Sympathetic blockade significantly improves cardiovascular alterations immediately after spinal cord injury in rats

Sympathetic blockade significantly improves cardiovascular alterations immediately after spinal cord injury in rats

Neuroscience Letters 319 (2002) 95–98 www.elsevier.com/locate/neulet Sympathetic blockade significantly improves cardiovascular alterations immediatel...

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Neuroscience Letters 319 (2002) 95–98 www.elsevier.com/locate/neulet

Sympathetic blockade significantly improves cardiovascular alterations immediately after spinal cord injury in rats G. Bravo a,*, E. Hong a, G. Rojas a, G. Guı´zar-Sahagu´n b a

Departamento Farmacobiologı´a, CINVESTAV, IPN, Calzada de los Tenorios 235, Col. Granjas Coapa, 14330 Mexico D.F., Mexico b IMSS-Proyecto Camina A.C., Mexico D.F., Mexico Received 1 November 2001; accepted 10 December 2001

Abstract Immediately after an experimental spinal cord injury (SCI) in rats, there is a large fall in mean arterial pressure (MAP) and heart rate (HR), followed by an abrupt increase in MAP. To better understand the mechanism involved in these early cardiovascular alterations, we tested the effect of treatment with ganglionic and sympathetic blockers in anesthetized rats subjected to T-5 SCI. Fall in MAP was partially diminished by propranolol and pentolinium, while increase in MAP was abolished by propranolol and pentolinium. Adrenalectomy did not diminish the fall in MAP and HR, however, the increase in MAP was significantly reduced. Likewise, propranolol and pentolinium completely abolished the effects in HR. These data suggest that the early cardiovascular alterations secondary to SCI results from an increased parasympathetic activity and a sympathetic withdrawal. q 2002 Published by Elsevier Science Ireland Ltd. Keywords: Spinal cord injury; Cardiovascular effects; Pentolinium; Propranolol; Adrenalectomy

Spinal cord contusion in the rat provides an animal model to study pathophysiological mechanisms following traumatic injury, as well as a tool for screening therapeutic agents of possible benefit to patients with spinal cord injury (SCI). SCI has been of interest because severe damage not only results in paraplegia or tetraplegia, but also in systemic and metabolic alterations secondary to autonomic dysfunction. The autonomic nervous system plays an important role in the regulation of cardiac function. Increased sympathetic activity causes acceleration in heart rate (HR) and atrioventricular conduction and, conversely, increased parasympathetic activity causes slowing of HR and atrioventricular conduction. Injuries above midthoracic segments disrupt both tonic and reflex sympathetic control of mean arterial pressure (MAP). Cardiovascular control is abnormal and unstable after SCI. Hypotension occurring immediately after injury has been attributed to the acute autonomic imbalance due to predominance of parasympathetic (vagus) activity and loss of sympathetic tone [1,4]. The studies presented here were designed to evaluate different drugs with regard to their cardiovascular actions after SCI. The present study was designed to evaluate the effect of pretreatment with ganglion and b blockers on cardiovascu* Corresponding author. Fax: 152-5-4832863. E-mail address: [email protected] (G. Bravo).

lar alterations secondary to SCI, in order to better understand the role of the autonomous nervous system on the changes in MAP and HR occurring immediately after an SCI. Animals: Male Wistar rats (body weight 150–180 g) were anesthetized with a mixture (i.m.) of ketamine (75 mg/kg) and xylazine hydrochloride (12.5 mg/kg). Animals were manipulated according to the ethical principles of our institutions and National Institute of Health (NIH) standards. The left femoral artery was cannulated and MAP recorded by a pressure transducer (P23ID, Gould Statham) linked to a Grass polygraph. HR was derived electronically from the upstroke of the arterial pulse pressure by a Grass tachograph. The left femoral vein was also cannulated for drug administration. MAP and HR were allowed to stabilize for 30 min before performing a laminectomy and spinal cord exposure. The body temperature of the animals was kept constant at 378C. After the animals had been in a stable hemodynamic condition for at least 15 min, a laminectomy was performed at T-5 level. The rats were then placed in a stereotaxic device to perform the spinal cord contusion as previously described [13]. Briefly, a stainless steel cylinder weighing 15 g and with a blunt tip of 2 mm diameter was dropped from a height of 14 cm through a guide tube onto the exposed dura, producing a severe injury with an inten-

0304-3940/02/$ - see front matter q 2002 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 1) 02 55 7- 5

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sity of 210 g/cm. Using this supra threshold intensity of lesioning, total and permanent paraplegia follows [8]. Groups of six rats each were used. Group 1 served as control (sham-lesion), being submitted only to laminectomy without injury. Groups 2–5 were submitted to SCI at the T5–T6 level. Group 2 received vehicle (i.v. bolus 0.3 ml of saline) 30 min before injury. Group 3 received an i.v. bolus of the ganglionic blocking agent, pentolinium 3 mg/ kg in a volume of 0.3 ml, 30 min before injury. Group 4 received an i.v. bolus of the b-adrenoceptor blocker propranolol, 1.5 mg/kg in a volume of 0.3 ml, 30 min before injury. Group 5 was adrenalectomized and treated as group 2. Adrenal medullectomy was performed 72 h prior to experiments in rats under ether anesthesia. The two adrenal glands were exposed through flank incisions and the medullas were extruded with gentle forceps pressure on the adrenal after slitting the capsule. The skin was sutured and rats were allowed to recover from anesthesia. Three days later, the rats were anesthetized again before SCI. For all groups, SCI was performed under stable hemodynamic conditions. MAP and HR were continuously monitored for 30 min immediately before the lesion and 60 min after it. Finally, rats were sacrificed using an overdose of anesthesia. Propranolol and pentolinium (Sigma Chemical, St. Louis, MO, USA) were used dissolved in sterile 0.9% saline. MAP and HR values were continuously recorded in all rats. Within each experimental group, maximal and minimal values of the control group (only with injury) were compared with all other experimental groups by analysis of variance followed by the Dunnett’s t-test. Differences were considered to be statistically significant when P , 0:05. Sham-injured animals exhibited constant values of MAP and HR over the 60 min observation period. Basal MAP and HR values were similar for all experimental groups (Table 1). In animals submitted to SCI (group 2), there was an

immediate fall of blood pressure accompanied by a significant decrease in HR. MAP was reduced by 78%, an effect that lasted up to 3 min after injury. MAP then rapidly recovered and exceeded basal values, in an overshoot fashion; it showed an increase of 67% over basal levels. Blood pressure then decreased gradually, to attain values similar to basal conditions in approximately 25–30 min after injury. Unlike blood pressure, HR did not show a biphasic behavior. HR decreased by 63% within 3 min after SCI, and then recovered rapidly during the next 2–3 min, but did not exhibit an overshoot, after the 6th min following SCI, HR recovered partially (Fig. 1a). Pretreatment with pentolinium (Fig. 1b) and propranolol (Fig. 1c) attenuated both, the fall and the overshoot in blood pressure; minimal and maximal MAP were significantly different from those observed in non-treated injured animals, while HR reduction was prevented. The pressor effect in MAP was prevented by adrenalectomy, however, the fall in MAP was not modified. The decrement in HR was not modified by this procedure (Fig. 1d). Our data show that in the acute phase after SCI in the rat, important changes in MAP and HR were observed during 20 min after injury, and after this time, both MAP and HR returned to basal values. Cardiovascular manifestations after SCI include bradycardia and hypotension followed by a transitory pressor effect. Hypotension occurring immediately after experimental SCI has been attributed to the loss of tonic supraspinal excitatory drive to spinal sympathetic neurons [6,14]. However, these phenomena may not be the only mechanisms involved in the earliest hemodynamic alterations after SCI, since a strong cholinergic input leading to NO release and vasodilation seems to account for the hypotension [7]. The frequency of discharge of the sinoatrial node is determined by its intrinsic automaticity as well as by the action of the sympathetic and parasympathetic nervous systems on this automaticity. Thus, control of HR may involve variations in the activities of both divisions of the autonomic nervous system [2], and it is likely that both

Table 1 Effects of SCI on MAP and HR in control conditions and after different treatments a Basal values

Before SCI

SCI Min

Max

MAP (mmHg) Sham Injury Pentolinium (3 mg/kg) Propranolol (1.5 mg/kg) Adrenalectomy

94 ^ 8 85 ^ 2 77 ^ 6 85 ^ 3 76 ^ 1

89 ^ 4 79 ^ 3 77 ^ 5 77 ^ 2 76 ^ 1

85 ^ 3 14 ^ 2 52 ^ 3* 53 ^ 4* 30 ^ 4*

87 ^ 3 131 ^ 5 88 ^ 5* 93 ^ 11* 93 ^ 6*

HR (beats/min) Sham Injury Pentolinium (3 mg/kg) Propranolol (1.5 mg/kg) Adrenalectomy

229 ^ 11 229 ^ 11 186 ^ 9 225 ^ 10 227 ^ 10

238 ^ 14 266 ^ 21 195 ^ 11 189 ^ 10 227 ^ 10

230 ^ 9 121 ^ 16 168 ^ 19 183 ^ 19* 131 ^ 12

234 ^ 14 216 ^ 7 201 ^ 16 193 ^ 8 211 ^ 14

a

Injury vs. treatment: *P , 0:05.

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Fig. 1. Effects of SCI on MAP and on HR in anesthetized rats. It shows after (a) sham ( ), injury (W); (b) pentolinium ( ) 3 mg/kg; (c) propranolol (X) 1.5 mg/kg; and (d) adrenalectomy (7). Each point represents the mean ^ s.e.m. of six rats. *P , 0:05 compared with injury rats without treatments.

sympathetic and parasympathetic may influence HR simultaneously [10]. The interaction between these two opposing nervous influences on HR is of considerable importance but has not been studied in great depth. In this work, pharmacological and surgical approaches were carried out to better understand the mechanisms involved in the very early cardiovascular changes secondary to SCI and associated with spinal shock. The cardiovascular changes (MAP and HR) were studied in response to three distinct conditions: (1) badrenoceptor blocker; (2) ganglionic blockade; and (3) adrenalectomy. The autonomic influences in the rat heart are of such nature that there is a stronger b-adrenergic than muscarinic predominance. Therefore, after SCI there are both a decrease of sympathetic discharge and an increase of parasympathetic stimulation [4]. These effects result in an initial decrease in MAP followed by an increment in MAP of larger duration (about 20 min) probably of reflex nature. Propranolol decreases HR in 27 beat/min, but has no effect on MAP; it diminished both, the fall and the increase of MAP, and blocked completely the decrement in HR induced by SCI. A plausible explanation for the smaller MAP decrease after SCI is that the sympathetic influence on the heart was already diminished by the blockade of b receptors, therefore when SCI interrupted the sympathetic influence on the heart, the decrement of blood pressure was lesser because the heart contractility was already diminished. The smaller pressor effect following the initial and

transient fall in MAP is probably related to the smaller fall in MAP, which produces a minimal reflex pressor effect. In the case of pentolinium, a ganglionic blocking agent affecting both sympathetic and parasympathetic pathways, its effects on MAP and HR were surprisingly smaller, however, it has been described that ketamine–xylazine anesthesia diminished the efferent sympathetic discharge [17], which may result in lower decreases of MAP [21] and HR [15] such as recorded in the present experiments. Therefore, the weak effect of pentolinium on MAP and HR and the modest effect of propranolol on HR could be explained by the decreased efferent sympathetic discharges produced by the anesthesia. Thus, when pentolinium was given, MAP and HR values were already low and consequently drug effects were smaller than those usually observed with other anesthesia. The reason for using ketamine–xylazine in the present study can be attributed to the fact that our group has carried out several multidisciplinary studies searching for the consequences of SCI using successfully the same anesthesia [3,4,11,12,19]. Pentolinium decreased the effects of SCI on blood pressure and produced an almost complete abolition of the bradycardic effect. The blockade of the pressor effect following SCI observed after pentolinium clearly indicates the importance of the sympathetic nervous system in such reflex pressor effect. Removal of the adrenal glands attenuated (22%) the pressor response to SCI. Thus, it appears that adrenal

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releases catecholamines in the circulation, produces vasoconstriction and stimulates the myocardium resulting in an elevation of MAP induced by SCI. The pressor effect was eliminated totally in the animals with adrenalectomy, but the fall in MAP or the fall in HR were not different from controls. The other important second messenger involved in mediating the actions of neurotransmitter in the cardiovascular and central nervous systems is NO [5,7,18]. We have previously shown the activity of nitric oxide synthase (NOS) using NADPH diaphorase histochemistry in sections of atria after SCI, and it seems that a transitory increase in NOS activity, apparently provoked by an excessive vagal stimulation, is produced in the endothelium and media of atrial intramural arteries in this rat model of acute SCI [3]. We suggest that our data may be similar to very early hemodynamical human alterations, however, we cannot probe it in view of missing data obtained in humans. Due to the nature of the different accidents in humans, it has not been possible to measure cardiovascular parameters immediately after the accident, however, bradycardia has been recorded in humans after variable intervals of the time [16]. Several cardiovascular disturbances had been reported in different species including dogs and primates [9,20] after SCI in the early phase and the results described are similar to those found in this work. The use of the present experimental model could become a useful tool to understand the pathophysiology of the hypotension that follows SCI and could lead to a more rational treatment in very early changes after injuries. The authors acknowledge A. Franco for the photographic material and H. Va´ zquez for the assistance in bibliography. This work was partially supported by Proyecto Camina, A.C. [1] Atkinson, P.P. and Atkinson, J.L., Spinal shock, Mayo Clin. Proc., 71(4) (1996) 384–389. [2] Berkowitz, W.D., Scherlag, B.J., Stein, E. and Damato, A.N., Relative roles of sympathetic and parasympathetic nervous systems in the carotid sinus reflex in dogs, Circ. Res., 24 (1969) 447. [3] Bravo, G., Larios, F., Rojas-Martı´nez, R., Hong, E., Salazar, L.A. and Guı´zar-Sahagu´ n, G., Early changes in nitric oxide synthase activity in atrial intramural arteries following experimental spinal cord injury in rats, Neurosci. Lett., 271 (1999) 37–40. [4] Bravo, G., Rojas-Martı´nez, R., Larios, F., Hong, E., Castan˜ eda-Herna´ ndez, G., Rojas, G. and Guı´zar-Sahagu´ n, G., Mechanisms involved in the cardiovascular alterations immediately after spinal cord injury, Life Sci., 68 (13) (2001) 1527–1534. [5] Bredt, D.S., Glatt, C.H.E., Hwang, P.M., Fotuhi, M., Dawson, T.M. and Snyder, S.H., Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of

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