Barbara K. Evans

A new method of destroying adrenergic nerves in adult animals using guanethidine

1 The structure of sympathetic neurones in the rat has been examined with histological, fluorescence histochemical and electron microscopical methods after chronic treatment for 6 weeks with guanethidine (25 or 30 mg/kg/day i.p.). 2 Less than 2% of the nerve cell bodies in the superior cervical ganglion remained at this time and in these cells the mitochondria were badly damaged. Few fluorescent adrenergic nerve fibres were found outside the central nervous system. This situation persisted even 4 months (the longest period studied) after cessation of treatment. 3 This procedure is proposed as a new method of producing sympathectomy. It has the advantage of being applicable to adult animals in a variety of experimental and pathological situations. It is uniquely advantageous for denervation of the male reproductive tract.

Intracranial injections of 6-OHDA. Comparison of catecholamine-depleting effects of different volumes and concentrations.

Fluorescence histochemistry was used to assess monoamine depletion after injections of 6-OHDA into selected brain areas. Two volumes (2 and 4 mul) and 4 concentrations (1, 2, 4 and 8 mug/mul) of 6-OHDA were injected into the olfactory tubercle, the posterior lateral hypothalamus and the lateral hyopthalamus. Selective destruction of catecholamine-containing neurons resulted from all injections of 6-OHDA with the exception of the 2 lowest doses (2 and 4 mul of 1 mug/mul) and the highest dose (4 mul of 8 mug/mul) which produced nonspecific damage of brain parenchyma. The results indicate that, in addition to the selection of an effective dose, it is also possible to choose a site of injection which will produce a maximal area of specific depletion. In cases where injections into terminal areas caused limited specific depletion the same dose injected into preterminal axons often caused a more widespread loss of fluorescence. With volume, concentration and anatomical location being important variables to consider, caution is needed in the interpretation of behavioural experiments. When using 6-OHDA it is necessary to show that specific depletion of catecholamines has been achieved.

Degeneration of adrenergic neurons following guanethidine treatment: an ultrastructural study.

SummaryThe degeneration of peripheral adrenergio neurons of the rat following chronic treatment with high doses of guanethidine has been described at the ultrastructural level. Neurons exhibiting three different degenerative appearances have been distinguished and evidence is presented that these represent sequential phases in the degeneration of individual neurons. These phases are referred to as“primary degeneration”, in which mitochondrial damage, extensive depletion of cytoplasmic organelles and swelling of the cell body occurred; the“transient growth phase”, characterised by proliferation of some organelles and by hyperplasia of neuronal processes; and“secondary degeneration”, in which the final breakdown and condensation of the neuronal contents occurred. Many of the characteristics of the “transient growth phase” and of “secondary degeneration” are comparable to those observed in studies of growth and degeneration respectively. Since the neuronal mitochondria represent a primary site of action of guanethidine, degeneration may be initiated by lowered energy metabolism within the neuron. This degenerative action of guanethidine is selective for adrenergic neurons, at least in the tissues studied; degeneration of other cell types, including cholinergic, purinergic and sensory neurons, was not observed.

Intracranial injection of drugs: Comparison of diffusion of 6-OHDA and guanethidine☆☆☆

Marked differences in extent of diffusion have been shown with the fluorescence histochemical method between guanethidine and 6-OHDA(64 mug in 2 mul) when injected acutely or chronically into the lateral hypothalamus, the substantia nigra or the amygdala of the rat brain. Cannulation damage up to 1 mm in diameter and attributed to the implantation of cannulae and placebo injection was observed. A further area of generalized damage occurred following the injection of drugs and was far greater for 6-OHDA (2 mm) than for guanethidien (0.3 mm). Guanethidine, but not 6-OHDA, caused specific damage to catecholamine-containing nerurons up to a distance of at least 3 mm and more from the cannula tip. These striking differences between the effects of intracranial injection of 6-OHDA and guanethidine are discussed in terms of the uptake and degradation of the two drugs and the anatomical features of the injection site; they are not explicable in terms of experimental conditions such as concentration, volume of injection, molecular weight or lipid solubility. The different patterns of damage would not easily be distinguished by biochemical analyses and the catecholamine specificity of 6-OHDA in studies of the central nervous system must be seriously questioned. Vascularization of chronically implanted cannula tracks and the presence of anatomical diffusion barriers are also discussed in relation to the diffusion of drugs injected intracranially.

Effects of chronic intracranial injection of low and high concentrations of guanethidine in the rat

Low (64 mug in 2 mul) or high 320-1280 mug in 2 mul) doses of guanethidine sulphate were injected daily for up to 19 days into the lateral hypothalamus, substantia nigra, locus coeruleus, dorsal raphe nucleus, or amygdala region of the rat brain. Effects on monoamine-containing neurons were determined using fluorescence histochemistry. The noradrenergic terminals of the hypothalamus were depleted over a diameter of 7 mm by both low and high doses of guanethidine whereas, even with high doses, the dopaminergic terminals of the median eminence, amygdala and acudate nucleus were only partially depleted. Fluorescence levels of dopaminergic cell bodies of the sub stantis nigra and 5HT-containing cell bodies of the dorsal raphe nucleus were unaltered by low doses of guanethidine. Low doses of guanethidine did not affect the fluorescence of the noradrenergic cell bodies of the locus coeruleus, however high doses caused a substantial reduction in fluorescence levels. Normal levels of fluorescence were observed in all catecholamine-containing neurons within 14 days from cessation of injections. Thus, the xon retraction and eventual degeneration of peripheral sympathetic adrenergic neurons, which occurs as a result of chronic intraperitoneal injections of guanethidine does not occur with the catecholamine-containing neurons in the central nervous system. The rapid recovery of centrat catecholamine-containing neurons is remarkable in view of the extensive areas of brain damage produced by chronic injection of such high concentrations of drug. Fluorescence in peripheral adrenergic nerves was unaffected by chronic injection of guanethidine into the lateral hypothalamus but adhesions of some internal organs were observed. Blood vessels in the vicinity of the cannula were heavily reinnervated by fluorescent fibres probably arising from intracranial catecholamine-containing neurons. Some of the advantages of intracranial injection of guanethidine compared to 6-hydroxydopamine for behavioral experiments are discussed.

Axon retraction following guanethidine treatment

SummaryVasa deferentia of rats chronically treated with high doses of guanethidine sulphate (30 or 60 mg/Kg/day i.p.) were examined using electron microscopic, fluorescence histochemical and pharmacological techniques. Counts of the axon population in segments of the proximal (urethral) end of the vas deferens showed a reduction to approximately 55% and 35% in the number of axon profiles after treatment for one week with the two dose levels respectively. In the same period only a few cell bodies in the hypogastric ganglion (from which most of the adrenergic innervation of the vas deferens arises) reached the stage of terminal degeneration. Although many axons showed some abnormalities, the number of axons observed in terminal stages of degeneration in treated tissue did not exceed, at any stage examined, the very small numbers observed in control tissue. Organ bath studies showed that the contractile response to transmural stimulation was lost fastest at the distal (epididymal) end of the treated vas deferens. These results have led to the conclusion that, in contrast to the degeneration of adrenergic axons produced by surgery or 6-hydroxydopamine, the sympathectomy produced by guanethidinein vivo involves theretraction of adrenergic axons prior to complete degeneration of the cell bodies.

Reinnervation following guanethidine-induced sympathectomy of adult rats.

SummaryThe reinnervation of various tissues by autonomic neurons has been studied in adult rats which had been sympathectomized by chronic guanethidine treatment (30 or 60 mg/kg/day for 8–15 weeks). In the heart, iris, ileum, mesentery, vas deferens and epididymis, fluorescence histochemistry reveals an almost complete disappearance of adrenergic fibres which is very longlasting. Even after 63 weeks few fluorescent fibres can be seen in these tissues and the density of innervation is not increased by incubation in α-methylnoradrenaline. The superior cervical ganglion represents an exception; large numbers of fluorescent fibres but few fluorescent ganglion cells were apparent during recovery. Axon counts carried out by electron microscopy on the vas deferens showed that after recovery for one year the number of axons was similar to that of controls; however, the pattern of innervation was abnormal, small granular vesicles were rarely seen and there was little uptake of 5-hydroxydopamine. On the basis of this evidence and of some pharmacological data it is suggested that there is a limited reinnervation by adrenergic axons accompanied by a great increase in the number of non-adrenergic, possibly cholinergic, axons.

The effects of intrahypothalamic injections of guanethidine on catecholamine fluorescence, food intake and temperature regulation in the rat

Abstract The effects of chronic injections of two dose levels of guanethidine sulphate into the lateral hypothalamus of the rat on eating, drinking, body temperature and on the levels of catecholamines as shown by fluorescence histochemistry were examined over a period of 12 days. During the guanethidine treatment, food and water intake were reduced, body temperature showed a significant rise, and the animals showed a wide loss of catecholamine fluorescence in the hypothalamic area. After cessation of injections, food and water intake as well as body temperature returned to preinjection base levels. Catecholamine fluorescence also returned during the 12 days after cessation of injections. These results are discussed in terms of current hypotheses concerning the hypothalamic adrenergic involvement in consummatory behavior and temperature regulation.

Effects of chronic guanethidine on the sympathetic nervous system of mouse and toad

Abstract 1. Chronic treatment of mice and toads with low (5–10 mg/kg per day) or high (25–150 mg/kg per day) doses of guanethidine depleted neuronal catecholamine stores, reduced adrenergic nervemediated responses and produced supersensitivity to noradrenaline. 2. No ultrastructural changes were seen in toad adrenergic cell bodies and only minor changes were seen in mouse noradrenergic cell bodies after treatment with high doses. Cells in later stages of degeneration were not observed. 3. After cessation of treatment, catecholamine levels and nerve-mediated responses increased considerably in both species. 4. Thus, the cytotoxic effects of chronically administered guanethidine vary considerably between species. Widespread degeneration of adrenergic neurons has so far been observed only in rats.

Effects of chronic bretylium treatment on the symphathetic neuron and the smooth musculature of the rat

The effects of chronic i.p. injection of high doses of bretylium on sympathetic nerves on the smooth musculature of the vas deferens of adult and newborn rats were examined using fluorescence histochemistry, light and electron microscopy and organ bath physiological techniques. Bretylium treatment caused mitochondrial swelling, loss of cristae and the formation of electron-dense inclusions in the mitochondria of sympathetic neurons. However, neuron degeneration was not observed and fluorescent histochemical appearance of adrenergic neurons was normal. A small transient supersensitivity of the isolated vas deferens of bretylium-treated rats to noradrenaline, but not to acetylcholine, occurred. There was, however, considerable increase in the maximal contractile response to both noradrenaline and acetylcholine. In high calcium concentrations acetylcholine-induced contractions of vasa deferentia from bretylium-treated rats were significantly greater than control; there was no difference in magnitude of noradrenaline-induced contractions.