Chemical sympathectomy of the cat with 6-hydroxydopamine.

HAEUSLER, G., W. HAEFELY AND H. THOENEN: Chemical sympathectomy of the cat with 6-hydroxydopamine. J. Pharmacol. Exp. Then. 170: 50-61, 1969. Cats were given 6-hydroxydopamine (6-Oll-DA; 2,4,5-trihydnoxyphenethylamine) i.v. according to a schedule which had previously been found to produce a selective and almost complete destruction of peripheral adnenergic nerve terminals. After varying intervals, spinal preparations, as well as the isolated medial smooth muscle of the nictitating membrane and the isolated perfused heart, were used to study the 6-OH-DA-induced alterations of the effects of sympathetic nerve stimulation and of the sensitivity to nonepinephrine. Twelve to 16 days after 6OH-DA, the norepinephnine content of heart, spleen, nictitating membrane and iris was below 10% of the normal value. At this time, the effects of sympathetic nerve stimulation on the nictitating membrane and heart were strongly reduced. Surgical denenvation was somewhat more effective than 6-OH-DA on the nictitating membrane. After both chemical and surgical sympathectomy, the sensitivity to norepinephrine of the isolated nictitating membrane had increased approximately 100-fold; both procedures seem to have induced the presynaptic as well as the postsynaptic types of supersensitivity. Only a 3-fold increase in the sensitivity to norepinephrine was observed in the isolated heart, and evidence was obtained for the absence of the postsynaptic type of supersensitivity in this organ. The damage to the adrenergic nerve terminals caused by 6-OH-DA was reversible; 3 to 4 weeks after chemical sympathectomy, there was a distinct increase in the norepinephnine content of the organs investigated, and after 14 weeks norepinephnine levels were normal again. The effectiveness of sympathetic nerve stimulation also returned gradually, but more rapidly than the levels of tissue norepinephrine. A single injection of 6-hydroxydopamine (6-OH-DA) markedly reduces the norepinephnine content in various sympathetically innervated organs of different species for several days or weeks (Porter et at., 1963, 1965; Laverty et at., 1965). In order to explain the long duration of the effect of 6-OH-DA, it was suggested that this amine or possible metabolites of it might replace the physiologic trasmitter or that it might irreversibly damage the storage sites for norepinephnine (Porter et at., 1963, 1965; Stone et ci., 1964; Laverty et at., 1965). In recent electron microscopic investigations, Received for publication April 11, 1969. Preliminany results have been presented at the Spring Meeting (Lausanne, 1968) of the Swiss Society for Physiology, Biochemistry and Pharmacologv. Send r pnint requests to: Dr. Guenther Haeusler, F. Hoffmann-La Roche & Co., Ltd., Basle. Switzerland. Tranzer and Thoenen (1967, 1968) observed a degeneration of adrenergic axon terminals, visible two to three days after treatment with 6-OH-DA. The degeneration selectively concerned the terminal part of the adrenergic neuron; its cell body and other tissue structures, especially cholinergic nerve endings, were morphologically left unaffected by 6-OH-DA. Thoenen and Tranzer (1968a,b) elaborated a treatment schedule for 6-OH-DA that leads to a generalized and virtually complete destruction of adrenergic nerve terminals and proposed the term “chemical sympathectomy.” The present investigation in cats deals with the consequences of chemical sympathectomy as regards the effect of sympathetic nerve stimulation and the sensitivity to norepinephnine. The experiments were performed on spinal cats, on isolated smooth muscles of the nictitating membrane and on isolated hearts of the cat at 1969 CHEMICAL SYMPATHECTOMY 51 various intervals after treatment with 6-OH-DA. On the isolated nictitating membrane, the effectiveness of surgical and chemical sympathectomy was compared. METHODS. Cats of either sex, weighing between 1.6 and 2.8 kg, were used. Isolated nictitating membrane. (See Haeuslen and Haefely, 1968.) The cats were anesthetized with ether, and spinal preparations were made as described by Burn (1952). The eyeball was removed, together with the orbital fascia, and trans-. ferred to a dish filled with ice-cold Tyrode’s solution. After separation from the eyeball, the orbital fascia was spread out, and the medial smooth muscle was freed from the surrounding tissue under a binocular dissecting microscope. Care was taken to preserve the fine sympathetic branch which arises from the infratrochlear nerve and innervates the medial smooth muscle. The cartilage on one side of the isolated medial smooth muscle was fixed to the bottom of a 10-mi organ bath, and the other end of the muscle was connected to a Statham force transducer. The temperature was maintained at 37#{176}C. A slightly modified Tyrode’s solution was used (in millimoles per liter): NaCl, 135; KC1, 3.8; CaCl2, 22; MgSO4, 0.9; NaH2PO4, 0.4; NaHCO8, 12; glucose, 53. The organ bath was gently bubbled with a 95% Or-5% CO2 mixture. A period of about one hour was allowed to elapse before starting the experiment. During this period, the modified Tyrode’s solution was replaced every five minutes. As a rule, the resting tone of the muscle, which was repeatedly readjusted to 2 g, reached steady conditions after 40 to 50 minutes. The infratrochlear nerve was pulled through shielded bipolar platinum electrodes for stimulation with monophasic rectangular pulses of 0.1msec duration and supramaximal voltage delivered by a Grass S5 stimulator. “Stimulus numberresponse curves” were obtained by recording the tension developed when a single shock on trains of 3, 9 and 27 shocks were applied at a stimulation frequency of 1.6/sec. For “frequency-response curves,” the cumulative effect of stimuli with a frequency progressively doubled from 02/sec up to 25/sec was determined. Cumulative dose-response curves for norepinephnine were obtained by adding norepinephrine to the bath fluid. Usually, 1 to 5 minutes after drug addition, the tension developed by the muscle reached a steady level. Each dose of norepinephrine was contained in 0.05 ml. Dilutions were prepared from a stock solution containing norepinephrine in a concentration of 10’ M dissolved in 0.02 N HC1. Only one dose-response curve was obtained in any single preparation since a large number of preliminary experiments had shown that, after a full dose-response curve, the sensitivity of the medial smooth muscle toward the same substance decreased somewhat. Pretreatment with reserpine. Cocaine causes a contraction of the normal nictitating membrane which is not observed after pretreatment with reserpine. In experiments with cocaine, cats therefore received an i.p. injection of 3 mg/kg of reserpine 16 hours prior to the preparation of the nictitating membrane. Isolated cat heart with sympathetic nerve supply intact. Isolation, perfusion and sympathetic nerve stimulation of the cat heart were carried out as described earlier (Haeusler et al., 1968b). In all cases atropine sulfate (final concentration, 106 g/ml) was added to the perfusion solution. Drug injections were made into the aortic cannula in the close vicinity of the aortic valves. Determination of the ED5O and the slopes of dose-response curves. All results are expressed as percentages of the maximal development of tension (isolated nictitating membrane) or of the maximal increase in heart rate (isolated cat heart). The horizontal shifts of dose-response curves are measured at the level of the ED5O. To facilitate the comparison with the results of Langer and Trendelenburg (1969), we expressed the slope of the dose-response curve in the same way (50/ log ED8O log ED3O). The significance of differences between the mean values of two groups was evaluated by Student’s t test and the range test, which both gave the same results. Spinal cats. Cats were anesthetized with ether and made spinal by section of the spinal cord at the first cervical level and mechanical destruction of the brainstem. The animals were under positive pressure respiration throughout the experiment. A polyethylene catheter was inserted into a femoral artery and connected to a Statham pressure transducer. A femoral vein was cannulated for i.v. injection. The head was fixed in a simplified stereotaxic head holder. Threads were pulled through the cartilagenous margin of the nictitating membranes and connected to a Statham transducer for semi-isometric recording. The resting tension was 4 g. Both cervical sympathetic trunks were cut low in the neck and laid on bipolar platinum electrodes for stimulation. Monophasic rectangular pulses of 0.5-msec duration at supramaximal voltage were delivered from Grass S4 stimulators. Stimulus-number and frequency-response curves were obtained as already described for the isolated nictitating membrane. The main deflection of an electrocardiogram (ECG) lead triggered a cardiotachometer for continuous recording of the heart rate. Arterial blood pressure, 52 HAEUSLER ET AL. Vol. 170 tension of the nictitating membranes and instantaneous heart rate were recorded on a Grass P7 polygraph. Chemical sympathectomy of cats by 6-OH-DA. For destruction of adrenergic nerve endings, the same schedule of treatment was used as described previously (Thoenen and Tranzer, 1968a,b; Haeusler et al.,1968a). Cats were given two doses of 20 mg/kg of 6-OH-DA hydrobromide i.v. (carresponding to two doses of 14 mg/kg of 6-OH-DA) on the first day, and one week later two doses of 50 mg/kg of 6-OH-DA hydrobromide (corresponding to two doses of 34 mg/kg of 6-OH-DA). 6-OH-DA hydrobromide was dissolved immediately before injection in 0.001 N HC1 which was gassed with nitrogen. Estimation of tissue norepinephrine. The organs were removed from cats anesthetized with pentobarbital and were frozen in petroleum ether at about -80#{176}C, weighed and homogenized in ice-cold 0.4 N HC1O4. The norepinephrine values are expressed in terms of micrograms per gram, with the exception of the iris, in which case the values are given in micrograms per organ. The fluorimetric determination of norepinephrine in the medial smooth muscle of the nictitating membrane was carried out following the method of H#{228}ggendal (1963) and in the other organs according to Bertler et al. (1958). The drugs used were: atropine sulfate (C. H. Boehninger und Soehne GmbH, Ingelheim, Germany), cocaine hydrochloride (Ph.H.V.), 1,1dimethyl-4-phenylpiperazinium iodide (DMPP, Parke, Davis and Company, Detroit, Mich.), 1norepinephrine, tyramine hydrochloride (Fluka AG, Buchs SG, Switzerland), and reserpine (Serpasil, Ciba Pharmaceutical Company, Basel, Switzerland). 2,4,5-Trihydroxyphenethylamine hydrobromide (6-hydroxydopamine) was synthesized by Dr. A. Langemann of the Chemical Research I)epartment of F. Hoffmann-La Roche & Co. Ltd., Basle, Switzerland. Concentrations or doses of norepinephrine and h-ramine are expressed as log molar concentrations or as log grams, respectively. Doses of norepinephrine and tyramine refer to the free base. Norepinephnine was diluted from a stock solution with saline containing ascorbic acid (1 mg/mi). RESULTS. ISOLATED NICTITATING MEMBRANE. Twelve to 16 days after the first injection of 6-OH-DA, stimulation of the sympathetic nerve with frequencies of up to 6.4 shocks/sec produced no contraction of the isolated nictitating membrane. At a stimulation rate of 12 or 25 chocke/see, a small response occurred (fig. 1A). The isolated nictitating membrane of cats whose superior cervical ganglia had been extirpated 12 to 16 days prior to the experiment did not respond to sympathetic nerve stimulation at any frequency (fig. 1A) . Accordingly, the norepinephrine content of smooth muscles of nictitating membranes denervated surgically was reduced virtually to zero, whereas that of chemically sympathectomized nictitating membranes was on the average 8% of the control value (fig. 1C). Similar results were obtained in the iris, in which the corresponding norepinephrine values were zero and 3%, respectively (fig. 2B). The medial smooth muscles of denervated nictitating membranes were supersensitive to norepinephrine. The ED5O of norepinephrine in surgically denervated membranes was shifted to the left by a factor of 96 as compared to the controls, and in chemically sympathectomized membranes by a factor of 71 (fig. 3; table 1). A precise comparison is, however, not possible since the slope of the dose-response curves of the denervated membranes was significantly smaller than that of the curve obtained in normal membranes (table 1). Dose-response curves for norepinephnine in the presence of cocaine (10 M) were determined in innervated nictitating membranes from reserpine-pretreated cats since this pretreatment abolishes the contraction caused by cocaine. As shown in figure 3 and table 1, cocaine caused the dose-response curve to shift to the left by a factor of 15. The dose-response curve determined in the presence of cocaine was significantly flatter than without cocaine but significantly steeper than those of denervated membranes (table 1). There was no statistically significant difference between any groups in the maximal tension produced by a supramaximal concentration of norepinephrine. ISOLATED CAT HEART. Twelve to 16 days after the first injection of 6-OH-DA, the effect of electrical sympathetic nerve stimulation on the increase in heart rate was abolished when low frequencies of stimulation were used and strongly reduced with frequencies between 6.4 and 25 shocks/sec (fig. 4A). The results were very similar to those obtained with isolated nictitating membranes. Sympathetic nerve stimulation (at any frequency) never produced a positive inotropic effect. Basal heart rate after 1969 CHEMICAL SYMPATHECTOMY 53