Central Versus Peripheral Origin of Vestibuloocular Reflex Recovery Following Semicircular Canal Plugging in Rhesus Monkeys

Bernhard J. M. Hess,1 Anna Lysakowski,2 Lloyd B. Minor,3 and Dora E. Angelaki4

 1Department of Neurology, University Hospital Zurich, CH-8091 Zurich, Switzerland;  2Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, Illinois 60612;  3Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins University, Baltimore, Maryland 21287; and  4Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110


    ABSTRACT
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ABSTRACT
INTRODUCTION
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Hess, Bernhard J. M., Anna Lysakowski, Lloyd B. Minor, and Dora E. Angelaki. Central Versus Peripheral Origin of Vestibuloocular Reflex Recovery Following Semicircular Canal Plugging in Rhesus Monkeys. J. Neurophysiol. 84: 3078-3082, 2000. We have previously shown that there is a slowly progressing, frequency-specific recovery of the gain and phase of the horizontal vestibuloocular reflex (VOR) in rhesus monkeys following plugging of the lateral semicircular canals. The adapted VOR response exhibited both dynamic and spatial characteristics that were distinctly different from responses in intact animals. To discriminate between adaptation or recovery of central versus peripheral origin, we have tested the recovered vestibuloocular responses in three rhesus monkeys in which either one or both coplanar pairs of vertical semicircular canals had been plugged previously by occluding the remaining semicircular canals in a second plugging operation. We measured the spatial tuning of the VOR in two or three different mutually orthogonal planes in response to sinusoidal oscillations (1.1 Hz, ±5°, ±35°/s) over a period of 2-3 and 12-14 mo after each operation. Apart from a significant recovery of the torsional/vertical VOR following the first operation we found that these recovered responses were preserved following the second operation, whereas the responses from the newly operated semicircular canals disappeared acutely as expected. In the follow-up period of up to 3 mo after the second operation, responses from the last operated canals showed recovery in two of three animals, whereas the previously recovered responses persisted. The results suggest that VOR recovery following plugging may depend on a regained residual sensitivity of the plugged semicircular canals to angular head acceleration.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Surgical occlusion of the membranous duct of a semicircular canal has been assumed to block completely the sensitivity to angular head acceleration. Based on this hypothesis, semicircular canal plugging has been used to study the contribution of individual semicircular canals to the vestibuloocular reflex (VOR) and to characterize its adaptive capacity to alterations of the peripheral vestibular input (Angelaki and Hess 1996; Angelaki et al. 1996; Baker et al. 1982; Böhmer et al. 1985; Broussard et al. 1999; Lasker et al. 1999; Yakushin et al. 1995, 1998). Recent pioneering work on the hydromechanics of the vestibular labyrinth has provided solid theoretical and experimental evidence that semicircular canal afferent responses may not only be due to endolymph flow but also to changes in macromechanical pressure (Rabbitt et al. 1994, 1995). This novel view of the semicircular canal mechanics has had further implications on previously widely accepted, yet unproven, assumptions. In fact, it has been clearly demonstrated in the toadfish that occlusion of a semicircular canal duct does not completely block the afferent response (Rabbitt et al. 1998, 1999). The residual response of plugged semicircular canal afferents, which is phase-shifted by approximately 90° relative to afferent responses from intact canals, has been explained by labyrinthine pressure gradients that lead to mechanical duct and cupula deformations (Rabbitt et al. 1999). In agreement with these novel findings on semicircular canal hydromechanics, Yakushin et al. (1998) have suggested that the high-frequency recovery of VOR in rhesus monkeys after semicircular canal plugging could be modeled by a simple change in the dynamics of the plugged canal afferents. In a previous study, we had put forward the hypothesis that the changes of VOR observed after plugging of selective semicircular canal planes could at least partly be due to signals originating from the remaining intact canals (Angelaki and Hess 1996; Angelaki et al. 1996). In fact, central adaptation can still exist even in the presence of a change in the peripheral dynamics of the plugged canals, particularly given the altered spatiotemporal response characteristics of the VOR in plugged animals (Angelaki and Hess 1996).

In this paper, we directly investigate this issue by studying VOR recovery in three rhesus monkeys after plugging of one or two coplanar vertical semicircular canal pairs. It is shown that the spatial tuning of partially recovered responses in a follow-up study of more than 1 yr remains virtually unchanged after additional plugging of the remaining semicircular canals. Our findings support the notion that recovery of vestibuloocular responses after complete histologically verified plugging of semicircular canals originates mainly from peripheral recovery processes and changes in the response dynamics of the semicircular canals and of vestibular-nerve afferents.


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INTRODUCTION
METHODS
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Animal preparation and eye movement recording

Experiments were performed in three juvenile rhesus monkeys (Macaca mulatta) that were chronically prepared with scleral dual-search coils for three-dimensional eye movement recording and with skull bolts to restrain the head during the experiments. Horizontal, vertical, and torsional eye positions were digitized at a sampling rate of 833 Hz and stored on a computer for off-line data analysis. Eye positions were expressed as rotation vectors relative to a right-handed coordinate system, where the y axis was aligned with the interaural axis, and the z and x axes were rotated forward and downward by 15° relative to the stereotaxic horizontal (positive directions were leftward, upward and forward, respectively). The eye angular velocity vector, Omega  = (Omega tor, Omega ver, Omega hor), was computed from the eye position rotation vector, E, as Omega  = 2(dE/dt + E Lambda  dE/dt)/(1 + |E|2). In one animal, eye velocity was also expressed relative to a rotated coordinate system that was obtained by rotating the standard x, y, z coordinates around the z axis through 45° to the left side. In these new coordinates (x' ~ perpendicular to the ralp plane, y' ~ perpendicular to the larp plane, z' = z), the velocity vector Omega  has the components: Omega x' Omega ralp = Omega tor/<RAD><RCD>2</RCD></RAD> + Omega ver/<RAD><RCD>2</RCD></RAD> along the intersection of the yaw and larp plane, Omega y' Omega larp = -Omega tor/<RAD><RCD>2</RCD></RAD> + Omega ver/<RAD><RCD>2</RCD></RAD> along the intersection of the yaw and ralp plane and Omega z' Omega hor along the z axis that did not change.

To investigate the origin of response recovery that we had observed previously in the VOR following canal inactivation, we plugged all the semicircular canals in the two ears in two surgical sessions that were separated in time by more than 1 yr. In two animals (JE, TW), the vertical semicircular canals of the right and left ear were first plugged ("all vertical canal-plugged animals"). In the third animal (NA), only one coplanar pair of vertical semicircular canals was plugged in the first operation, namely the right-anterior semicircular canal on one side and the left-posterior canal on the other side ("ralp canal-plugged animal"). After a first recovery period during which the spatial tuning of the VOR was systematically tested (see following text), the animals underwent a second surgery in which the remaining semicircular canals were occluded. That is, the lateral canals were occluded in all vertical canal-plugged animals (JE, TW), whereas the lateral and remaining vertical canals were plugged in the ralp-canal plugged animal (NA). For the surgical occlusion, the respective semicircular canals were exposed on both sides, and a small hole was drilled in the bony wall of the canal. The membranous duct was then cut with the tip of a sharp knife. Subsequently the hole was filled with bone chips and fascia as previously described (Angelaki et al. 1996).

Experimental protocols

Postoperative testing and follow-up experiments were performed in each animal on the first postoperative day and at regular intervals over 12-14 mo following the first operation and 2-3 mo following the second operation. During the experiments, animals were seated in a primate chair with the head restrained at 15° nose-down relative to the stereotaxic horizontal (defined as "upright" position) to place the lateral semicircular canals approximately earth-horizontal. The animals were placed inside a motorized three-dimensional turntable that was completely surrounded by a light-tight sphere to study the eye movements in complete darkness. To maintain a constant level of alertness, a small dose of D-amphetamine (1.5 mg orally or 1 mg im) was administered before each experimental testing. In all experimental protocols, the axis of rotation was earth-vertical to avoid any dynamic contribution of the otolith-ocular system.

Identical protocols were delivered in complete darkness both before and at regular intervals after occlusion of the semicircular canal ducts. The spatial organization of the VOR was investigated by repositioning the animals relative to the axis of rotation and oscillating about different head axes in the pitch and roll plane (JE, TW) as well as in the left-anterior right-posterior semicircular canal plane (NA). The orientation of the axis of rotation relative to the animal's vertical axis in each of the roll, pitch, and larp planes was defined with a polar angle, alpha , according to the right-hand rule. For the roll plane, for example, a stimulus angle of 0° described a rotation in upright position, a stimulus angle of 90° a rotation in left ear-down position and a stimulus angle of -90° a rotation in right ear-down position (see Fig. 1, inset). Sinusoidal oscillations were delivered at 1.1 Hz, ±5° (35°/s peak velocity). Responses were evaluated by fitting a sine function to stimulus velocity and to each component of the desaccaded eye velocity trace using a nonlinear least-squares algorithm based on the Levenberg-Marquardt method. The gain of the horizontal, vertical and torsional response components was expressed as the ratio of the response amplitude and peak head velocity.



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Fig. 1. Recovery of vestibuloocular reflex (VOR) following consecutive plugging of the vertical and lateral semicircular canals (animal JE). A: response gain of horizontal (Omega hor), vertical (Omega ver), and torsional (Omega tor) slow-phase eye velocity as a function of head orientation in the roll plane 1 day () and 14 mo (*) after plugging of all vertical semicircular canals. B: persistence of recovered response 1 day () and 2 mo (diamond ) after plugging of the remaining lateral semicircular canals in a 2nd operation in the same animal. For comparison, the recovered response 14 mo after the 1st operation is also shown (*). - - -, tuning response curves in normal animals. Inset shows schematically the definition of the tilt angle (alpha ) to describe the roll tilt during earth-vertical axis oscillations. Head oscillations were always around an earth vertical axis at 1.1 Hz, ±5° (±35°/s).

An ANOVA with repeated measures was done on the differences between the recovered responses shortly before the second operation ("control") and the acute responses 1 day after all remaining canals had been plugged.

Histological verification of semicircular canals occlusion sites

After termination of the experimental tests, animals were deeply anesthetized with pentobarbital sodium and perfused transcardially with a phosphate-buffered 10% Formalin solution. The temporal bones were subsequently removed for histological examination. They were decalcified in EDTA, embedded in celloidin, and sectioned at 80 µm. Sections were then stained with hematoxylin and eosin, cleared with alpha -turpineol, and mounted on slides. The six plugging sites were identified in each animal, and complete plugging of the semicircular canal lumina was confirmed by inspection of histological sections at the level of light microscopy. The sensory epithelia of all canals and surrounding tissues were intact. In one animal (NA), the plugs for the horizontal (2nd operation) and posterior canals (1st operation) on the left side appeared to be touching the membranous ampulla. It is possible that the ampulla became fibrosed to the plug, and that this may explain the gradual loss of function that was observed in this animal after the second operation. A second possible explanation in this animal is that the wall between the vestibular and the facial nerve canal was very thin, almost nonexistent, representing a perilymphatic fistula.


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Spatial VOR tuning in animals with the vertical canals plugged

Acutely after plugging of all vertical semicircular canals, vertical eye movement responses were abolished during rotations about all axes. In contrast, the horizontal response was normal or only slightly affected (see Fig. 1A). A significant torsional response was also elicited in both vertical canal plugged animals during rotations about axes in the roll and pitch plane. This response component was cosine tuned with a peak gain of about 0.20-0.25 in upright position and 0 at left (right) ear-down head position (Fig. 1A). Because no such component can be recorded in control animals, this response must be interpreted as an unmasked input from the lateral semicircular canals to the vertical eye muscles that is normally compensated by a similar input from the vertical semicircular canals.

During the follow-up period of more than 1 yr after the first surgery, a twofold change in the spatial VOR response characteristics could be observed in these animals. First, there was a progressive recovery of the vertical response component that reached gains of ~0.2-0.4 during earth-vertical axis oscillations at right or left ear-down positions 14 mo after surgery (Fig. 1, A and B). Second, the torsional response component that was present immediately after plugging with a peak in upright position disappeared in the roll plane completely over time (Fig. 1). Similarly in the pitch plane, the torsional response observed acutely after the operation shifted its peak over time from upright to supine/prone position (for the chronic stage, see Fig. 2B).



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Fig. 2. Recovery of VOR following consecutive plugging of the vertical and lateral semicircular canals (animal TW). A: comparison of response gains of horizontal (Omega hor), vertical (Omega ver), and torsional (Omega tor) slow-phase eye velocity as a function of head roll orientation 14 mo (*) after plugging of all vertical semicircular canals and 1 day () and 3 mo (diamond ) after plugging of the remaining lateral semicircular canals. B: response gains measured in the same animal for different pitch orientations. - - -, tuning curves in normal animals. Insets show schematically the angle (alpha ) of head orientation defining the tilt in the roll and pitch plane. Head oscillations were always around an earth vertical axis at 1.1 Hz, ±5° (±35°/s).

Spatial VOR tuning in animals with partially recovered VOR after plugging of the remaining semicircular canals

To test the hypothesis whether the partially recovered torsional and vertical responses in these animals were due to some form of central adaptation or due to a residual response of the plugged canals, we plugged the remaining semicircular canals in these animals 12-14 mo after the first operation. Acutely after the second surgery, responses in the plane of the newly plugged canals were completely abolished. However, there was little change in the partially recovered tuning characteristics of the VOR during rotations in the planes of the previously plugged canals. In the two all vertical canal-plugged animals, the horizontal VOR gain dropped below ~0.1 on the first postoperative day (Figs. 1B and 2), indicating "successful" plugs of the remaining lateral semicircular canals. In both animals, the partially recovered vertical and torsional responses retained their spatial tuning characteristics, partially overlapping the responses obtained during rotations about different axes in the roll and pitch plane before the second surgery (Figs. 1B and 2). In the follow-up period after the second operation, the horizontal VOR recovered partially in both animals, reaching peak gains of about 0.25-0.30 after 3 mo.

The recovery of the spatial tuning was also tested in a third animal in which the right anterior/left posterior canals were previously plugged. Over a period of 12 mo, the torsional and vertical VOR partially recovered to reach gains of ~0.2-0.3 during rotation about axes in the larp plane. This combined torsional-vertical response recovery becomes more clearly apparent when the VOR is expressed in a 45° rotated coordinate system, which decomposes the response into a velocity component perpendicular to the ralp, larp and yaw plane (Omega ralp, Omega larp, Omega hor), as shown in Fig. 3. Following the second surgery, in which the two lateral and remaining vertical canals were plugged, little changes were observed in the partially recovered torsional-vertical response. This animal gradually lost all VOR responses over the course of 2 mo after the second plugging procedure.



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Fig. 3. Persistence of recovered VOR after plugging of the right-anterior and left-posterior semicircular canals (animal NA). Gain of slow-phase eye velocity, in rotated coordinates with components Omega ralp (~ perpendicular to ralp-plane), Omega larp (~ perpendicular to larp-plane), and Omega hor, is displayed as a function of head tilt in the larp-plane. The new coordinate system was defined by rotating the standard x, y, z coordinate system around the z axis through 45° to the left. Responses obtained 12 mo after plugging the semicircular canals in the ralp-plane (*) are compared with data 1 day () and 12 mo (diamond ) after plugging of the remaining vertical and lateral semicircular canals. Inset shows schematically the tilt angle (alpha ) for different head tilts in the larp-plane. Head oscillations were always around an earth vertical axis at 1.1 Hz, ±5° (±35°/s).

An ANOVA with repeated measures between the responses 12-14 mo after the first operation and the acute responses on the first day after the second operation of all three animals yielded no significant difference in the responses [F(1,47) = 1.7, P = 0.2]. Thus the occlusion of the remaining canals in the three animals had no significant effect on the recovered responses.


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This study shows that the frequency-specific partial recovery that can be observed in the VOR of rhesus monkeys after plugging of selective semicircular canal planes persists after additional surgical occlusion of the remaining semicircular canals. In two animals, we found that the recovered vertical responses following plugging of all four vertical semicircular canals remained virtually unchanged after surgical occlusion of the remaining lateral semicircular canals. Not only did the previously partially recovered vertical and torsional VOR persist, but there was also a consecutive partial recovery of the horizontal responses. In another animal, a similar response recovery following inactivation of a single pair of coplanar vertical semicircular canals also remained unchanged after plugging of the lateral and remaining vertical semicircular canals. This animal exhibited a progressive loss in the VOR gain over the weeks following the second surgery, perhaps due to fibrosis of the ampulla.

Until recently, it has been tacitly assumed that a complete and permanent obliteration of the canal lumen at the site of the plugging would eliminate the sensitivity of the semicircular canals to angular head acceleration. Recent studies in the toadfish have challenged this traditional view of rendering semicircular canals nonfunctional by surgical occlusion of their membranous duct (Rabbitt et al. 1998, 1999). Rather, plugging attenuates low-frequency stimuli (less than 0.1 Hz) by two or more orders of magnitudes and advances the response phase of primary afferent responses in the toadfish (Rabbitt et al. 1999). These results were explained on the basis of a hydrodynamical model where semicircular canal afferents respond not only to endolympth flow but also to dilatational pressure (Rabbitt et al. 1994, 1995, 1999). In line with these observations, Yakushin et al. (1998) reported that the high-frequency recovery of the VOR in canal-plugged macaques could be modeled as a reduction of the dominant time constant of the plugged canal.

In an earlier report, we noted that the horizontal VOR was minimal acutely after plugging during yaw rotations (Angelaki and Hess 1996; Angelaki et al. 1996). Over time, however, the gain of the horizontal VOR increased gradually in these animals, reaching values of about 0.4-0.5 during 1.1-Hz yaw oscillations 5 mo after lateral canal inactivation. This frequency-specific recovery was suggested to be due to a central adaptation in the semicircular canal-ocular pathways, although a peripheral effect was not excluded (Angelaki and Hess 1996; Angelaki et al. 1996). The present results point against a central adaptation effect. Thus the previously reported changes in the VOR properties seem to be due to spatiotemporal interactions of signals from the intact semicircular canals with a slowly recovering phase-shifted response from the orthogonal plugged canals (Angelaki and Hess 1996).

It remains unclear why in the first 1-2 wk after surgery the VOR appears to be completely abolished in the plugged semicircular canal plane. In the toadfish, it has been observed that rapid compression of the membranous duct causes a detachment of the cupula and loss of canal sensitivity to rotational stimuli (Rabbitt et al. 1999). In this preparation, afferent modulation was maintained only if the compression of the canal proceeded slowly over the course of about 3-5 min. This suggests that the initial suppression of VOR gain in the first 1-2 wk after plugging reflects cupula detachment from the apex of the crista ampullaris, which has been suggested to serve as a "relief valve" to accommodate excess transcupular pressure (Hillman 1974).

It is interesting to point out that plugging appears to result in a greater reduction of time constants in squirrel than in macaque monkeys. For example, canal plugging in squirrel monkeys is associated with a much lower recovery of gain although the same frequency-specific characteristics were noted (Lasker et al. 1999). Geometric factors such as the relationship between the transluminal diameter of the canal and its circumference may account for this difference. Alternatively, differences in the plugging techniques may have a role. Lasker et al. (1999) opened the bony canal and compressed the membranous canal with fascia and bone chips without transecting it. In the present and previous studies performed in macaques, the membranous duct was cut with a sharp knife and the fenestrum in the bony canal was filled with fascia and bone chips (Angelaki and Hess 1996; Angelaki et al. 1996; Yakushin et al. 1995, 1998). The findings in this study further demonstrate that canal plugging using this technique in macaques is not an optimal method to investigate central adaptation as we had originally assumed (Angelaki and Hess 1996; Angelaki et al. 1996).


    ACKNOWLEDGMENTS

We thank J.-I. Suzuki and S. Newlands, who performed the first plugging surgery of these animals, and S. Price for excellent histological preparation.

This study was supported by Swiss National Science Foundation Grant 31-47287.96 and National Institutes of Health Grants EY-10851 and DC-02521.


    FOOTNOTES

Address for reprint requests: B.J.M. Hess, Dept. of Neurology, University Hospital, Frauenklinikstrasse 26, CH-8091 Zurich, Switzerland.

Received 8 May 2000; accepted in final form 15 August 2000.


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