(Hypertension. 1997;30:632.)
© 1997 American Heart Association, Inc.
Articles |
Heart Rate Variability and Baroreceptor Function in Chronic Diabetic Rats
From the Departments of Physiology (M.F.D.M., H.C.S.) and Pharmacology (G.B., M.C.O.S.), School of Medicine of Ribeirão Preto, University of São Paulo; and Biological Sciences, School of Medicine of Triângulo Mineiro, Uberaba, (R.F.), Brazil.
Correspondence to Dr Helio C. Salgado, Department of Physiology, School of Medicine of Ribeirão Preto (USP), 14049-900 Ribeirão Preto, SP, Brazil. E-mail hcsalgad{at}fmrp.usp.br
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Abstract |
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Abstract In conscious chronic (12 to 18 weeks) streptozotocin diabetic rats, we examined the changes in basal heart rate, with particular attention to heart rate variability assessed by evaluating the standard deviation (bpm) of the lengths of adjacent pulse pressure. We also investigated in anesthetized rats the ability of the aortic baroreceptors to acutely (30 minutes) reset to hypertensive levels. For this purpose, pressure-nerve activity curves for the baroreceptors were obtained, and gain (slope of the curve) and mean arterial pressure at 50% of maximal baroreceptor activity were calculated. The shift of the pressure-nerve activity curve was used as an index of resetting. Conscious diabetic rats (n=6) exhibited lower mean arterial pressure (93±6 versus 109±4 mm Hg), heart rate (272±25 versus 359±11 bpm), and heart rate variability (18±7 versus 36±6 bpm) than control rats (n=7). Under anesthesia, diabetic rats (n=7) and control rats (n=8) exhibited similar mean arterial pressure (113±6 versus 109±7 mm Hg in control rats ), mean arterial pressure at 50% of maximal baroreceptor activity (117±5 versus 107±6 bpm), gain (1.66±0.08 versus 1.81±0.05%/mm Hg), and extent of resetting (44±12 versus 49±9%) to hypertensive levels. The present study demonstrated that conscious chronic diabetic rats presented lower heart rate variability than control rats. On the other hand, chronic diabetes was not associated with alterations in baroreceptor function or its ability to rapidly reset to hypertensive levels.
Key Words: diabetes • streptozotocin • baroreceptor resetting • baroreflex • heart rate variability
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Introduction |
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Patients with diabetes mellitus often have autonomic dysfunction, and their reduced ability to regulate heart rate (HR) is attributed to an impairment of cardiac and/or central parasympathetic function. The diabetic injury to the parasympathetic nervous system may reduce heart rate variability (HRV), which is predominantly determined by parasympathetic control and is a valuable index of cardiac parasympathetic nerve functional integrity.1 2 3 4 Although extensive clinical investigations on cardiac autonomic neuropathy in diabetic patients have been performed, scanty information is available on whether chronic experimental diabetes is associated with cardiac parasympathetic nerve abnormalities. Thus, the first goal of the present study was to examine HRV in conscious chronic diabetic rats.
In addition to causing autonomic neuropathy, chronic diabetes is also associated with vascular alterations such as atherosclerosis,5 6 which decreases blood vessel distensibility and might impair baroreceptor function.7 8 9 Baroreceptor function has been studied in single fibers in an isolated aortic arch–aortic nerve preparation from streptozotocin-diabetic rats10 and in a multifiber preparation of the aortic nerve of chronic alloxan-diabetic rabbits,11 and it has been shown to be unaltered. The ability of the baroreceptors to reset rapidly during acute changes in mean arterial pressure (MAP) has not hitherto been investigated in chronic diabetic animals. Therefore, the second goal of the present study was to examine in chronic streptozotocin-diabetic rats, by means of electroneurographic recordings of the whole aortic nerve, baroreceptor function and its ability to rapidly reset to a prompt and sustained (30 minutes) rise in arterial pressure.
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Methods |
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The experiments were performed on male Wistar rats (250 to 300 g). Diabetes was induced by a single intravenous injection of streptozotocin (50 mg/kg, Sigma) into the penile vein of the animals after an overnight fast. The diabetic state was confirmed by high plasma glucose levels, excessive daily water intake, and a large increase in urinary volume. Nonfasting blood glucose (mmol/L) was determined with a glucose analyzer (Beckman Instruments, Inc) at the time of the experiment, 12 to 18 weeks after streptozotocin. Control rats received vehicle (0.01 mol/L citrate buffer, pH 4.5) injected into the penile vein and were submitted to the same protocol as the diabetic rats.
Basal Hemodynamics of Conscious Rats
Twenty-four hours before the experiment, the rats (control n=7;
streptozotocin n=6) were anesthetized with sodium pentobarbital
(40 mg/kg IP) and a catheter was inserted into the femoral
artery for arterial pressure recording. On the day
of the experiment, the rats were left in the experimental room at least
1 hour before the beginning of the experiment. The arterial
catheter was then connected to a pressure transducer (Hewlett-Packard,
model 21080A) and the signal was amplified and digitally recorded
by means of an analog to digital interface (Lynx Tecnologia
Eletrônica) and recorded (1 kHz) for 30 minutes on a
microcomputer (IBM/PC-AT 486) for later analysis. During the
experiment, absolute silence was maintained inside the room to avoid
any interference with MAP or HR. The variables were
analyzed using software developed in our laboratory to detect
beat to beat systolic/diastolic
arterial pressure and HR. HRV was assessed by evaluating
the standard deviation (SD) of the lengths of adjacent pulse
pressures.12 The high sample frequency used in these
experiments to record the pulsatile arterial pressure
permitted a highly accurate determination of the
parameters.
Baroreceptor Recording in Anesthetized Rats
On the day of the experiment, the animals (control n=8;
streptozotocin n=7) were anesthetized with sodium pentobarbital
(40 mg/kg IP) and catheters were inserted into the right carotid
artery for measurement of arterial pressure and into the
right femoral artery for withdrawal and reinfusion of blood. A
pneumatic cuff was placed around the abdominal aorta immediately below
the diaphragm in order to sustain the rapid increase in MAP for
baroreceptor resetting. The left aortic nerve was identified in the
neck, carefully isolated from connective tissue, and placed on a
bipolar stainless steel pair of electrodes. The nerve activity was
amplified using a differential high impedance preamplifier
(Princeton Applied Research, model 113). The signal was passed
through a customized band-pass active filter (gain 10 x, lower
cutoff 100 Hz, higher cutoff 3 kHz). Nerve activity was displayed on an
oscilloscope (model 5113, Tektronics, Inc) and monitored with a
loudspeaker. Action potentials that exceeded the background noise level
were counted using a nerve traffic analyzer (model 605C,
University of Iowa Bioengineering). The output from the spike counter
was in spikes per second (rate mode) with a time constant of 200 ms.
This activity was digitally recorded with an analog to digital
interface and recorded (100 Hz) on a personal computer
simultaneously with the pulsatile carotid pressure. To
assess baroreceptor firing range, the rats were submitted to rapid
changes (20 to 30 seconds) in arterial pressure by
withdrawal and reinfusion of blood into the femoral artery. To avoid
the influence of hysteresis, only the values obtained during reinfusion
of blood (a rate of about 6 to 7 mm Hg/s) were used. Three
pressure-nerve activity curves were obtained for each animal during the
withdrawal and reinfusion of blood. The animals were then submitted to
a prompt and sustained (30 minutes) increase in MAP by means of aortic
constriction caused by the pneumatic cuff. At the end of this period,
three other pressure-nerve activity curves were determined. The
systolic pressure threshold (SPth) for baroreceptor
activation and the pressure-nerve activity curve were determined.
Aortic nerve activity (spikes/s) was transformed to relative units (%
of maximal activity) and plotted against MAP (mm Hg) to obtain
pressure-nerve activity curves. Using nonlinear regression based on the
Levemberg-Marquadt algorithm,13 the average gain of the
pressure-nerve activity curve and the mean arterial
pressure at 50% of maximal nerve activity (MAP50) were
calculated. The ratio between the shift in MAP50 and the
shift in basal MAP after aortic constriction was used as an index of
the extent of baroreceptor resetting.
Statistical Analysis
MAP, HR, SD of HR, baroreceptor gain, MAP50,
SPth, and the extent of baroreceptor resetting to
hypertensive levels were compared between control and diabetic rats by
Student’s unpaired t test. The pressure-nerve activity
curves from the two groups were compared by two-way MANOVA for repeated
measures. Changes were considered significant at P<.05.
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Results |
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All rats given streptozotocin developed severe hyperglycemia (24±1.2 versus 6.5±0.4 mmol/L in control group) associated with a decrease in body weight (231±29 versus 418±10 g in control group). The diabetic rats showed reduced basal MAP (93±6 versus 109±4 mm Hg) and HR (272±25 versus 359±11 bpm). As illustrated in Fig 1, the diabetic rats presented a smaller HRV, ie, reduced SD during 30 minutes of beat to beat recording of pulse pressures. The SD of the diabetic rats was 18±7 bpm obtained from a basal HR measured in 8164±210 pulses, whereas the SD of the control rats was 36±6 bpm obtained from a basal HR measured in 10769±360 pulses.
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Under sodium pentobarbital anesthesia, both groups
exhibited similar MAP (109±7 versus 113±6 mm Hg in diabetic
rats), MAP50 (107±6 versus 117±5 mm Hg in diabetic
rats), SPth (87±4 versus 92±5 mm Hg in diabetic
rats), and gain of the baroreceptors (1.81±0.05 vs
1.66±0.08%/mm Hg). The pressure-nerve activity curves did not differ
between groups before (Fig 2, top) or
after (Fig 2, bottom) a prompt and sustained (30 minutes) hypertensive
challenge (
MAP of 36±3 versus 34±5 mm Hg in diabetic rats)
elicited by aortic constriction. Both groups presented a
similar displacement of the pressure-nerve activity curve to the right
(Fig 2, bottom), indicating a similar extent of partial baroreceptor
resetting (49±9% versus 44±12% in diabetic rats) following the rise
in pressure.
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) rats. Variability markers design
SEM. |
Discussion |
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The high levels of blood glucose confirmed the efficacy of streptozotocin in producing an experimental chronic (12 to 18 weeks) diabetes in rats. To our knowledge, this is the first report in the literature showing a decrease in HRV in rats with chronic experimental diabetes. The lower HRV is an indication of cardiac parasympathetic nerve function impairment and is consistent with clinical observations showing decreased HRV in long-term diabetic patients exhibiting autonomic neuropathy.14 15 16 Studies comparing cardiac autonomic function tests and HRV indices (based on 5-minute or 24-hour electrocardiographic recordings) demonstrated that HRV is also decreased in patients without abnormal function tests,14 17 indicating that cardiac parasympathetic nerves are affected early in the development of autonomic neuropathy associated with chronic diabetes. The relative bradycardia observed in our chronic diabetic rats could be related to a decrease in cardiac ß-adrenergic receptors18 or to cardiac sympathetic nerve impairment, since it has been shown that a sympathetic nerve dysfunction as severe as the parasympathetic one can occur in chronic diabetic patients.1 19
Experimental20 21 22 and clinical studies23 24 25 have revealed an impairment of reflex regulation of HR in chronic diabetes. Data from alloxan-induced diabetic rabbits indicated that baroreflex-mediated bradycardia is impaired, whereas reflex tachycardia is preserved.22 In addition, the same authors suggested that the impairment was caused by a defect in the activation of central parasympathetic pathways.11
The present study demonstrated that chronic diabetic rats presented a pressure-nerve activity curve identical to that of control rats. The lack of changes in gain of the curves, MAP50, and in SPth is consistent with previous studies of baroreceptor function in chronic streptozotocin diabetic rats10 and alloxan-diabetic rabbits.11 Therefore, despite that clinical studies have evidenced morphological alterations in the carotid sinus nerve of diabetic patients,26 the present findings demonstrate that baroreceptor activity is preserved in chronic diabetic rats exhibiting signs of cardiac parasympathetic neuropathy as reflected by the reduced HRV.
It is well known that sustained changes in pressure, for minutes or hours, lead the baroreceptors to reset rapidly following the new conditioning pressure.27 28 Under these circumstances, the pressure-nerve activity curve shifts in the direction of pressure change to operate at a new set point on the steep portion of the reset curve in order to allow the baroreceptors to buffer fluctuations of arterial pressure in a more effective manner.9 Although baroreceptor function was found to be unchanged in chronic diabetes (present study10 11 ), it is well known that this pathological condition commonly causes vascular diseases such as atherosclerosis.5 6 Accordingly, changes in vascular distensibility that occur in pathological states might influence the extent of resetting in response to a sustained rise or fall in conditioning pressure.7 9 In the present study, we demonstrated that the aortic baroreceptors of diabetic rats exhibited the same extent of resetting to hypertensive levels as the control rats when submitted to an acute and sustained (30 minutes) rise in MAP. Although we did not examine possible morphologic changes caused by chronic streptozotocin diabetes, vascular changes that may have occurred in this experimental model did not affect the extent of resetting.
In conclusion, the present study demonstrated that chronic streptozotocin-diabetic rats presented lower HRV, indicating a functional cardiac parasympathetic neuropathy. On the other hand, chronic diabetes was not associated with alterations in baroreceptor function or in its ability to rapidly (30 minutes) reset to hypertensive levels.
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Acknowledgments |
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This research was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, grant 1995/4685-8), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), CAPES (Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior), and FINEP (Financiadora de Estudos e Projetos, grant 357/96-PRONEX). The authors gratefully acknowledge the excellent technical assistance of Jaci A. Castania and Mauro de Oliveira.
Received March 15, 1997; first decision April 17, 1997; accepted May 7, 1997.
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