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
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ABSTRACT |
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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.
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INTRODUCTION |
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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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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, = (
tor,
ver,
hor), was computed from the eye position
rotation vector, E, as
= 2(dE/dt + E
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
has the components:
x' =
ralp =
tor/
+
ver/
along the
intersection of the yaw and larp plane,
y' =
larp =
tor/
+
ver/
along the
intersection of the yaw and ralp plane and
z' =
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, , 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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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
-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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RESULTS |
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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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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
(ralp,
larp,
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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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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DISCUSSION |
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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
).
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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