[1] 9w Prevention of Ethanol-Induced Sympathetic Overactivity and Degeneration by Dexmedetomidine

[1] 191w The effects of dexrnedetomidine, a selective alpha2-adrenoceptor agonist, on rat sympathetic neurons were studied during a I2-day, heavy ethanol exposure. Adult male Wistar rats were given ethanol or isoealoric sucrose three times a day by intragastric intubation. Both acute (a single dose of 300 t*g/kg p.o.) and chronic (100/zg/kg x 2 P.O. throughout the experiment) effects of dexrnedetomidine were tested. The superior cervical ganglia (SCG) of the ethanol-exposed, non-dexmedetomidine-treated rats showed an abnormally high overall level of tyrosine hydroxylase immunoreactivity (TH-IR) and catecholamine histofluorescence. However, a subpopulation of neurons had apparently lost their catecholamine synthetic activity, as they exhibited no TH-IR or catecholamine fluorescence. The ethanol-exposed ganglia also showed structural alterations (e.g., decreased neuronal size and increased occurrence of vacuolated neurons). In the ethanol-exposed, chronically dexmedetomidine-treated group, by contrast, the SCG exhibited TH-IR and catecholamine fluorescence intensities comparable to those seen in the control ganglia. All the structural parameters studied, as well, were at the control level in the chronically dexrnede~tomidine-treated group. The single dose of dexmedetomidine offered only marginal protection against the ethanol-induced ;flterations. These results suggest that chronic dexmedetomidine treatment may prevent ethanol-induced overactivity and degeneration of catecholaminergic neurons.

[1] 92w Due to individual differences in ethanol tolerance, the total dose of ethanol during the 12-day exposure varied slightly from animal to animal. The cumulative ethanol doses or blood ethanol concentrations did not differ significantly between the three ethanol-exposed groups (Table 1). The control rats lost an average of 6°70 _+ 1 °70 of their body weight, whereas weight loss in the ethanol-exposed groups was significantly higher, 13070 +_ 1070 [F(1, 28) = 33.58, p < 0.001]. Weight loss did not differ between the three ethanol-exposed groups (EtOH, EtOH 4-dex, EtOH + sd).

[2] 148w During the last 5-6 clays of the experiment, the rats were severely intoxicated for most of the day, and in the morning they exhibited withdrawal symptoms of varying severity (piloerection, rigidity, tremor, irritability, convulsions). The dose of dexmedetomidine used in the chronic treatment (100 t~g/kg × 2 P.O.) had only a marginal sedative effect on the rats, which means that the E~:OH + dex rats were only slightly less active than the EtOH rats, did not sleep any more than the EtOH rats, and responded normally to handling. The acute dose of 300/~g/kg was more clearly sedative, but did not cause a loss of the righting reflex. The severity of ethanol withdrawal symptoms was not measured in the present study, but the dexmedetomidine-treated rats (EtOH + dex) were easier to handle and did not exhibit withdrawal seizures, suggesting a less severe withdrawal reaction compared to the nondexmedetomidine-treated ra~ts.

[3] 214w Formaldehyde-induced catecholamine histofluorescence (FIF) revealed remarkable differences between the groups in the SCG fluorescence intensities (i.e., neuronal concentrations of noradrenaline; Figs. l and 2). The overall level of FIF intensity was markedly higher in the EtOH ganglia than in the control or EtOH 4-dex ganglia (Figs. 1, 2A, 2B, and 2C). In the EtOH + sd group (Fig. 2D), the overall FIF intensity was comparable to, or even higher than in the EtOH SCG. The strongly fluorescent neurons in the EtOH and EtOH 4-sd ganglia also had strong TH-IR, suggesting a high catecholamine turnover in these cells (Fig. 2B, 2D, 2F, and 2H). A combination of intense TH-IR with weak or moderate FIF was also seen in several neurons of the EtOH and EtOH + sd ganglia (Fig. 2D, 2H, 2J, anti 2K). This combination probably reflects an extremely high norepinephrine turnover (i.e., large amounts of norepinephrine being synthesized and rapidly transported to the periphery). At the same time, a considerable population of neurons in the EtOH ganglia seemed to have lost their catecholamine synthetic activity, judging by the lack of both TH-IR and FIF (Fig. 2B, 2F). The SCG of the chronically dexmedetomidine-treated control rats (Fig. 2I) did not differ from the nontreated control ganglia, except for the increased proportion of TH-negative neurons.

[4] 86w A distinct population of vacuolated neurons (Fig. 2J, 2K) was seen in the SCG of the ethanol-exposed, non-dexmedetomidine-treated rats. The vacuolated neurons (VN) typically exhibited one to four large cytoplasmic vacuoles and a high TH-IR with low or moderate FIF intensity. Occasional VN also appeared in the SCG of the control rats (Sucr, Sucr + dex), and in the ganglia of the EtOH + dex rats. In the EtOH + sd ganglia, the number of VN did not differ significantly from the EtOH group (Fig. 3).

[5] 79w The morphometric results are shown in Fig. 3. One-way ANOVA revealed significant differences between the groups in the proportion of vacuolated neurons [F(4, 55) = 12.39, p < 0.001] and TH-negative neurons [F(4, 55) = 16.45, p < 0.001], as well as in neuronal size [F(4, 2995) = 7.80, p < 0.001], and neuronal density [F(4, 55) = 4.75, p < 0.01]. The statistically significant differences between the groups in pairwise comparisons (Bonferroni-corrected t-tests) are shown in Fig. 3.

[1] 173w The present study showed that the adverse effects of chronic ethanol exposure on rat sympathetic neurons could be prevented by dexmedetomidine, a novel alpha2-adrenoceptor agonist. Dexmedetomidine is the pharmacologically active (23,37) dextro enantiomer of medetomidine, a highly selective and potent alpha2-adrenoceptor agonist (38,44). Medetomidine is currently used in veterinary practice for its analgesic and sedative properties. The dextroisomer dexmedetomidine has been tested in human clinical trials, as an anesthetic adjuvant (1,2). The interactions of dexmedetomidine and ethanol have been previously studied in terms of acute effects on motor performance, body temperature, and brain monoamine metabolites (15,40). Dexmedetomidine, at the dose of 300 #g/kg i.p., was found to enhance the ethanol-induced hypothermia, sedation and motor impairment of mice, whereas the dose used by us (P.O.) in the chronic treatment, 100 #g/kg i.p., did not significantly potentiate the acute effects of ethanol. Contrary to the enhanced behavioral effects of ethanol, dexmedetomidine inhibited the ethanol-induced increase in brain MHPG levels (i.e., noradrenaline turnover seemed to remain at control level) when ethanol and dexmedetomidine were administered together.

[2] 167w Chronic dexmedetomidine treatment significantly reduced the morpho-functional alterations in the SCG after the 12-day ethanol exposure. The proportion of TH-negative neurons was approximately twofold (mean 11.3°/0 vs. 6.4070) in the EtOH ganglia compared to the control ganglia, whereas the EtOH 4-dex or the EtOH + sd ganglia did not differ from the controls in this respect. The fact that a single dose of dexmedetomidine normalized the proportion of TH-negative neurons suggests that the loss of TH-IR need not be a sign of irreversible damage to a neuron, but rather a reversible alteration of the functional state of the neuron. The proportion of TH-negative neurons was also increased in the dexmedetomidine-treated control group compared to the control group with no medication. This suggests that a chronic inhibition of catecholamine release by dexmedetomidine may suppress the synthesis of the neurotransmitter in "healthy" peripheral adrenergic neurons. In the ethanol-exposed ganglia, on the contrary, the administration of dexmedetomidine restored the abnormally high functional activity of the neurons to the control level.

[3] 87w The proportion of vacuolated neurons in the SCG was increased ca. sevenfold (0.7°70 in the EtOH group vs. 0.1°70 in the control groups) by the 12-day ethanol exposure applied in the present study. VNs have been reported to occur normally in several parts of rat peripheral nervous system, specifically in the superior cervical, coeliac, and pelvic (hypogastric/paracervical) sympathetic ganglia, as well as in the ganglion nodosum of the vagus nerve [see (28,32)]. The appearance of VN in sympathetic ganglia has been regarded as a sign of neurosecre-

[4] 218w FIG. 1. Left: Pseudocolor video prints representing the formaldehyde-induced catecholamine histofluorescence in the superior cervical ganglion of a control rat (Sucr = sucrose fed), an ethanol-exposed rat (EtOH), and an ethanol-exposed rat given dexmedetomidine treatment throughout the 12-day ethanol exposure (EtOH + dex). Right: Tyrosine hydroxylase immunoreactivity (TH-IR) on the same sections. Note the intense catecholamine histofluorescence and TH-IR in the ethanol-exposed ganglion, and the fluorescence and TH-IR intensities in the EtOH + dex ganglion, which are comparable to the control ganglion. Bar = 50/~m throughout. 444 JAATINEN ET AL. 33)]. Repeated immobilization stress, which is known to increase the activity of the sympathoadrenal system, has been shown to induce extensive vacuolation in rat SCG (26). Chronic ethanol exposure and ethanol withdrawal, in particular, also increase noradrenerglc activity in both central and peripheral nervous system (see Introduction). Increased functional activity of SCG neurons was apparent in the present experiment, as well: most of the SCG neurons exhibited high levels of catecholamine fluorescence and TH-IR, including the vacuolated neurons. Therefore, the vacuolation may represent a particular response of a population of sympathetic neurons to excessive stimulation. Compatible with this interpretation, the EtOH + dex ganglia, which exhibited normal levels of the catecholamine markers, also showed neuronal vacuolation comparable to the control ganglia, ca. one VN per 1000 SCG neurons.

[5] 101w We have previously (18) shown the proportion of VN to increase up to 1.6% of SCG neurons after a 3-week ethanol exposure, and up to 2.7% after 4 weeks of ethanol feeding. Thus, there is an apparent positive correlation between the length of ethanol exposure and the extent of neuronal vacuolation in rat SCG. Our previous studies have indicated that most of the VN disappear during the first few days after the ethanol exposure has been finished. It is presently not known which proportion of the VN degenerate and die, and which are capable of retaining normal functional activity and morphology.

[6] 133w Neuronal packing density was higher in the EtOH ganglia than in the control ganglia, which seemed to be mainly due to the decreased neuronal size in the ethanol-exposed SCG (11% decrease in neuron cross-sectional area vs. 15% increase in packing density compared to the control group). There was a tendency towards normalization of SCG neuronal density in the ethanol-exposed groups given either acute or chronic dexmedetomidine treatment. As the perikaryal area of SCG neurons in the acute dexmedetomidine group (EtOH + sd) was comparable to that of the EtOH group, the difference in neuronal density between these groups must be due to changes in the nerve fibers and/or interstitial elements. A better water balance (less dehydration) in the EtOH + sd group compared to the EtOH group may have contributed to this difference.

[7] 294w No attempt was made in thepresent study to estimate the total number of SCG neurons, because, on the basis of previous studies, it was highly improbable that significant neuronal loss should take place within a 12-day ethanol exposure. Thus, neuronal density was here considered a measure of ganglion structure, and not a measure of total neuron number. As the neuron counts were not corrected according to neuron size (which influences the probability of a neuron being seen and counted in a given section), the proportion of large neurons (e.g., the vacuolated neurons) may be slightly overestimated. The mean size of the VN was, however, equal in all groups, and the ca. 6% decrease in the average neuronal diameter of the EtOH and EtOH + sd ganglia cannot account for the four-to sevenfold increase in the proportion of VN. There-FIG. 3. Morphometric data on the SCG of rats exposed to ethanol for 12 days, and of pair-fed control rats (mean + SEM). Dex = chronic dexmedetomidine treatment, sd = a single dose of dexmedetomidine; see text for detailed methods. *, **, *** = Significantly different from the sucrose group (* = 0.05 level; ** = 0.01 level; *** = 0.001 level); +, + +, + + + = significantly different from the ethanol group, respectively; Bonferroni-corrected t-tests (all groups were compared with the sucrose group, and EtOH + dex and the EtOH + sd groups were compared with the EtOH group). fore, the observed increase in the proportion of VN in the ethanol-exposed rats reflects a true increase in the number of VN, and not an increase in their (relative) size. Neither was there any difference in the size distribution of TH-positive and TH-negative neurons (i.e., the proportions of TH-positive and negative neurons were unbiased).

[8] 142w Previous attempts to produce pharmacological neuroprotection against ethanol-induced neuronal degeneration in vivo have mostly proved unsuccessful. Exogenous gangliosides, which have been shown to exert neuroprotective action against a variety of neural insults [see (14)], did not affect the morphology of hippocampal granule or pyramidal neurons after a 12-month ethanol exposure to adult rats (6). However, a positive effect was found on the symptoms of alcoholic sensory polyneuropathy with mixed brain gangliosides ( 25), but no morphological data was reported on this clinical study. There is no direct evidence so far, on a neuroprotective effect of antioxidants, although free radical-induced oxidative damage has been implicated in the to~dcity of ethanol to nervous tissue [see (27)]. Vitamin E supplementation failed to prevent the learning deficit caused by a .';-month ethanol feeding to mice, although brain lipofuscin content was decreased by the vitamin supplementation (11).

[9] 139w Alpha2-adrenoceptor antagonists (atipamezole, idazoxan, RX 821002) have been shown to attenuate the behavioral effects of acute ethanol intoxication (7,21). For example, antagonism of ethanol-induced hypothermia, ataxia, and reduction of exploratory behavior by alpha2-antagonists has been reported in mice. Although the acute intoxicating effects of ethanol may be reduced by aipha2-antagonists, neuroprotection by these agents against long-term ethanol toxicity is unlikely to be established. Inhibition of alpha2-adrenoceptor activity would increase the catecholarainergic activity, which is already elevated during chronic eth~Lnol intoxication or withdrawal. The increased norepinephrine release might aggravate ethanol withdrawal symptoms and contribute to ethanol-induced neuronal damage either directly, or via interactions with other excitatory neurotransmitter systems, like the corticotropin releasing factor (CRF) activity [see (30)]. The clinical utility of alpha2-adrenoceptor antagonists, particularly in the treatment of chronic alcoholics, may further be compromised by their potential proconvulsant properties (21).

[10] 182w The present findings show that the ethanol-induced overactivity and morpho-functional degeneration (shrinkage, loss of TH-IR, vacuolization) of rat peripheral sympathetic neurons can be prevented by dexmedetomidine. This neuroprotective effect was not due to an altered metabolism of ethanol, as blood ethanol levels were similar in all the ethanol-exposed groups (dexmedetomidine-treated or nontreated). No pharmacokinetic interaction of dexmedetomidine and ethanol was found in a previous study (40), either. Interestingly, a neuroprotective effect of dexmedetomidine against ischemic brain damage has been previously reported (13,24). The improvement of neurologic and histopathologic outcome after incomplete ischemia with dexmedetomidine was attributed to a decrease in sympathetic activity; the effect was mediated by aipha2-adrenoceptors, as it was reversed by atipamezole, a specific alpha2-antagonist. Increased sympathetic activity may also contribute to the pathogenesis of aicohofic brain damage, especially during repeated withdrawal episodes. Cadete-Leite et al. (4,5) have shown that ethanol withdrawal after long periods of alcohol intake worsens the degenerative processes in rat central nervous system. The repeated withdrawal phases in the present experiment have most likely contributed to the neuropathology observed, in addition to ethanol intoxication per se.

[11] 147w Alpha2-adrenoceptor agonists, mainly clonidine and lofexidine, have been used in the treatment of the ethanol withdrawal syndrome [see (22)]. Our preliminary results (19) show that dexmedetomidine significantly relieves ethanol withdrawal symptoms (rigidity, tremor, irritability) in the rat, even at doses that do not produce any measurable sedative effect. In clinical studies (1,2,39), dexmedetomidine has been shown to relieve anxiety and to diminish harmful cardiovascular responses (tachycardia, hypertension) before and during surgical anesthesia. These cardiovascular and sedative/anxietyrelieving effects of dexmedetomidine could be applied to the treatment of withdrawal reactions in humans, as well. The present study indicates that in addition to a withdrawal symptom-relieving effect, dexmedetomidine might possess a neuroprotective action on catecholaminergic neurons. Further studies are needed to elucidate the possible effects of dexmedetomidine in the central nervous system, and to find out, whether the neuroprotective effects of dexmedetomidine apply to human subjects (detoxified alcoholics), as well.

[1] 250w Thirty 4-month-old male Wistar rats were used in the study. The rats were housed individually under standard conditions (+21 _+ I°C, with lights on between 0800 and 2100), and were carefully habituated to handling before starting the experiment. The rats were divided into five experimental groups as shown in Table 1. To ensure a high and accurate dosage of ethanol, intragastric intubation of 25°70 ethanol plus 5070 sucrose was used three times a day (0800, 1400, and 2000). The daily dose of ethanol was gradually increased to 10 g/kg as absolute alcohol, the duration of the exposure being 12 days. To avoid deaths during the course of the experiment, severely intoxicated animals were given reduced doses of ethanol; severely atactic animals received 1-2 g/kg, and rats having lost their righting reflex were intubated with isocaloric sucrose only. The ethanol-exposed rats (EtOH, EtOH + dex, and EtOH + sd groups) had free access to standard laboratory rat food and tap water. The control rats were intubated with similar amounts of isocaloric sucrose three times a day and were given food according to the consumption of the respective ethanol-exposed group (pair-feeding), to control for the possible impact of malnutrition in the neuropathology observed. Dexmedetomidine hydrochloride (Orion Corporation Orion-Farmos, Finland) was given P.O. in connection with the ethanol/sucrose intubations, either 100/~g/kg twice a day (0800 and 2000) throughout the experiment (EtOH + dex and Sucr + dex groups), or as a single dose of 300/~g/kg 2-4 h before sacrifice (EtOH + sd group).

[2] 45w Blood ethanol concentration (BEC) was determined by gas chromatography (9) from a capillary blood sample (0.05 ml), which was taken from the tip of the tail 2 days before the exposure was finished, before (BEC-0) and 1 h after (BEC-Ih) the morning dose of ethanol.

[3] 230w The rats were killed by decapitation under light ether anesthesia between 1000 and 1200, and the superior cervical sym-pathetic ganglia (SCG) were rapidly prepared and frozen in liquid nitrogen. After storing in liquid nitrogen, the ganglia were processed for histochemical detection of catecholamines by the standardized formaldehyde-induced fluorescence (FIF) method (10), as described in detail previously (18). Every tenth section (thickness 10 ttm) was viewed through an Olympus Vanox-T fluorescence microscope (Olympus, Inc., Tokyo, Japan) equipped with a special filter combination for the detection of monoamines (filter block "V," excitation light wavelength 395 to 415 nm, emission light 455 nm and up). Two randomly selected sections from the middle part of the ganglion were processed for pseudo color video prints by a Mitsubishi CP 100E video printer (Mitsubishi Electric Corporation, Tokyo, Japan) to attain an objective image of the neuronal FIF-intensity. Tyrosine hydroxylase immunoreactivity (TH-IR) in the sympathetic ganglia was demonstrated by using the peroxidase-antiperoxidase (PAP) technique of Sternberger (43) on the sections previously studied by fluorescence microscopy. The dilution of the TH-antiserum (Eugene Tech. International, Allendale, N J) was 1 : 1000 and the incubation time was 24 h at +4°C. Goat anti-rabbit serum (1 : 80) and PAP (1 : 80) incubations were carried out for 30 min at room temperature. Diaminobenzidine was used as chromogen. In the control incubations, the TH-antiserum was replaced by normal rabbit serum.

[4] 120w All the ganglia were sectioned longitudinally, and the sections for the morphometric measurements were randomly selected from a corresponding set of sections in the middle part of each SCG. Neuronal density, and the proportions of THpositive, TH-negative and vacuolated neurons were measured from two randomly selected TH-immunostained sections of each ganglion. All nucleated neuronal profiles on each section were included in these measurements. Neuronal size (crosssectional area) was measured from 50 nucleated neuronal profiles (either TH-positive or TH-negative) at both the cranial and the caudal end of one randomly selected section per animal (i.e., from 600 neurons in each group). The morphometric measurements were carried out with the aid of a Hamamatsu ARGUS-10 image processor (Hamamatsu Photonics, Hamamatsu City, Japan).

[5] 73w The results are expressed as mean +_ SEM. One-way analysis of variance was used to identify overall effects (treatment as a factor). The effects of the treatments were further ana- *BEC-0 = blood ethanol concentration before the morning dose of ethanol, BEC-Ih = blood ethanol concentration 1 h after the morning dose of ethanol; tl00/~g/kg x 2 P.O., throughout the experiment = 12 days; ~/300 /~g/kg x 1 P.O., 2-4 h before sacrifice.

[6] 50w lysed by Bonferroni-corrected t-tests. All groups were compared with the control group, and the EtOH 4-dex and EtOH + sd groups were, in addition, compared with the EtOH group. A probability of < 0.05 (p < 0.0083, when six comparisons were made) was considered to indicate a statistically significant difference.

[1] 122w Ethanol Chronic exposure Dexmedetomidine Degeneration Withdrawal CHRONIC ethanol exposure has been reported to cause overactivity of noradrenergic neurons in the central (3,35,41,42) and the peripheral nervous system (16,(34)(35)(36). The noradrenergic overactivity is even more pronounced during ethanol withdrawal (12,20,35), as reflected by the typical ethanol withdrawal symptoms, such as anxiety, tremor, sweating, hypertension, and tachycardia. Reduced alpha2-adrenoceptor sensitivity may be, in part, responsible for the increased noradrenergic neuronal activity after chronic ethanol ingestion (31). As stimulation of the presynaptic alpha2-receptors on noradrenergic neurons reduces the release of norepinephrine, alphaz-receptor subsensitivity increases norepinephrine release. Pharmacological interventions with alphaz-adrenoceptor agonists (especially clonidine, lofexidine) have been shown to improve this inhibitory dysfunction, and to alleviate the signs of sympathetic overactivity during ethanol withdrawal [see (22)1.

[2] 68w The prolonged noradrenergic overactivity may contribute to the morpho-functional degeneration of the sympathetic nervous system, which has been reported in chronic alcoholics (8,29), as well as in experimental animals chronically exposed to ethanol (16)(17)(18). Significant neuronal loss and cytoplasmic vacuolation were observed in rat peripheral sympathetic ganglia after 4 weeks of ethanol feeding (16,18); the alterations were age-dependent and correlated with the functional overactivity of the sympathetic ganglia.

[3] 95w The present study was designed to find out whether the overactivity and degeneration of the noradrenergic nervous system associated with chronic ethanol exposure and withdrawal could be prevented by dexmedetomidine, a selective Requests for reprints should be addressed to Pia Jaatinen, University of Tampere, School of Public Health, P.O. Box 607, FIN-33101 Tampere, Finland. 440 JAATINEN ET AL. alpha2-adrenoceptor agonist. Here we report the results concerning the neuropathology of the sympathetic neurons in the superior cervical ganglion (SCG). Parts of the present study were presented at the Seventh Congress of ISBRA, Queensland in 1994 (19).