Journal of Vestibular Research. Vol. 8. No.3. pp. 201-207. 1998 Copyright (!;) 1998 Elsevier Science Inc. Printed in the USA. All rights reserved

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Original Contribution

THE EFFECTS OF STEROIDS ON VESTIBULAR COMPENSATION AND VESTIBULAR NUCLEUS NEURONAL ACTIVITY IN THE GUINEA PIG

C. Alice A.E. Paul,* Andrew J. Sansom,*t Karyn Maclennan,* Cynthia L. Darlington,t and Paul F. Smith*

-Department of Pharmacology, School of Medical Sciences, University of Otago Medical School; and tDepart­ment of Psychology and the Neuroscience Research Centre, University of Otago, Dunedin, New Zealand

Reprint address:@r. Paul F. Smith, Dept. of Pharmacology, School of Medical Sciences, University of Otago Medical School, Dunedin, New Zealand. Tel: +64-3-479-7253; Fax: +64-3-479-9140;

E-mail: paul.smith@stonebow.otago.ac.nz

D Abstract-Recent studies have suggested that steroids such as dexamethasone and methylpred­nisolone might be useful in the treatment of vestib­ular disorders, irrespective of whether inflamma­tory processes are involved. The aim of this study was to investigate the effects of systemic adminis­tration of dexamethasone on vestibular compensa­tion of spontaneous nystagmus (SN) in guinea pig, and the effects of dexamethasone and methylpred­nisolone on extracellularly recorded spontaneous activity of medial vestibular nucleus (MVN) neu­rons in brainstem slices in vitro. In the behavioral study, none of the 3 doses of dexamethasone (5, 10, or 40 mglkg ip, delivered at 0, 12, 24, and 36 h fol­lowing a unilateral surgical labyrinthectomy (UL)) resulted in a significant change in the frequency or compensation of SN, relative to the vehicle control group. In the in vitro study, only a minority ofMVN neurons showed any response to 1 j.LM dexametha­sone (lout of 9 neurons), or 10 oM (3 out of 13), or 0.1 j.LM methylprednisolone (3 out of 7). These re­sults suggest, contrary to previous evidence, that dexamethasone may not accelerate compensation of SN following surgical UL and that dexamethasone and methylprednisolone may have a direct action only on a minority of MVN neurons. © 1998 Elsevier Science Inc.

D Keywords - vestibular compensation; medial vestibular nucleus; vestibular disorders; steroids; methylprednisolone.

Introduction

The glucocorticoid steroids, hydrocortisone and corticosterone, are secreted by the adrenal cor­tex, and their synthesis and release are under the control of adrenocorticotrophic hormone (ACTH), released by the anterior pituitary gland. Gluco­corticoids influence carbohydrate and protein metabolism, but also have significant anti­inflammatory and immunosuppressive activity, for which they are often used therapeutically. Among the most commonly used glucocorti­coids, which have minimal mineralcorticoid effects, are prednisolone, methylprednisolone, dexamethasone, and betamethasone (see 1-3 for reviews).

Methylprednisolone is routinely used in the treatment of acute spinal cord trauma (4), where it is thought to reduce inflammation and intra­cellular calcium concentrations, thereby limiting secondary injury (5,6). Ariyasu and colleagues (7) have also reported that short-term methyl­prednisolone treatment can reduce vertigo due to conditions such as peripheral vestibular neu­ritis, raising the possibility that glucocorticoid steroids may be of general use in the manage­ment of vestibular disorders. Although, ini­tially, the anti-inflammatory properties of meth­ylprednisolone seemed to be the most likely explanation for its beneficial effects in the treat-

RECEIVED 6 January 1997; ACCEPTED 18 January 1997.

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ment of acute vestibular vertigo, recent animal studies have suggested another possible mecha­nism of action (8,9).

Yamanaka and colleagues (8) reported that sys­temic (iv) administration of dexamethasone fol­lowing unilateral labyrinthectomy (UL) in rab­bits resulted in a dose-dependent acceleration of vestibular compensation. This effect was blocked by a selective type IT glucocorticoid receptor antagonist (RU38486). In labyrinthine-intact anesthetized rabbits, Yamanaka and colleagues also found that iontophoretic application of dex­amethasone produced a dose-dependent in­crease in the fIring rate of type I medial vestibu­lar nucleus (MVN) neurons, which could be blocked by RU38486. Jerram and colleagues (9) reported that systemic (sc) administration of methylprednisolone, before and at 4 h post-UL, reduced the frequency of spontaneous ocular nystagmus (SN) in guinea pigs. However, there were three main differences between the results of Jerram and colleagues (9) using methylpred­nisolone and those of Yamanaka and colleagues (8) using dexamethasone. First, although the frequency of SN was reduced by methylpred­nisolone, the rate at which it compensated was not increased as it was by dexamethasone. Sec­ond, whereas dexamethasone also accelerated the compensation of yaw head tilt and roll head tilt in rabbits, methylprednisolone had no bene­fIcial effects on these postural symptoms in guinea pigs. Third, while the benefIcial effects of dexamethasone increased in a dose-depen­dent fashion from 1 to 5 mglkg iv, the effects of methylprednisolone followed an inverted U­shaped dose-response function, with 30 mglkg sc being more effective than 15 or 60 mglkg sc. In addition to other differences between the two studies (that is, timing and frequency of injec­tions), methylprednisolone is known to have a higher affinity for glucocorticoid receptors than dexamethasone (1-3). Interestingly, in both stud­ies, the UL symptoms did not increase following the discontinuation of the steroid treatment.

In a further study using labyrinthine-intact anesthetized cats, Yamanaka and colleagues (10) found that iv administration or iontophore­sis of prednisolone resulted in a dose-dependent increase in the resting activity and response to horizontal rotation of type I and type IT MVN

c. A. A. E. Paul at al

neurons. The increase in fIring rate was gener­ally greater for type I neurons and was, on aver­age, similar for resting activity and response to head movement. Using iontophoresis, Yamanaka and colleagues found that the increase in fIring rate produced by prednisolone was not blocked by either a nonselective glutamate receptor an­tagonist (glutamic acid diethylester) or the cal­cium channel antagonist, cobalt chloride. The authors suggested that the cellular effects of prednisolone were not produced via modulation of glutamate release, postsynaptic glutamate re­ceptors, calcium channels, or cytosolic or nu­clear steroid receptors, but possibly via an ex­tracellular steroid receptor that is independent of the steroid binding site on the GABAA recep­tor (11). These results suggested the possibility that steroids like methylprednisolone and dexa­methasone might enhance recovery from vestib­ular disorders via an action on vestibular nu­cleus neurons themselves.

The aim of the present study was twofold: 1) to investigate the effects of dexamethasone on vestibular compensation of SN following UL in guinea pig, and 2) to investigate the effects of dexamethasone and methylprednisolone on guinea pig MVN neurons in brains tern slices in vitro.

Methods

Behavioral Experiment

Data were obtained from 20 male and female pigmented guinea pigs (300 to 950 g) housed in pairs in an animal holding rpom with a 12-h light/dark cycle; food and water were available ad libitum. Prior to each experiment, animals were brought into the laboratory and housed in­dividually in boxes with perspex windows at the front to facilitate video recording. Following the UL, they remained in these boxes for the re­mainder of the experiment.

The animals were randomly divided into 4 groups: Group 1 (n = 5) received an injection of 5 mglkg dexamethasone in a cyclodextrinl distilled water vehicle (Sigma) at 0, 12,24, and 36 h post-UL; Group 2 (n = 5) received 10 mg! kg dexamethasone in cyclodextrin at the same times; Group 3 (n = 5) received 40 mglkg dexa-

Steroids and Vestibular Compensation

methasone in cyclodextrin at the same times; Group 4 (n = 5) received 0.5 mL/kg of the cy­clodextrin vehicle (157.5 mglmL) at the same times post-UL. The injection schedule was in­tended to be an approximation of that used by Yamanaka and colleagues (8) in their study us­ing rabbits. All injections were delivered ip, and the injection volume was 0.5 mLlkg in all cases except for the 40 mg/kg dose used in Group 3, in which the injection volume was 1.5 mLlkg, due to solubility problems. Previous studies us­ing similar ip injection volumes have shown that volumes between 1 and 2 mL do not affect the compensatiofl process per se (12).

Prior to labyrinthine surgery, animals were anesthetized with 0.4 mUkg im fentazin (0.4 mg/mL fentanyl citrate, 58.3 xylazine HCI, 3.2 mglmL azaperone; Parnell, New Zealand) (13). Wound margins and pressure points were in­fused with 2 % procaine, and heart rate was monitored using ECG electrodes inserted in the forelimb muscles. The right temporal bone was exposed using blunt dissection and the vestibu­lar labyrinth was visualized using a dental drill with a fine burr under microscopic control. The ampullae of the horizontal and anterior semicir­cular canals were opened and the contents aspi­rated; the utricle, saccule, and posterior canal ampulla were also probed, aspirated, and de­stroyed. The UL was deemed complete when all of these structures had been removed and clear ipsilateral deviation was observed in the right eye. At the end of the surgery, antibiotic cream (Bactroban (mupriocin 2%» was topically ap­plied to the opened labyrinth to prevent infec­tion, and the temporal bone was sealed with dental cement. The wound was sutured and the animal allowed to recover in light.

Only the frequency of spontaneous ocular nystagmus (SN) was quantified, as we have found this to be the most reliable behavioral in­dex of static compensation in the guinea pig (for example, 12). All measurements were per­formed by an experimenter who was blind to the experimental conditions, and all solutions were coded to ensure this. Measurements were made at 10, 25, 30, 45, and 50 h post-UL. SN frequency was measured by gently retracting the skin behind the animal's left eye to expose the sclera and counting the number of SN quick

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phases in a 15-s interval ("beats/ISs"), as de­fined by an electronic timer that emitted an au­dio signal at the end of the specified period. This procedure was performed 5 times at each measurement time and the means obtained. SN was then videotaped using a video camera (pa­nasonic NV-M7) and the frequency verified. The SN quick phase is easily seen as a rapid, large amplitude, mainly horizontal eye move­ment, contralateral to the UL (12,13). Measure­ments were made only when the animal's head was stationary to avoid contamination of SN by vestibulo-ocular reflex nystagmus induced by head movement. Animals were allowed to choose a natural posture and were not restrained in any way since stress induced by restraint has been observed to increase SN frequency. We estimate that the measurement error involved in using the video method of analyzing SN fre­quency is of the order of 1 beat/IS s (9,12, 13).

A single 2-factor analysis of variance (ANOV A) with repeated measures on time was performed on the data (Statview 512+ package). Factor A represented the drug effect on SN fre­quency; Factor B, the repeated measure, repre­sented time; and the interaction (AB) repre­sented the change in the rate of compensation as a result of drug treatment (12). As Factor B, the repeated measure, was usually significant (that is, indicating that compensation had occurred), it will not be discussed further. The significance level was set at 0.05 for all comparisons.

In Vitro Experiment

Data were obtained from a total of 29 MVN neurons in brainstem slices from 10 labyrin­thine-intact pigmented and albino guinea pigs (300 to 500 g), which were anesthetized with ether and decapitated. The brainstem was rap­idly dissected and submerged in chilled artifi­cial cerebrospinal fluid (ACSF, see below). Coronal slices containing the MVN, approxi­mately 500 !J.m thick, were cut using a Stoelting tissue chopper. The MVN was easily identified because within the rostrocaudal co-ordinates that we used, the MVN in a coronal slice is bor­dered laterally by the lateral vestibular nucleus, medially by prepositus hypoglossi, ventrally by

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the reticular formation, and dorsally by the IVth ventricle (14).

Slices were incubated in an immersion slice chamber at 28 to 30°C for 1 to 2 h prior to re­cording and were continuously superfused with ACSF (in mM): NaCl (126.0), KCI (5.0), KH2P04 (1.25), MgS04 (1.3), NaHC03 (26.0), glucose (10.0), CaCh (2.5). The ACSF was continuously bubbled with 95% O2 and 5% CO2

and maintained at a pH of 7.4. The time from decapitation until immersion in the slice cham­ber was always less than 12 min (14). During recording, the chamber temperature was main­tained at 33 to 35°C using a temperature control unit. ACSF flow rate was maintained at 2.0 to 2.5 mL/min using a flow meter, resulting in a chamber turnover time of approximately 2 min.

Spontaneous action potentials from single MVN neurons were recorded extracellularly us­ing glass micropipettes filled with 2 M NaCI (impedance 3.0 to 6.0 Mil) and fast green FCF, to facilitate visualization of the electrode tip. The electrode was advanced through the slice in 1 to 2-j.Lm steps using a Narishige nanostepper. Signals were amplified (X 10) using a Dagan 8100-1 amplifier (low-pass filtered at 30 kHz), displayed on an Iwatsu digital storage oscillo­scope and monitored using a Grass audiomoni­tor. For the purposes of analyzing action poten­tial waveforms, signals were sampled from the Dagan amplifier by a MacLab data acquisition system (20 kHz sampling frequency) and dis­played on a Macintosh LC ill computer using a Chart program. In order to analyze action po­tential frequency, signals from the Dagan am­plifier were fed into a MacLab bioamplifier (fil­tered at 0.3 Hz and 2.5 kHz, with a notch filter set at 50 Hz) and displayed on the second chan­nel of the oscilloscope. Action potentials from a single neuron were isolated using a window dis­criminator, and the action potential frequency was converted to a voltage using a custom­made frequency-to-voltage converter. A voltage proportional to action potential frequency (ac­curacy ± 0.5 Hz) was displayed on the LC ill computer using the MacLab data acquisition system. Histograms of action potential fre­quency over time were displayed using a Chart program and printed using a laser writer dedi­cated to the recording system (14).

C. A. A. E. Paul et al

Dexamethasone in a cyclodextrin vehicle (Sigma) was dissolved in ACSF to a concentra­tion of 1 j.LM. Methylprednisolone (6 a-methyl­prednisolone 21-hemisuccinate, sodium salt; Sigma) was dissolved in ACSF to a concentra­tion of 10 nM or 0.1 j.LM; both dexamethasone and methylprednisolone were applied to the slice by superfusion. These concentrations were chosen on the basis of previous studies which have shown that, applied in vitro, such steroids produce their maximal responses in the nM to j.LM range (15-18). Concentrations of methyl­prednisolone that were lower than those of dex­amethasone were used because the former is known to have a higher affinity for glucocorti­coid receptors than the latter (1-3). For each neuron, baseline firing rate was recorded for ap­proximately 4 min during superfusion of ACSF alone, then the drug was applied for 4 min (that is, twice the turnover time), followed by a re­turn to the control ACSF solution for 4 min. In cases in which a neuron stopped firing com­pletely, the drug solution was turned off imme­diately and the ACSF solution turned on again. Firing frequency was considered to have in­creased or decreased from baseline when a change of greater than or equal to 20% of base­line occurred. This conservative criterion has proven useful to exclude smaIl changes in firing rate caused by extraneous variables, particularly for neurons with low firing rates (14). Only neurons that showed reversible changes in fir­ing frequency were analyzed in order to exclude changes produced by cell damage.

Results

In the behavioral study, none of the 3 doses of dexamethasone resulted in a significant change in the frequency of SN or the rate of SN com­pensation, relative to the vehicle control group (see Figure 1).

In the in vitro study, only 1/9 MVN neurons exhibited a change in firing rate in response to 1 j.LM dexamethasone, and this neuron showed a 100% decrease in firing. Only 3 out of 13 neu­rons tested with 10 nM methylprednisolone re­sponded, one with a decrease (that is, 100% de­crease from baseline) and two with increases in

Steroids and Vestibular Compensation

20

'U -0- veh II III -.- dexlS II) 15 -- dexl10 ... -.- dexl40 .!! III II e. 10 >-0 s:: II ::I cr 5 ~ z Ul

0

+ 10+ +25 30 35+ 45 50

tIme post-UL (hs)

Figure 1. CompensatIon of spontaneous nystagmus (SN) frequency In guinea pigs receiving Ip vehicle in­Jections or one of 3 doses of dexamethasone. Sym­bols represent means, bars :t 1 SO (n = 5 per group).

firing rate (33% and 43% increase from base­line, respectively). Three out of seven neurons responded to 0.1 f.LM methylprednisolone, one with a decrease (100% decrease from baseline) and two with increases (150% and 167% in­crease from baseline, respectively (see Figure 2).

Discussion

In contrast to the results of Yamanaka and colleagues (8) using rabbits, we found no ef­fects from 3 different doses of dexamethasone on the compensation of SN in guinea pig. A number of possible explanations for this dis­crepancy must be considered. First, it is possi­ble that because we used ip rather than iv ad­ministration, even the highest dexamethasone dose (40 mg/kg, ip) used in our study resulted in a lower blood concentration of dexamethasone than the highest dose used in the study by Ya­manaka and colleagues (5 mg/kg, iv). In partic­ular, since ip injections are subject to flrst-pass metabolism by the liver, the dexamethasone that we administered could have been partially metabolized before reaching the systemic circu­lation. However, this explanation is very un­likely. According to previous studies in lower mammalian species, the bioavailability of dex­amethasone following oral or ip administration is between 66% and 81 % (19-25); therefore, an

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ell lncntalla ~ dec:reaaa o nochange

80

~ c '6 c &. 60 .. ! .. c e :0 40 .. c '0 ;/I.

20

0 10 nM math 0.1 11M math 1 liM dax

Figure 2. Percentage of MVN neurons responding to 10 nM (n = 13) or 0.1 JLM (n = 7) methylprednisolone ("meth"), or 1 JLM dexamethasone ("dex") (n = 9), with an Increase, decrease, or no change In firing rate.

ip dose of 40 mglkg would have to be equiva­lent to at least 26 mg/kg iv, which is still 5 times higher than the maximal dose used by Ya­manaka and colleagues (8). Guinea pigs that re­ceived 40 mg/kg ip dexamethasone also exhib­ited the restlessness and irritability that is characteristic of animals receiving high doses of steroids. A second possible explanation is that the acceleration of vestibular compensation that Yamanaka and colleagues observed with dex­amethasone requires a longer period of time post-UL: whereas SN compensation in guinea pigs takes 2 to 3 days, it takes 4 to 5 days in rab­bits (8). However, as in Yamanaka and col­leagues' study, we spaced the injections through­out the compensation period so that blood plasma levels of the drug were maintained. A third possibility is that the discrepancy is due to the different methods of vestibular deafferenta­tion employed in the two studies: whereas Ya­manaka and colleagues (8) used a chemical UL, we used a surgical UL. SN compensation has been shown to have different time courses fol­lowing chemical and surgical ULs (for exam­ple, compare references 8 and 12); furthermore, if the injection of an ototoxic drug into the mid­dle or inner ear produces an inflammatory re­sponse, then steroids such as dexamethasone might reduce this inflammation. Finally, it is possible thal a ::.pecies difference accounts for

206

the discrepancy between our results and those of Yamanaka and colleagues (8). Although Jer­ram and colleagues (9) found that methylpred­nisolone reduced the initial frequency of SN following UL in guinea pig, it should be noted that 1) no acceleration of SN compensation oc­curred: 2) this effect was obtained only when the methylprednisolone was administered be­fore as well as following the UL; and 3) unlike the linear, dose-dependent effect reported by Yamanaka and colleagues (8), the effects of methylprednisolone in guinea pig followed an inverted U-shaped dose-response function.

We also obtained electrophysiological results that were different from those of Yamanaka and colleagues (8,9). Whereas Yamanaka and col­leagues found that prednisolone and dexa­methasone produced a dose-dependent increase in MVN neuron firing rate in the anesthetized rabbit, we found that, in most cases, methyl­prednisolone (14 out of 20 tests) and dexa­methasone (8 out of 9 tests) had no effect on guinea pig MVN neurons in vitro; in 2 out of 6 cases in which neurons did respond to methyl­prednisolone, and 1 of 1 cases for dexametha­sone, the response was a decrease in spontane­ous flring rate rather than the increase reported by Yamanaka and colleagues. Using the in vitro brainstem slice, it was not possible for us to identify type I and type IT MVN neurons; there­fore, we cannot be certain that we were testing the same neuronal categories as Yamanaka and colleagues (8,9). It is also possible that MVN neurons in vitro and in vivo respond differently to steroids. However, we think that a likely ex­planation for the discrepancy is the difference in the drug concentrations used in the two experi­ments. Yamanaka and colleagues (8,9) used iontophoretic application of a 100 mM concen-

c. A. A. E. Paul at al

tration of dexamethasone and prednisolone, which we suggest is far beyond the physiologi­cal concentration range that would nonnally be present in the brain. Most electrophysiological studies have shown that steroid concentrations in the nM to 11M range are sufficient to produce responses (15-18), and in other studies we have observed some responses to methylpredniso­lone even at pM concentrations (Maclennan, Smith and Darlington, unpublished observa­tions). It is also possible that nonspeciflc effects of iontophoresis are responsible for the rapid in­creases in flring rate observed by Yamanaka and colleagues (26,27); superfusion, on the other hand, is associated with few artifacts.

The question of whether steroids such as me­thylprednisolone and dexamethasone accelerate vestibular compensation generally and whether they do so via a peripheral action or via action on the vestibular nucleus, is clinically impor­tant. Our results, in contrast to those of Ya­manaka and colleagues (8,9), suggest that nei­ther dexamethasone nor methylprednisolone accelerates SN compensation (although methyl­prednisolone may reduce initial SN frequency (9)) following surgical UL, and that neither of these drugs has a consistently excitatory action on MVN neurons. These data are consistent with the conclusion that steroids may be useful only in the treatment of vestibular disorders arising from inflammatory causes (7), and not following vestibular deafferentation in general.

Acknowledgments - This research was supported by a Project Grant from the New Zealand Neurological Foundation (to PS). We thank the technical staff of the Departments of Pharmacology and Psychology for their excellent assistance, and Annabelle Jerram for her role in some of the earlier experiments.

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