Download Displacement of the medial rectus pulley in superior oblique

Reports
IOVS, January 1998, Vol 39, No. 1
Displacement of the Medial
Rectus Pulley in Superior
Oblique Palsy
Robert A. Clark,1 Joel M. Miller,2 and
Joseph L Demer13
PURPOSE. The rectus extraocular muscles pass through
fibromuscular connective tissue pulleys that stabilize muscle paths and control the direction of muscle pull. The
authors investigated whether abnormal forces associated
with superior oblique palsy can cause displacement of
pulleys and muscle paths.
Coronal magnetic resonance imaging (MRI)
showing significantly reduced superior oblique cross-sectional areas and lack of contractile changes with vertical
gaze confirms that seven subjects had superior oblique
palsies. Binocular misalignment was quantified using the
Hess test. In those seven subjects with palsies and in 18
normal orbits, coronal MRI scans corrected to standardized head position were analyzed digitally to determine
muscle paths in primary gaze. Horizontal and vertical
coordinates of the pulleys, known histologically to lie just
posterior to the equator in primary gaze, were inferred
from these muscle paths.
METHODS.
Normal pulley coordinates were highly uniform.
Compared with both normal orbits and fellow orbits,
orbits with superior oblique palsies showed a statistically
significant 1.1 mm superior displacement of the medial
rectus pulley. No other pulley was displaced significantly
from normal. Computer simulation using a biomechanical
model of ocular statics showed that, in each case, the
pulley position shifts alone were insufficient to reproduce
the clinical pattern of strabismus.
RESULTS.
CONCLUSIONS. The excyclotorsion of the globe that accompanies superior oblique palsy does not systematically displace the pulleys of all the rectus muscles. The only
significant rectus muscle path change is for the medial
rectus muscle, and it may arise as a mechanical consequence of the atrophy of the adjacent superior oblique
muscle belly. Biomechanical modeling suggests that this
displacement of the medial rectus pulley alone does not
account for the pattern of strabismus observed in superior
oblique palsy. (Invest Ophthalmol Vis Set. 1998;39:
207-212)
From the Departments of 'Ophthalmology and 'Neurology, University of California, Los Angeles, and 2 Smith-Kettlewell Eye Research
Institute, San Francisco.
Supported by National Eye Institute consortium grant EY-08313
(JD, JM); core grant EY-OO331 (Department of Ophthalmology, University of California, Los Angeles); and core grant EY-06883 (SmithKettlewell Eye Research Institute). RAC is a Rosalind W. Alcott Fellow.
Submitted for publication June 19, 1997; accepted August 20,
1997.
Proprietary interest category: N.
Reprint requests: Joseph L. Demer, Jules Stein Eye Institute, UCLA,
100 Stein Plaza, Los Angeles, CA 90095-7002.
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207
ur understanding of the mechanics of rectus extraocular muscles (EOMs) and their surrounding connective tissues has been enhanced by the recent re-examination of orbital histology and by high-resolution magnetic
resonance imaging (MRI) in alert subjects. In the region of
posterior Tenon's capsule, behind the equator of the globe,
cross-sectional coronal orbital histology and immunohistochemistry demonstrate that each EOM passes through a connective tissue sleeve composed of collagen, elastin, and
smooth muscle.' These tissue sleeves have been shown by MRI
to stabilize EOM bellies relative to the orbit, permitting only
the insertional ends of muscle paths to move with globe rotations.2'3 Even large transpositions of EOM insertions fail to shift
posterior muscle paths.4 Functionally, these tissue sleeves act
as pulleys and become the mechanical origins for EOM action.
The positions of all EOM pulleys have been found to be
stereotypic in normal subjects, as might be expected for important mechanical structures.2'3 In addition, significantly abnormal pulley positions are associated with incomitant strabismus.3'5 The implications of pulley location for binocular
alignment can now be understood using computer simulations.
Displacement of the EOM pulleys radially (as would be associated with a large or small orbit) is not predicted to affect
binocular alignment, but displacement of the pulley location
perpendicular to the muscle's plane of action (i.e., horizontal
displacement of vertical EOM pulleys or vertical displacement
of horizontal EOM pulleys) is predicted by computer simulation to cause incomitant strabismus.3'5 These analyses suggest
that heterotopic pulleys may be an important cause of incomitant strabismus.3'5
O
Pulley displacement may also be a secondary effect of
ocular torsion associated with incomitant strabismus.6 In subjects with significant cyclotorsion, the mechanical forces exerted by the cyclotorted EOM insertions on the pulleys might
displace them systematically, creating heterotopic pulleys. To
examine the effect of ocular torsion on EOM pulley position,
we analyzed subjects with known superior oblique (SO) palsy
as a primary cause of excyclotorsion (displacement of the
superior fundus temporally) to determine whether their EOM
pulleys were significantly displaced.
METHODS
Seven subjects (14 orbits) confirmed to have unilateral or
bilateral SO palsy by SO muscle belly atrophy and impaired
contractility on dynamic MRI were compared with 10 normal
volunteers (18 orbits). Each normal volunteer was examined to
verify normal binocular alignment and the absence of strabismus. Each subject with SO palsy was tested for ocular torsion
measured using double Maddox rods. Each subject underwent
a Hess screen test to quantify binocular alignment in 21 fixation positions over a ±30° field for each eye.
After obtaining written, informed consent according to a
protocol conforming to the Declaration of Helsinki and approved by the Human Subject Protection Committee at the
University of California, Los Angeles, all subjects underwent
high-resolution, T,-weighted MRI using a superconducting 1.5
T General Electric Signa (Milwaukee, Wl) or Picker Vista
(Cleveland, OH) scanner according to techniques described in
detail elsewhere.2"5'7 In brief, each subject's head was stabilized using foam cushions and tape. Then a surface coil was
placed over the scanned orbit, and multiple contiguous coro-
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Reports
nal images 3 mm thick were obtained with a 256 X 256 matrix
over an 8- or 10-cm-square field of view, giving pixel resolutions of 312 or 390 jam, respectively. Fixation targets for
approximately 23° of eccentric gaze were provided inside the
scanner magnet. Images in straight-ahead gaze, elevation, and
depression were obtained for all subjects.
The digital MRI images were transferred to Macintosh
computers (Apple Computer, Cupertino, CA), converted into
8-bit tagged image file format (TIFF) using locally developed
software and quantified using the program NIH Image (W.
Rasband, National Institutes of Health; available by ftp from
"zippy.nimh.nih.gov" or on floppy disc from NTIS, 5285 Port
Royal Road, Springfield, VA 22161, part number PB955OO195GEI)- Images of left orbits were digitally reflected to the
orientation of right orbits to allow uniform analysis of EOM
positions.
Image position and orientation were normalized to facilitate quantitative comparisons across subjects. To normalize
position in the coronal plane, all rectus EOM positions were
translated to place the coordinate origin at the area centroid of
the orbit. Orientation in the coronal plane was normalized by
rotating the image to align the interhemispheric fissure of the
brain with the scanner-defined vertical meridian. Finally, the
anterior-posterior position was normalized by selecting the
image plane 3 mm anterior to the globe- optic nerve junction
for analysis in each subject. A more anterior image plane,
nearer the globe equator, would have better transected the
densest pulley regions, but the flatness of muscle tendons and
the density of connective tissue in the more anterior image
planes made it difficult to distinguish contours by which muscle or pulley position could be judged. The image plane 3 mm
anterior to the globe- optic nerve junction was the most anterior in which all rectus EOM bellies could be identified clearly
in every subject. The average locations and standard deviations
of the area centroids of EOM bellies in this plane were then
calculated for normal subjects. Pulley positions were estimated
as the positions of EOM area centroids. The normal EOM pulley
locations were then compared with EOM pulley locations in
subjects with SO palsy.
In normal subjects and in those with SO palsy, the crosssectional area of the SO muscle was measured in straight-ahead
gaze, elevation, and depression using analysis techniques described elsewhere.7 In brief, to determine SO muscle area and
contractility, the area of the SO muscle was measured sequentially in image planes posterior to the globe- optic nerve junction to isolate the image plane containing the largest crosssectional area in primary gaze. Then, the change in muscle
belly area from depression to elevation was measured in this
image plane (Fig. 1) and geometrically corrected to obtain a
true cross-section perpendicular to the long axis of the muscle.
Biomechanical modeling was performed using the Orbit
Gaze Mechanics Simulation program (vl.7, Eidactics, San Francisco, CA; hereafter referred to as "Orbit")9 running on Macintosh computers. Orbit simulates binocular alignment using
static force balance equilibrium equations involving innervations, eye positions, muscle forces, and such eye parameters as
the EOM insertions, contractilities, elasticities, lengths, stiffnesses, and pulley positions. The program then calculates the
mechanical state of the eyes based on equations and methods
given previously, in part, by Miller and Robinson.8
Pulley positions based on histologic studies' in Orbit's
description of a normal eye were taken as the starting point for
each subject.3'5 To simulate heterotopic pulleys, lateral-medial
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IOVS, January 1998, Vol 39, No. 1
and superior-inferior coordinates of Orbit's pulleys were altered to match simulated muscle paths to paths observed in the
MRI scans. To simulate SO palsy, SO contractility was reduced
(e.g., to zero), and SO elasticity was reduced based on contractility and atrophy observed by MRI. Small-length changes (~1
mm) were made to the horizontal muscles to improve thefitof
simulation to clinical alignment data; these may reflect small
horizontal heterophorias, which are prevalent in the normal
population. Because Orbit requires that the fixing eye obeys
Listing's Law, we only ran simulations with the palsied eye
following.
RESULTS
No normal subject had SO palsy as determined by MRI
criteria (normal maximum average cross-sectional area in primary gaze of 0.19 cm2 and minimum contractile change in
cross-sectional area from 23° elevation to 23° depression of
0.03 cm2).7 Each of two normal subjects had one heterotopic
EOM pulley, defined as a pulley displacement greater than two
standard deviations from normal.5 Based on a normal distribution of pulley position, this number is not unexpected. In one
subject, the right medial rectus (MR) pulley was shifted inferiorly 2.3 mm from normal. In the other subject, the right lateral
rectus (LR) pulley was shifted superiorly 1.8 mm from normal.
In both subjects, no other EOM pulley was displaced more than
one standard deviation from normal. Neither subject had any
clinical abnormality on examination or Hess screen test. In
computer simulation using the measured pulley positions, the
maximum calculated deviation in primary gaze for both subjects was 0.6° (1.1 prism diopters (pd)) of horizontal deviation,
1.0° (1.8 pd) of hyper deviation, and 1.3° of cyclotorsion, all
well within the range of binocular fusion.
The clinical characteristics of subjects with SO palsy are
summarized in Table 1. In two of the three subjects whose
history showed they had congenital SO palsy, the SO muscle
was absent in one orbit on MRI (Table 1, Fig. 1 inset). Of the
four subjects whose history showed they had acquired SO
palsy, one subject had bilateral SO atrophy and impaired contractility on MRI (Table 1). The other three subjects had unilateral SO atrophy and impaired contractility. Excyclotorsion of
subjects with SO palsy by double Maddox rod testing averaged
9.6° (range, 2-20°).
The average positions of the EOM pulleys of normal subjects and subjects with SO palsy are summarized in Figure 2.
Normal pulley coordinates were highly uniform, with maximum standard deviations of only 0.9 mm. The only statistically
significant difference in pulley position between the two
groups was superior displacement of the MR pulley 1.1 mm (JP
< 0.001). This superior displacement of the MR Pulley was
found in all posterior image planes as well (P < 0.01) for all
posterior image planes. The superior rectus (SR) pulley was
laterally displaced an average of 0.6 mm in subjects with SO
palsy, a distance that was not significant (JP = 0.08). The LR and
inferior rectus OR) pulleys were virtually identical to the normal position.
There was no difference in MR pulley position between
acquired (four subjects, MR pulley averaging 1.2 mm superior)
and congenital (three subjects, MR pulley averaging 1.1 mm
superior) SO palsies. There was no difference in MR pulley
position between subjects with torsion less than 10° (MR
pulley averaging 1.2 mm superior) and torsion greater than 10°
(MR pulley averaging 0.9 mm superior).
IOVS, January 1098. Vol 39. No. 1
Normal Right Orbit
Reoorts
Paretic Left Orbit
.
it-
^
J.UI
FIGURE 1. Coronal magnetic resonance imaging (MRT) through the orbit 9 mm posterior to the
globe- optic nerve junction, comparing the size and contractility of the right and left superior
oblique (SO) muscles in subject AT who had acquired left SO palsy. Note the decreased size
of the left SO muscle compared with the normal right SO muscle in primary gaze and the
diminished contractile change of the left SO muscle compared with the normal right SO
muscle in downward gaze, the field of action of the SO muscle, (center inset) Coronal MRI
through the posterior orbit of subject JB with congenital right SO palsy showing absence of the
right SO muscle belly. LPS = levator palpebrae superioris; MR = medial rectus; SR = superior
rectus; LR = lateral rectus; IR = inferior rectus; SO = superior oblique.
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209
210
Reports
TABLE
1. Profiles of Subjects with Superior Oblique Palsy
JOVS, January 1998, Vol 39, No. 1
Maximum SO
Area (cm2)
Age
Subject (years) Sex
18
22
22
32
83
43
19
KA
JB
RS
DD
HK
MM
AT
Clinical Diagnosis
Excyclotorsion
MRI Findings
(DMR)
Congenital LSO palsy
Congenital RSO palsy
Congenital LSO palsy
Acquired RSO palsy
Acquired LSO palsy
Acquired RSO palsy
Acquired LSO palsy
M
F
M
M
F
M
M
2°
4°
10°
5°
20°
20°
7°
Left SO palsy
Absent RSO
Absent LSO
Right SO palsy
Bilateral SO palsy
Right SO palsy
Left SO palsy
SO Contractile
Change (cm2)
Right SO
Left SO
Right SO
Left SO
0.19
0.12
0.15
0.06
0.03
0.08
0.19
0.10
0.10
0.08
0.21
0.19
0.10
0.20
0.11
0.14
0.05
0.03
0.02
0.11
0.09
0.03
0.07
0.01
Note that two of the three subjects diagnosed with congenital superior oblique (SO) palsy actually had an absent SO muscle on magnetic
resonance imaging (MRI). Also note that two of the subjects with acquired SO palsy had 20° of excyclotorsion. In one of those cases (HK), both
SO muscles were clearly atrophic and showed impaired contractility from upgaze to downgaze. In the other case (MM), only the right SO muscle
was atrophic and showed impaired contractility. The other SO muscle was clearly normal in both cross-sectional area and contractility. RSO = right
superior oblique; LSO = left superior oblique; DMR = double Maddox rod.
Three subjects with SO palsy had heterotopic pulleys, 5 all
in the palsied orbit. Subject MM had three of four EOM pulleys
displaced in an excyclotorted fashion in the palsied, right orbit
(MR superior 1.9 rrtm, SR lateral 3-5 mm, and LR inferior 2.6
mm). Interestingly, subject MM had unilateral SO palsy by MRI
T
O
^SR
E
2
II
o
5-
LR
9-O 0-
Normal OrbitY
X
MR 12.0 ±0.6,
SR -1.4 ±0.8,
LR -11.9 ±0.5,
2.1 ±0.9,
IR
•
0.6 ±0.9
12.6 ±0.7
-0.6 ±0.9
-11.8 ±0.9
MR
hCH
1
SO Palsy Orbit
X
Y
MR 11.8 ±0.7, 1.7 ±0.8*
SR -2.0 ±2.2, 12.5 ±1.0
LR -12.0 ±0.3, -0.7 ±1.6
IR
2.211.6,-11.911.2
*O-5-
5
-10-
IR
-10
T
-5
0
5
Horizontal Displacement from
Orbital Center (mm)
10
FIGURE 2. Average positions (relative to orbital area centroid
and viewed as if facing the subject) of area centroids of rectus
extraocular muscles for primary gaze in the coronal image
plane 3 mm anterior to the globe-optic nerve junction for
normal subjects and subjects with SO palsy. Left orbits have
been digitally reflected to the configuration of right orbits to
facilitate comparison. The positive x coordinate values represent medial displacement. The positive y coordinate values
represent superior displacement. Error bands shown represent ± 1 SD. Note significant superior displacement of the
medial rectus muscle belly and slight lateral displacement of
the superior rectus muscle belly in subjects with SO palsy. The
lateral rectus and inferior rectus muscle bellies are almost
exactly at the normal positions. Abbreviations repeat those
used in Figure 1.
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criteria but measured 20° of excyclotorsion on double Maddox
rod testing (Table 1). Subject DD had lateral displacement of
the right SR pulley, and subject HK had medial displacement of
the left IR pulley. All other pulleys were normally placed in
both orbits of all three subjects.
Computer simulation using measured pulley positions
showed that, in every case, abnormal pulley position alone was
not sufficient to reproduce the clinical pattern of strabismus. In
addition, elevating the MR pulley 1.1 mm in an otherwise
normal orbit, without changes in other pulley positions, does
not cause clinically significant strabismus, with a maximum
vertical or horizontal deviation from binocular alignment of
1.6°.
For subject MM, computer simulation of the heterotopic
pulley positions alone, without postulating any SO abnormalites, was sufficient to generate a "V" pattern strabismus
(eyes deviated outward more on upward gaze than downward
gaze) and greater than 4° of excyclotorsion in primary gaze
(Fig. 3), but did not accurately reflect the subject's measured
Hess screen test. Similarly, simulation of only a right SO palsy
without heterotopic pulleys did not accurately reflect the subject's measured Hess screen test (Fig. 3). Superimposing both
the heterotopic pulleys and right SO palsy, however, did generate a simulation that accurately reflected the measured Hess
screen test (Fig. 3).
DISCUSSION
Large rectus EOM pulley abnormalities have been postulated to
cause incomitant strabismus. In craniosynostosis syndromes,
such as Apert, Crouzon, and Pfeiffer, laterally rotated orbits and
abnormally located EOMs are associated with a marked Vpattern exotropia. 9 Similarly, in heavy-eye syndrome, high axial
myopia and a large inferior displacement of the LR muscle are
associated with esotropia and hypotropia, because the normal
abducting action of the LR is converted to depression. 10 In
addition, smaller heterotopic displacements of the EOM pulleys are postulated to cause incomitant strabismus, simulating
the presence of oblique muscle dysfunction.5 Because many of
these subjects with heterotopic pulleys have significant ocular
torsion, however, an argument can be made that the change in
position of the pulleys could be secondary to the torsion. 6 For
Reports
IOVS, January 1998, Vol 39, No. 1
Right Eye (Left Eye Fixing)
Heterotopic
Pulleys Only
z
AD -40 -30 -20 -10 0
Right SO
Palsy Only
10 20 30 40 AB
d
10 20 30 40 AB
UP
V
e
r
t
Right Eye (Left Eye Fixing)
Heterotopic z
Pulleys and e
Right SO Palsy d
e
g
i
a
t
DNAD -40 -30 -20 -10 0
10 20 30 40 AB
3. Orbit 1.7 computer simulations for subject MM. For
all three simulations, the simulated Hess screen is represented by
solid lines, and the measured Hess screen is represented by dotted
lines. The top Hess screen shows the V pattern that would be
predicted from measured extraocular muscle pulley position abnormalities alone over a ±30° gazefield.The simulated deviation
underestimates the measured deviation but generates almost 4° of
excyclotorsion in primary gaze. The middle Hess screen shows
the V pattern that would be predicted with a right superior
oblique (SO) palsy alone, with right SO contractility of 0% of
normal and elastic strength of 50% of normal, without superimposing the measured heterotopic pulley positions. The simulated
deviation once again underestimates the measured deviation. The
bottom Hess screen shows the "V" pattern that would be predicted from measured pulley position abnormalites combined
with a right SO palsy. This simulation accurately predicts the
measured Hess screen data.
FIGURE
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211
example, perhaps during acute SO palsy, the resultant excyclotorsion of the globe might drag the EOM pulleys in an
excyclotorted direction, where they might remain after the
acute episode of palsy resolves.
If simple excyclotorsion were the explanation, however,
all EOM pulleys would be systematically displaced to the same
degree. In addition, greater degrees of torsion would result in
greater degrees of displacement of the EOM pulleys. In the
current study of orbits exhibiting torsion caused by SO palsy,
only the MR pulley was displaced to a significant degree, and
that displacement was similar for small (less than 10°) and large
(greater than 10°) amounts of excyclotorsion. No other EOM
pulley was significantly displaced.
Two additional factors argue against simple torsion causing
displacement of the EOM pulleys. First, on histology, the MR
pulley has the densest connective tissue suspension and should
be the EOM pulley most resistant to displacement by torsional
forces.1 Second, simple geometric analysis indicates that an excyclotorsion of 10° in a 24-mm globe would generate less than 2 mm
of displacement of the anterior tendinous insertion of the EOMs.5
This value represents the maximum theoretical displacement of
the posterior EOM bellies if the pulleys have no intrinsic stiffness
and are displaced freely with the EOM tendons. Even after muscle
transposition surgery of greater than 6 mm, however, the posterior muscle bellies do not demonstrate significant displacement
from normal.4 It is unlikely that torsion, which displaces the
muscle insertions by a much smaller amount, could significantly
displace the EOM pulleys.
A more likely explanation for the superior displacement of
the MR pulley is simple mechanical displacement of the posterior MR muscle belly caused by atrophy of the adjacent SO
muscle belly. In the posterior orbit, the two muscle bellies are
in proximity, and a significant decrease in SO muscle size could
allow passive superior migration of the adjacent orbital structures, including the MR muscle belly (Fig- 1).
Although the superior displacement of the MR pulley 1.1
mm in SO palsy is statistically significant, biomechanical modeling demonstrates that it is unlikely to be clinically significant.
Larger pulley displacements, on the order of 2 mm or greater,
are more likely to introduce significant unbalanced forces to
disrupt binocular alignment.5
Subject MM represents a special case of heterotopic
pulleys combined with SO palsy. Usually, when excyclotorsion exceeds 10°, the possibility of bilateral SO palsy is
considered clinically. Bilateral SO palsy was present in subject HK, the only other subject with greater than 10° of
excyclotorsion (Table 1). In subject MM, only one SO muscle was paretic by MRI characteristics (Table I). 7 The other
SO muscle was clearly normal (Table I). 7 The heterotopic
pulleys in the paretic orbit, however, did, on computer
simulation, generate a V pattern and excyclotorsion without
presuming any SO dysfunction. It is likely that the heterotopic pulleys combined with the unilateral SO palsy produced a much larger amount of excyclotorsion than would
have been predicted from a unilateral SO palsy.
In conclusion, SO palsy is associated with a statistically
significant elevation of the ipsilateral MR pulley by an average
of 1.1 mm. Although statistically significant, the small superior
displacement is unlikely to be clinically significant or to produce strabismus by itself. This displacement is also unlikely to
result from ocular torsion alone because no other EOM pulley
was significantly displaced, and the amount of torsion did not
correlate with the amount of pulley displacement. Finally, the
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Reports
combination of unilateral SO palsy and heterotopic pulleys can
produce a clinical pattern that resembles bilateral SO palsy,
with excessive excyclotorsion compared with a unilateral SO
palsy in the setting of normal EOM pulley position.
References
1. Demer JL, Miller JM, Poukens V, Vinters HV, Glasgow BJ. Evidence
for fibromuscular pulleys of the recti extraocular muscles. Invest
Ophthalmol Vis Sd. 1995:36:1125-1136.
2. Miller JM. Functional anatomy of normal human rectus muscles.
Vision Res. 1989;29:223-240.
3. Clark RA, Miller JM, Demer JD. Location and stability of rectus
muscle pulleys: muscle paths as a function of gaze. Invest Ophthalmol Vis Sci. 1997:38:227-240.
4. Demer JL, Miller JM, Rosenbaum AL. Effect of transposition surgery
on rectus muscle paths by magnetic resonance imaging. Ophthalmology. 1993:100:475-487.
Establishment and
Characterization of a Retinal
Muller Cell Line
Vijay P. Sarthy,1 Sevan J. Brodjian,1
Kamla Dutt,2 Breandan N. Kennedy?
Randall P. French,1 and John W. Crabb5
Primary cultures of Miiller cells have proven
useful in cell biologic, developmental, and electrophysiological studies of Miiller cells. However, the limited
lifetime of the primary cultures and contamination from
non-neural cells have restricted the utility of these cultures. The aim of this study was to obtain an immortalized
cell line that exhibits characteristics of Muller cells.
PURPOSE.
METHODS. Primary Muller cell cultures were prepared from
retinas of rats exposed to 2 weeks of constant light. Cells
were immortalized by transfection with simian virus 40.
Single clones were obtained by repeatedly passaging cells
using cloning wells. Immunocytochemical and immunoblotting studies were carried out with glial nbrillary acidic
protein (GFAP)-specific and cellular retinaldehyde-binding
protein (CRALBP)-specinc antibodies. Transient transfections with CRALBP-luciferase constructs were performed
by electroporation.
Oncogene transformation resulted in the establishment of a permanent cell line that could be readily
propagated. Immunocytochemical and immunoblotting
RESULTS.
From the 'Department of Ophthalmology, Northwestern University Medical School, Chicago, Illinois; the 2Department of Pathology,
and Cell Biology and Anatomy, Morehouse Medical School, Atlanta,
Georgia; and the 3W. Alton Jones Cell Center, Lake Placid, New York.
Supported by National Eye Institute grants EY-03523 and EY06603 and by an unrestricted award from Research to Prevent Blindness Inc.
Submitted for publication January 14, 1997; revised June 10, 1997;
accepted September 19, 1997.
Proprietary interest category: N.
Reprint requests: Vijay Sarthy, Department of Ophthalmology,
Tarry 5-715, Northwestern University Medical School W113, 300 E.
Superior St., Chicago, IL 606ll.
Downloaded From: http://iovs.arvojournals.org/ on 04/30/2017
IOVS, January 1998, Vol 39, No. 1
5. Clark RA, Miller JM, Rosenbaum AL, Demer JL. Heterotopic muscie
pulleys or oblique muscle dysfunction? J Am Assoc Pediatr Ophthalmol. Stabismus. 1998. In press.
6. Guyton DL, Weingarten PE. Sensory torsion as the cause of
primary oblique muscle overaction/underaction and A- and Vpattern strabismus. Binoc Vis Eye Muscle Surg Q. 1992;9:209236.
7. Demer JL, Miller JM. Magnetic resonance imaging of the functional
anatomy of the superior oblique. Invest Ophthalmol Vis Sci. 1995;
36:906-913.
8. Miller JM, Robinson DA. A model of the mechanics of binocular
alignment. Comput Biomed Res. 1984; 17:436-470.
9. Cheng H, Burdon MA, Shun-Shin GA, Czypionka S. Dissociated eye
movements in craniosynostosis: a hypothesis revived. Br J Ophthalmol. 1993:77:563-568.
10. Krzizok T, Wagner D, Kaufmann H. Elucidation of restrictive motility in high myopia by magnetic resonance imaging. Arch Ophthalmol. 1996;115:1019-1027.
studies demonstrated that the Muller cell line, rMC-1,
expressed both GFAP, a marker for reactive gliosis in
Muller cells, and CRALBP, a marker for Muller cells in the
adult retina. Transient transfection assays showed that
promoter-proximal sequences of the CRALBP gene were
able to stimulate reporter gene expression in rMC-1.
CONCLUSIONS. Viral oncogene transformation has been successfully used to isolate a permanent cell line that expresses Muller cell phenotype. The rMC-1 cells continue
to express both induced and basal markers found in primary Muller cell cultures as well as in the retina. The
availability of rMC-1 should facilitate gene expression studies in Muller cells and improve our understanding of Miiller cell-neuron interactions. (Invest Ophthalmol Vis Sci.
1998;39:212-2l6)
M
uller cells are the most abundant non-neuronal cells in
the vertebrate retina, and they perform diverse functions that support the activity of retinal neurons, hi
recent years, the availability of dissociated cell preparations and
primary Miiller cell cultures has greatly facilitated cell biologic,
biochemical, developmental, and electrophysiological studies of
Muller cells. Miiller cell cultures have been obtained from neonatal and adult retinas, andfromretinas with inherited dystrophy or
constant light damage.1"8 However, primary Muller cell cultures
have certain problems that limit their utility: the cells have a
limited life span and undergo senescence with passage; the cultures are usually contaminated with astrocytes and microglia3'9;
unless a large number of eyes is used, only a small number of
cultures can be obtained3; and the small culture size restricts their
use to morphologic, immunocytochemical, and electrophysiological studies.
Some problems associated with primary cultures can be
overcome by establishing permanent cell lines through immortalization of primary cells with viral oncogenes. During gene
regulation studies using transfection assays, we found that the
small number of cells and the low transfection efficiency in
primary Muller cell cultures were serious drawbacks.10 This
motivated us to establish a Muller cell line. This report describes the isolation and immunochemical identification of a
Muller cell line (rMC-1) from adult rat retina.