M.Sc. in SPACE STUDIES 2004/2005
Contralateral reversible labyrinth lesion in gerbils to probe
critical periods during vestibular compensation
Dr. Anis Karim
Individual Project I Report submitted to the International Space University in partial
fulfillment of the requirements of the M.Sc. Degree in Space Studies
August, 2005
Internship Mentor:
Dr. Shawn Newlands, Dr. Galen Kaufman, Dr. Sheryl Bishop
Host Institution:
University of Texas Medical Branch, Galveston, Texas, USA
ISU Academic Advisor:
Prof. Nikolai Tolyarenko
Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
ABSTRACT
Introduction: This study is an attempt to investigate the relationship between short and
long term plasticity in recovery from vestibular deficits, such as vestibular lesions and
microgravity, using rodent animal models (gerbils). Methods: The research protocol uses
Dr. Kaufman's rodent vestibular apparatus, which involves studying the vestibular
responses of rodents (gerbils) with a rodent centrifuge, using infrared video oculography
(non-invasive technique). First, Tetrodotoxin (TTX) is applied to the middle ear to
reversibly block vestibular information from reaching the brainstem. This, for instance,
mimics periodic exposure to microgravity. Later, this technique is combined with longterm vestibular lesions, like labyrinthectomy, to investigate the relationship between short
and long term plasticity in recovery from vestibular deficits, such as vestibular lesions and
microgravity. Another reason for these studies is to determine whether there is a critical
period for labyrinth input during vestibular compensation. Results: The observations
suggest that preventing contralateral vestibular input during vestibular compensation
delays the recovery of VOR gain. Moreover, there is a residual TTX effect which remains
for more than 24 hours. Blocking the “good” ear with TTX well after compensation causes
a severe phase disturbance. Conclusions: These experiments support consensus work about
the expected behavior of VOR gain and symmetry following surgical and reversible (TTX)
labyrinth lesions. These results also support the emerging evidence for a dynamic interplay
between sensory modalities and sources of compensatory input, and time, in the
compensation process. They suggest that acute contralateral vestibular input does support
acute compensation, but that this input might in fact delay chronic compensation.
Keywords: vestibular compensation, vestibulo-ocular reflex, .tetrodotoxin, microgravity.
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
TABLE OF CONTENTS
ABSTRACT…………………………………………………………………....i
INDEX OF FIGURES………………………………………………………..iii
1
INTRODUCTION………………………………………………….…...1
2
METHODS……………………………………………………………..2
3
RESULTS……………………………………………………………...7
4
CONCLUSIONS………….………..........................................…..14
5
REFERENCES……………………………………………………….15
6
APPENDIX A……………………………………………….………...16
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
INDEX OF FIGURES
Fig. 2-1: Gerbil skull………………………………………………………………….........2
Fig. 2-2: Dr. Kaufman’s rodent vestibular apparatus…………………….………………..3
Fig. 2-3: Gerbil placed inside the apparatus for testing…………………………...……….3
Fig. 2-4: Gerbil eye seen on the monitor with the pupil tracked by ISCAN system…….....4
Fig. 3-1 – 3-3: Result graphs……………………………………………………….……7-9
Fig. 3-4 – 3-10: Case by case example graphs…………………………………..……10-13
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
1
INTRODUCTION
When man first set his foot into spacedom, mankind was hurled into an exciting field of
study, research and application of spaceflight, enabling him to overcome the constraints in
space exploration. Since this ‘giant leap’, he has been going through a continuous and
dynamic course of scientific advancements.
1.1 THE NEED
Gravity – the very force essential for man’s survival on Earth – and its absence in space,
opens up opportunities to understand the adaptive and compensatory mechanisms of the
human body. All the sensory systems are affected; but the vestibular system which relies
on gravity shows the most acute response. Although the current space neuroscience
research is focused on this aspect, there is still a dearth of information on the underlying
vestibular compensatory mechanisms. Moreover, the long term effects of neurovestibular
dysfunction are relatively unknown. So there is a definite need for further research in this
field.
An understanding of the basic vestibular physiology would enable us to develop better and
effective countermeasures for human spaceflight. Moreover, it would also lead to better
management of vestibular lesions in patients.
1.2 THE OBJECTIVE
This study is an attempt to investigate the relationship between short and long term
plasticity in recovery from vestibular deficits, such as vestibular lesions and microgravity,
using rodent animal models (gerbils).
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
2
METHODS
2.1 EXPERIMENT OVERVIEW
The research protocol uses Dr. Kaufman's rodent vestibular apparatus, which involves
studying the vestibular responses of rodents (gerbils) with a rodent centrifuge, using
infrared video oculography (non-invasive technique). First, Tetrodotoxin (TTX) is applied
to the middle ear to reversibly block vestibular information from reaching the brainstem.
This, for instance, mimics periodic exposure to microgravity. Later, this technique is
combined with long-term vestibular lesions, like labyrinthectomy, to investigate the
relationship between short and long term plasticity in recovery from vestibular deficits,
such as vestibular lesions and microgravity. Another reason for these studies is to determine
whether there is a critical period for labyrinth input during vestibular compensation.
2.2 EXPERIMENTAL SUBJECTS
14 male gerbils (Meriones unguiculatus) weighing 60-80 grams were the subjects of this
study. Gerbils have a large and easily accessible vestibular labyrinth, which makes
surgeries easier. Their neurovestibular system is primitive and less complex, compared to
primates. These aspects make gerbils excellent animal models for studying vestibular
plasticity.
Fig. 2-1: Gerbil skull
2.3 DR. KAUFMAN’S RODENT VESTIBULAR APPARATUS
The rodent rotating apparatus (centrifuge) was used to provide the vestibular and
optokinetic stimulation to the gerbils. It was capable of movement in four independent
rotational axes:
International Space University, MSS ‘05
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
•
•
•
•
The main earth vertical rotor
The eccentric earth vertical rotor
A pitch or roll platform (±30 degrees) where the animal and camera were mounted
A horizontal optokinetic drum for visual stimulation
Fig. 2-2: Dr. Kaufman’s rodent vestibular apparatus
Fig. 2-3: Gerbil placed inside the apparatus for testing
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
The apparatus is controlled by a custom LabView (National Instruments) software
MotionControl. Another custom program, DataStore, was used for data storage. A
computer with two monitors was used for software run. Two television monitors captured
the left and right eye images from the ISCAN cameras in the apparatus. ISCAN (RK-426)
Pupil/Corneal Reflection Tracking System tracked the eye position in response to a
stimulus. Another small monitor displayed the image of the gerbil in the centrifuge. All
these are set up in a dark room to prevent stray illumination from reaching the gerbil’s field
of view.
Fig. 2-4: Gerbil eye seen on the monitor with the pupil tracked by ISCAN system
2.4 TETRODOTOXIN
Tetrodotoxin (TTX) is a heterocyclic, small, organic molecule that acts directly on the
electrically active sodium channel in nerve tissue (neurotoxin). It blocks diffusion of
sodium through the sodium channel, preventing depolarization and propagation of action
potentials in nerve cells. All toxicity is secondary to the action potential blockade.
Tetrodotoxin acts on the central and the peripheral nervous systems (ie, autonomic, motor
and sensory nerves). It also stimulates the chemoreceptor trigger zone in the medulla
oblongata and depresses the respiratory and vasomotor centers in that area.
The amount of TTX used for every injection in these experiments was 10 microliters of 0.3
mM TTX in PBS of pH 5. In a previous study in the rat, it was 75 microliters of 0.1 M
(Saxon)
2.5 EXPERIMENT PROTOCOL
The experiment had 3 phases:
Phase
I
Procedure
Obtaining baseline control data for the gerbils
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
II
III
TTX Protocol (Performing labyrinthectomy + Application of TTX + Data
collection)
Data analysis
Phase II involved 14 gerbils divided into the following 4 groups:
•
•
•
•
TTX dose/response
Surgical Unilateral Labyrinthectomy (UL)
UL + 1 day contra TTX block
UL + 5 day contra TTX block
The VOR testing (Vestibulo-Ocular Reflex using video-oculography) was done at the
following time-points:
Group
TTX dose/response
Surgical Unilateral Labyrinthectomy (UL)
UL + 1 day contra TTX block
UL + 5 day contra TTX block
VOR Testing Time-points
3, 7, 24, 36, 48 hours and 3, 7, 14 days
1, 3, 7, 14 days
3, 7, 24, 36, 48 hours
3, 7, 24, 48 hours and 3, 4, 5 days
The following is the status of the gerbils at the end of the experiments:
(The gerbils are designated AK1 – AK14)
Animal
AK1
AK2
AK3
AK4
AK5
AK6
AK7
AK8
AK9
AK10
AK11
AK12
Procedures done
TTX dosing
TTX dosing
6/6/05 – TTX dosing
6/10/05 – Bilateral TTX
6/9/05 – TTX dosing
6/14/05 – TTX Left TM injection
6/14/05 – Right UL
7/1/05 – TTX Right TM injection
6/14/05 – Right UL
7/1/05 – TTX Left TM injection
6/29/05 - Right UL, TTX Left TM injection
8/5/05 - TTX Left TM injection
6/29/05 - Right UL, TTX Left TM injection
Fate
Died after injection
Died after injection
Perfused on 6/14/05
Perfused
Alive
Had to be sacrificed on
7/3/05 due to separation of
head bolt
7/6/05 - Right UL, TTX Left TM injection Had to be sacrificed on
7/9/05 due to separation of
head bolt
7/6/05 - Right UL, TTX Left TM injection Died after injection
7/29/05 - Right UL, TTX Left TM injection Alive
7/29/05 - Right UL, TTX Left TM injection Alive
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
AK13
AK14
8/1/05 - Right UL, TTX Left TM injection
8/2/05 - TTX Left TM injection
8/1/05 - Right UL, TTX Left TM injection
8/2/05 - TTX Left TM injection
Died after injection on
8/2/05
Died after injection on
8/2/05
2.6 DATA ANALYSIS
Two custom software, DataView and Unit Rates, written in LabView were used to convert
the raw eye position data to the final processed data (gain, phase shift). Then, statistical
programs like SPSS were used for systematic data analysis.
The vestibulo-ocular reflex (VOR) data was analysed to calculate the half-cycle gains. The
“perfect” gain is 1.0, which is close to what is observed when a head stimulus is applied
with a visual surround acting synergistically. This is vestibulo-ocular reflex + optokinetic
reflex (VOR + OKR). In the dark, VOR is ~0.8 typically.
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
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RESULTS
Although the number of gerbils tested was low (N=14), the results obtained were
promising.
Figure 3-1 shows right eye, leftward head movement, VOR gains in the dark (0.5 Hz, 60
d/s) for six different treatment groups, and reveals most of the expected behavior due to the
experimental manipulations. Three time points are shown: “0” hours, or the response before
any manipulation has been done (pre), and 3 and 24 hours following surgical vestibular
lesion, TTX injection, or both. All groups are merged for the pre values and revealed a
VOR gain value of ~0.85. At 3 hours post-treatment, UL animals are still gradually
declining their VOR gain until a nadir at 24 hours (data not shown, but see Shinder et al,
2004,5). However, TTX alone, and a TTX lesion on the “good” side after chronic
compensation (blue, TTXlatecontra) acts quickly to lower the gain to almost zero at 3 hours.
TTX given on the lesion side chronically has no effect on gain (green).
At 24 hours, the group that was given TTX on the side contralateral to surgical lesion
remained lower in VOR gain response than the UL group with lesion alone. This finding
appears to be consistent for the other directions of head movement and the left eye in most
of the data to date. The observation suggests that preventing contralateral vestibular input
during vestibular compensation delays the recovery of VOR gain.
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
However, Figure 3-2 (VOR gain in the dark across frequencies and velocities) suggests
that over time, the UL+TTX group might actually improve VOR gain compared to the UL
group. This preliminary data has far-reaching implications for the importance of peri-lesion
inputs.
The persistence of the TTX effect is missing, since data beyond 24 hours is not available
in the gerbil. However, while spontaneous nystagmus generally was gone by 24 hours in
TTX-only treated gerbils, the very low VOR gain, and the observation that additional TTX
application (day 2 in the 5 day group) kills the animal, suggest that a residual TTX effect
remains for more than 24 hours.
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
Figure 3-3 shows the phase relationship (in degrees) between stimulus (0.5 Hz, 60 d/s
maximum velocity horizontal sines) and the slow-phase eye response from the same data
as shown above. A typical phase response is a slight lag, but here the UL+TTX group
appears to have a slightly greater lag relative to UL or TTX alone. Blocking the “good” ear
with TTX well after compensation causes a severe phase disturbance (blue).
3.1 CASE BY CASE EXAMPLES
The following 7 figures use a plot of eye velocity response versus head stimulus velocity
to show the asymmetry of the eye response to different directions of head movement.
Negative rotator values correspond to leftward or counter-clockwise horizontal rotation.
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
Figure 3-4 shows the relatively symmetric response in the pre group, or the normal
response in the dark at 0.5 Hz, 60 d/s sinusoidal stimulation.
Figure 3-5 shows that the half-cycle response to leftward head movement after a left TTX
injection 24 hours previously is severely reduced. Movement rightward still had a strong
VOR gain.
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
Figure 3-6 shows a flat rightward response in an animal that received surgical lesion to the
right side 24 hours previously. The head left VOR gain is poor but relatively healthy.
Figure 3-7 shows an almost flat response at 24 hours following surgical vestibular lesion
on the right side, and an injection of TTX on the left side at the time of surgery.
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
Figure 3-8: Bilateral TTX application at 3 hours
Figure 3-9: Bilateral TTX application at 24 hours
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
Figure 3-10: TTX injected through the contralateral tympanic membrane (TM) after
chronic compensation to a right surgical lesion. The weak leftward and absent rightward
responses suggest a reorganization of the commissural VOR reflexes due to the
compensation process that flip the expected response in an acute situation.
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
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CONCLUSIONS
These experiments support consensus work about the expected behavior of VOR gain and
symmetry following surgical and reversible (TTX) labyrinth lesions. The use of TTX in
gerbils is novel, but generates responses expected from other studies. The gerbil appears to
be much more sensitive to TTX than other rodents, possibly due to thinner bone and
membrane structures around the ear. The pharmacodynamics of the TTX effect, and its
possible effect on extra-labyrinth structures, remains controversial.
These results also support the emerging evidence for a dynamic interplay between sensory
modalities and sources of compensatory input, and time, in the compensation process. They
suggest that acute contralateral vestibular input does support acute compensation, but that
this input might in fact delay chronic compensation. While more data and statistical power
will be required to bear out these findings, the use of reversible labyrinth lesion has opened
a new window into the mechanism of vestibular compensation.
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
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REFERENCES
Kaufman, G. D. (2002). "Video-oculography in the gerbil." Brain Res 958(2): 472-87
Kaufman, G. D., M. Ramanathan, et al. (2005). "Microarray analysis of vestibular
compensation in the gerbil." Journal of the Association for Research in Otolaryngology In
preparation
Kaufman, G. D., T. Weng, et al. (2005). "VOR adaptation and CNS Fos expression
following centripetal Coriolis stimuli in the gerbil." J Vestib Res In Press
Kaufman, G. D., M. E. Shinder, et al. (2000). "Convergent properties of vestibular-related
brain stem neurons in the gerbil." J Neurophysiol 83(4): 1958-71
Kaufman, G. D., M. E. Shinder, et al. (1999). "Correlation of Fos expression and circling
asymmetry during gerbil vestibular compensation." Brain Res 817(1-2): 246-55
Kaufman, G. D., J. H. Anderson, et al. (1993). "Otolith-brain stem connectivity: evidence
for differential neural activation by vestibular hair cells based on quantification of FOS
expression in unilateral labyrinthectomized rats." J Neurophysiol 70(1): 117-27
Kaufman, G. D., M. J. Mustari, et al. (1996). "Transneuronal pathways to the
vestibulocerebellum." J Comp Neurol 370(4): 501-23
Saxon, D.W., J.H. Anderson and A.J. Beitz (2001) Transtympanic tetrodotoxin alters the
VOR and Fos labeling in the vestibular complex. NeuroReport, 12: 3051-3055
Saxon, D.W. (2003a) Asymmetric Fos labeling in lobule X of the cerebellum following
transtympanic tetrodotoxin (TTX) in the rat. Neurosci. Lett. 339: 57-61
Shinder, M. E., A. A. Perachio, et al. (2005b). "Adaptation of the Gerbil VOR and the Fos
response." Brain Res in review
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Contralateral reversible labyrinth lesion in gerbils to probe critical periods during vestibular compensation
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APPENDIX A
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