weight in most tissues measured.

Studies of changes in organ weight as a result of obesity are limited. Most studies recognize cardiac hypertrophy as common in obesity^{11 18 30 33} ; this increase in heart weight in obesity was recognized as early as 1928.³⁴ In a later review Alexander³⁰ stated that the increase in heart weight is primarily in the left ventricle and that the kidneys were also somewhat larger and heavier in obese subjects. Our data in obese rabbits confirm organ hypertrophy in both the kidney and left ventricle and expand these observations to suggest increased organ weight in many other body organs as well. We have previously demonstrated in the rabbit that cardiac hypertrophy associated with obesity-associated hypertension is not related to fatty infiltration but is primarily due to hypertrophy,³⁵ similar to results seen in humans³⁶ and rats.³⁷ We have also shown in this model that most of the obesity-associated increases in organ weight in the lungs and gastrocnemius muscle are lean mass.²⁴ Similarly, the increase in weight in the renal parenchyma is almost exclusively lean mass and water; however, there are accumulations of fat in the renal sinus that may contribute to increased renal interstitial pressure and thus to the pathogenesis of obesity-associated hypertension.²⁴ Finally, obesity-associated increases in liver weight are primarily due to increases in lean mass, and although there are significant increases in fat, fat still represents a small proportion of the weight of the organ.²⁴

Adipose Tissue Flow

There have been several reports of decreases in normalized flow^{18 19} and increases in normalized resistance¹⁹ in adipose tissue in obese animals; our results demonstrate a similar adaptation in adipose tissue in obesity-associated hypertension. Early speculations were that although flow to adipose tissue in obesity might be less per unit weight of tissue, the increases in mass of the fat depots and the blood supply to them might become sufficiently large to cause significant increases in resting cardiac output.³⁰ Accordingly, we estimated total flow to the adipose mass in lean and obese rabbits using a mean value of adipose flow measured in the present study (0.125 and 0.045 mL/min per gram, respectively) and total fat mass measured previously in this rabbit model (360 and 1900 g, respectively) using the same dietary protocol, which resulted in similar increases in body weight.²⁴ This calculation assumed that all the fat depots measured contributed equally to the total fat mass and that fat depots not measured had comparable flows; nevertheless, it likely provides a good estimation of the magnitude and direction of difference in total adipose flow in the lean and obese groups. These calculations yielded an approximate total adipose flow in lean and obese rabbits of 45 and 86 mL/min, respectively, representing an estimated 7% and 11%, respectively, of cardiac output. Although the 7% estimate for the lean rabbits in the present study is slightly higher than values measured previously,^{6 15 38} differences in the animals’ sex and/or body weight may account for the variation. Thus, despite the reduced flow per gram in adipose tissue in the obese rabbits, a greater absolute amount of the overall flow is directed to adipose tissue by virtue of the large increase in fat mass; overall resistance to the adipose mass would consequently be reduced. Therefore, although most of the increase in cardiac output in obesity is directed toward lean tissue, adaptations in flow and resistance in adipose tissue of obese animals are also important in the hemodynamics of the development of obesity-associated hypertension.

In summary, our results demonstrate that obesity-associated hypertension in the rabbit is associated with increases in organ weight, an increase in cardiac output, and a decrease in overall peripheral resistance. Alterations in flow and resistance were most striking in the left ventricle, kidney, and adipose tissue. These results indicate that even relatively short periods of obesity are associated with marked overall and regional hemodynamic changes. These results also demonstrate that the rabbit model mimics many of the cardiovascular changes noted in obesity-associated hypertension in humans and other animal models. Thus, the rabbit model may prove useful in the study of nutritional, cardiovascular, and metabolic problems related to obesity-associated hypertension.