Development of a Multi-Electrode Electroretinography System
and
Characterization of meERG Signals in Rat
BY
YELENA KRAKOVA
B.S., University of Illinois at Chicago, Chicago, 2005
THESIS
Submitted as partial fulfillment of the requirements for the degree of Master of Science in
Bioengineering in the Graduate College of the University of Illinois at Chicago, 2012
Defense Committee:
John Hetling, Advisor
James Patton
Simon Alford, Neuroscience
ii
TABLE OF CONTENTS
Chapter
Page
1. Brief overview………………………………………………………………………………1
1.1. Problem statement …………………………………………………………………..... 1
1.2. Specific Aims……………………………………………………………………….....1
1.3. Summary of results………………………………………………………………........ 2
2. Introduction ………………………………………………………………….....................3
3. Background ……………………………………………………………………………… 4
3.1. Overview of anatomy / physiology of the eye………………………………………. 4
3.2. The ERG and its origins……………………………………………………………... 6
3.3. A- and B-wave……………………………………………………………………….. 6
3.4. ISCEV standard…………………………………………………………………….... 8
3.5. Current electrode types……………………………………………………................. 9
4. Existing methods for mapping retinal function …………………………………………..10
4.1. Perimetry – HVF, Goldman………………………………………………………….11
4.2. mfERG ……………………………………………………………………………….11
4.3. Potential advantages of meERG-based approach to mapping retinal function……... 12
4.4. Commercially available ERG stimulus sources……………………………………...12
Methods and Results by Specific Aim
5. Specific Aim 1………………………………………………………………………….....14
5.1. Specific Aim 1 methods……………………………………………………………...15
A. Initial design of the stimulus source……………………………………………....15
B. Star stop …………………………………………………………………………..17
5.2. Specific Aim 1 Results……………………………………………………………….18
A. Light source calibration………………………………………………………...…18
B. Luminance profiles through time………………………………………………….19
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iii
TABLE OF CONTENTS (continued)
6. Specific Aim 2…………………………………………………………………………….22
6.1. Specific Aim 2 methods……………………………………………………………22
A. Lens assembly procedure………………………………………………………....24
B. Revision of lens design…………………………………………………………....25
i.
Analysis of rat eye size vs. age…………………………………………....26
ii. Measurement Procedure using DinoCapture 2.0 version 1.3.2…………. .26
iii. Measurement Procedure using ImageJ……………………………………28
iv.
Depth of lens cup………………………………………………………….29
v.
Width of lens voids………………………………………………………..30
C. Lens Filling Procedures………………………………………………………….. 31
i.
Saline………………………………………………………………………31
ii. Saline lens filling procedure………………………………………………32
iii. Saline lens cleaning and storage procedure……………………………….33
iv.
Poly-Ethylene Glycol (PEG)………………………………….....................33
v.
PEG fabrication and lens filling procedure……………………………….34
vi.
PEG lens storage procedure………………………………………………35
6.2
A.
B.
C.
D.
Specific Aim 2 results……………………………………………………………..35
Analysis of rat eye size vs age…………………………………………………….35
Depth of lens cup………………………………………………………………….37
Width of lens voids………………………………………………………………..38
Lens filling procedures……………………………………………………………38
7. Specific Aim 3 ……………………………………………………………………………40
7.1. Specific Aim 3 methods……………………………………………………………40
A. Recording protocol………………………………………………………………..41
B. Analysis protocol………………………………………………………………….43
7.2. Specific Aim 3 results……………………………………………………………...44
A. Typical meERG response…………………………………………………………44
B. Inter-animal variability……………………………………………………………49
i. A-wave by ring…………………………………………………………….49
ii. B-wave by ring…………………………………………………………….52
C. Spatial Differences……………………………………………………………...…54
D. Test-retest variability (same animal)…………………………………………........56
8. Discussion…………………………………………………………………………………60
iii
iv
TABLE OF CONTENTS (continued)
8.1. Summary of Results by Specific Aim………………………………………………..61
A. Summary of Results: Specific Aim-1……………………………………………..61
B. Summary of Results: Specific Aim-2……………………………………………..62
C. Summary of Results: Specific Aim-3……………………………………………..64
9. Cited Literature……………………………………………………………………………66
10. Appendix………………………………………………………………………………….68
10.1. Standard operating procedures………………………………………………………68
A.
Lens Assembly………………………………………………………………………..68
B.
Measurement Procedure using DinoCapture 2.0 version 1.3.2 ………………………68
C.
Measurement Procedure using ImageJ ……………………………………….............69
D.
Saline lens filling procedure…………………………………………………………..69
E.
Saline lens cleaning and storage procedure…………………………………………..70
F.
PEG lens filling procedure……………………………………………………............70
G.
PEG fabrication procedure……………………………………………………............71
H.
PEG lens storage procedure…………………………………………………………..71
I.
Recording Protocol …………………………………………………………………...72
J.
Analysis Protocol……………………………………………………………………..72
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LIST OF FIGURES
Figure
Page
1.
Anatomy of the human eye……………………………………………………………5
2.
A typical flash-evoked corneal ERG …………………………………………………7
3.
Schematic current source model of the eye…………………………………………...7
4.
Photograph of Burian-Allen and DTL electrodes…………………………………….10
5.
Schematic of the spatially –homogenous light –flash stimulus source……………….16
6.
Star stop ………………………………………………………………………………16
7.
Light diffuser dome…………………………………………………………………..16
8.
Star stop with ring pattern overlay……………………………………………...........17
9.
Star stop flow chart……………………………………………………………………18
10.
Luminance profiles through time……………………………………………………..21
11.
Photo and schematic of rat lens……………………………………………………….23
12.
Photo of the lens assembly table……………………………………………………...25
13.
Screen capture of the measurement procedure using DinoCapture…………………..28
14.
Schematic of base lens and a photo of the base lens…………………………………30
15.
Rat eye radius measurements plotted from the age of 24 to 66 days…………………36
16.
Photos of the modified and original depth of lens……………………………………37
17.
Impedance of channel 77 on the CLEAr Lenstm..........................................................40
18.
A photograph of an anesthetized rat during a recording……………………………..43
19.
Position plot and electrode map………………………………………………………45
20.
Normalized standard deviation of the amplitudes from Table 3 …………………….47
21.
A wave amplitudes from 22 experiments averaged together…..................................48
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LIST OF FIGURES (CONTINUED)
22.
The mean divided by the standard deviation of Figure 21 plotted……………………48
23.
Normalized a- wave amplitude ratios from 8 animals at 6 and 7 weeks of age………51
24.
Normalized b-wave amplitude ratios from 8 animals at 6 and 7 weeks of age ………53
25.
Probability distribution of a-wave amplitude ratios for 18 experiments …………….55
26.
Probability distribution of a-wave amplitude ratios for 18 experiments averaged
together……………………………………………………………………………….56
27.
A wave amplitude ratios from 8 animals at 6 and 7 weeks of age …………………..58
28.
Averaged a-wave amplitude ratios from 8 animals at 6 and 7 weeks of age…………60
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LIST OF TABLES
Table
Page
1.
Light source calibration data…………………………………………………………..20
2.
Light source calibration table with light from filters…………………………………..41
3.
Mean a- and b-wave amplitudes with all electrodes averaged………………………...46
vii
viii
LIST OF EQUATIONS
Equation
Page
1. Normalized ring averages………………………………………………….
2. Spatial ring ratios……………………………………………………………
viii
ix
LIST OF ABBREVIATIONS
ERG
Electroretinogram
mfERG
Multi-focal electroretinogram
meERG
Multi-electrode electroretinogram
SNR
Signal to noise ratio
PMMA
Polymethyl-methacrylate
PEG
Poly-ethylene-glycol
Sc cd s m-2
Scotopic candela seconds per meter squared
CLEAr Lens™
Contact lens electrode array
ISCEV
International Society for Clinical Electrophysiology of Vision
NEVL
Neural engineering vision laboratory
HVF
Humphrey visual field
ix
1. Brief overview
Multi-electrode electroretinography (meERG) is a new technique being developed in the Neural
Engineering Vision Laboratory at UIC. The meERG is a simultaneous measure of potentials at
several locations on the corneal surface in response to a photic stimulus. It is anticipated that
analysis of these potentials will reveal spatial information about the health of the retina, and thus
may prove useful in early diagnosis of eye diseases, such as glaucoma and diabetic retinopathy.
1.1 Problem statement
While the meERG technique is being developed for clinical use, there is a great deal that can be
learned in animal studies. In particular, the NEVL lab is working to characterize the sensitivity
and specificity of the technique for detecting laser-induced retinal lesions in rats. The main goal
of this thesis was to support this effort by improving the design of the stimulus source and the
contact lens electrode array, and by doing the initial characterization of the meERG signals
recorded from normally-sighted rats. With this in mind, the following aims were addressed.
1.2 Specific Aims
I.
To optimize a full-field, spatially-homogenous light-flash stimulus source suitable for use
with rats.
-1-
-2-
II.
To improve the design and function of the rat-sized meERG Contact Lens Electrode Array
(CLEAr Lens™), with the goals of increasing the yield of functional electrodes on each lens
and improving the quality of the recorded signal.
III.
To record the meERG signal from normal rats and to characterize this relatively novel signal
with regard to test-retest and inter animal variability and to quantify spatial differences in
meERG potentials across the rat cornea
Note: CLEAr Lens™ is a trademark of RetMap, Inc., a start-up company founded by NEVL
alumni to facilitate development and distribution of meERG technology.
1.3 Summary of results:
A full-field light flash stimulus source for use in rats was optimized and characterized, and found
suitable for the planned experiments (it is currently in routine use). The lens design, as well as
the procedure for preparing the lens for recording, were both significantly improved; the lens
preparation procedures developed here have been adopted for the human-version of the CLEAr
Lens™ as well. The meERG responses were analyzed for spatial differences in amplitudes; the
typical standard deviation of amplitudes across the corneal surface was 2-3% of the mean,
indicating fairly uniform potnetials for normally-sighted rats. Inter-animal variability between aand b-wave amplitudes was found to be high, but consistent with conventional ERG results.
Regarding test-retest variability, a measure related to the spatial distribution of a-wave
amplitudes was found to be significantly different in three of eight animals when comparing
measurements made one week apart; this seems to indicate a degree of procedural variability
inherent in this relatively new measurement.
2. Introduction
There is a high prevalence of degenerative retinal diseases that affect the peripheral retina, such
as glaucoma and retinitis pigmentosa, that currently have limited means of early detection.
Methods that are available today include the Humphrey Visual Field test and the multi-focal
electroretinogram (ERG). These methods are only capable of detecting the disease once it
progresses into the central visual field and either have a long duration time or require the patient
to have good visual acuity. The meERG has the potential to overcome these challenges by
probing the entire retina in a short duration time and being independent of good visual acuity.
The purpose of the work presented here is to support the development of the new multi-electrode
electroretinogram (meERG) technique by improving the design of the full-field spatiallyhomogeneous stimulus source and characterizing the meERG signals collected from rats to begin
the formation of a normative data set. The meERG is a means of detecting potentials on the
cornea at several locations simultaneously. The characterization of the meERG helps set the
foundation for the spatial differences present in corneal a and b wave amplitudes in normal rats.
Simultaneously recorded amplitudes at multiple locations on the cornea have been analyzed on a
limited basis in the literature; spatial variance will be the focus in this thesis.
-3-
3. Background
3.1 Overview of anatomy/physiology of the eye
The retina is a layer of several cell types that lines the inner surface of the eye. The human retina
is approximately 0.5 mm thick (webvision, 2012), and contains approximately 55 different cell
types, each with a different function (Masland, 2001). A cross section of the retina shows
photoreceptors, ganglion cells, muller cells, amacrine cells, horizontal cells and bipolar cells.
Figure 1 shows these major cell types. There are three layers of nerve cell bodies and two layers
where synapses occur. The layer that is furthest from the cornea contains the rods and cones
which make up the two types of photoreceptors. In most mammalian species, rods outnumber
cones by approximately 20-fold (Masland, 2001). Starting at the outer most part of the retina, the
rods and cones are followed by the outer nuclear layer which contains bipolar and horizontal
cells. Amacrine cells make up the inner nuclear layer which is followed by the ganglion cells and
their axons. The nerve fiber layer forms the vitreous surface of the retina and is the first retinal
layer to receive light entering the eye.
-4-
-5-
A)
B)
Aqueous
humor
Vitreous
humor
Figure 1: (A) Cross section of a human eye (B) Schematic cross section of a mammalian retina. The outer most part
of the retina contains the rods and cones followed by the outer nuclear layer, outer plexiform layer, inner nuclear
layer and the inner plexiform layer (webvision, 2012).
When light enters the eye, it must pass through the cornea, aqueous humor, lens, vitreous humor
and several cell layers present in the retina before reaching the photoreceptors. The central part
of the retina has a high density of cones while the peripheral part of the retina is rod dominated
(Purves, 2001). The cones are responsible for color vision at higher light levels while the rods are
capable of functioning in minimal light and hence are primarily responsible for night vision
(webvision 2012). When a photon enters the eye and reaches the retina the molecule rhodopsin, a
photo pigment in the rod photoreceptors, undergoes a conformation change that through a signal
cascade results in the hyperpolarization of the cell. The hyperpolarization reduces the release of
the neurotransmitter glutamate at the synaptic cleft. Glutamate causes the depolarization of the
ON-bipolar cells and the hyperpolarization of the OFF bipolar cells. OFF and ON bipolar cells
occur in approximately equal numbers (Masland, 2001). The signal then travels to the ganglion
cells where an action potential occurs to be further propagated through the optic nerve into the
primary visual cortex in the brain for additional processing.
3.2 The ERG and its origins
The electroretinogram is a bioelectric signal originating at the retina that can be recorded at the
surface of the cornea. It is a test used in basic research to assess the state of the retina in diseased
as well as healthy eyes. It is used in human patients as well as laboratory animals. Typically, an
electrode is placed on the cornea and a light flash is produced which elicits a recordable
waveform. A full-field, spatially homogenous stimulus source such as the Ganzfeld dome and
electrodes types such as the Burian-Allen, JET and DTL are commonly used for recording the
ERG (Figure 4). The ERG is recorded following the ISCEV standard (Hood et al., 2012). This
standard states that the recording electrode impedance must be measured and the patient dark
adapted based on the type of recording that is to be performed. The appropriate stimulation levels
for recording rod or combined rod-cone responses are given with appropriate interval times
between stimuli. The standard includes guidelines for averaging as well as reporting the recorded
information.
3.3 A- and B wave
Several cell types contribute the recordable ERG waveform; for this introduction a-wave and bwave origins are described below. Figure 2 is an example of a typical flash evoked corneal ERG
showing the a- and b-wave. The a-wave represents the light response of the photoreceptors and
reflects the change in the circulating currents due to light absorption. The initial negative slope of
the ERG is the a-wave and would have a much longer duration if the photoreceptors were to be
isolated from the rest of the retina. Due to the intrusion of the b-wave, only a small portion of the
a-wave is typically visible (Lamb & Pugh, 1992). With the decrease in glutamate post light
stimulus, the ON-bipolar cells activate and depolarize, responsible for the positive peak seen in
the ERG waveform.
1200
b-wave
1000
800
µV
600
400
200
0
-200 0
-400
-600
50
100
150
200
250
300
350
a-wave
ms
Figure 2: A typical flash-evoked corneal ERG recorded with the CLEAr Lens™ in rat. Waveform recorded on
electrode channel 83 at flash strength of 116 sc cd s m-2 delivered at 50.4 ms. (experiment 20111123-A)
Figure 3: Schematic current source model of the eye, (webvision, 2012).
400
A mammalian eye can be represented with the circuit diagram in Figure 3. A light stimulus
produces an electrical current that can be divided into local and remote pathways as illustrated by
pathway A and B. Hence two electrodes are used to record the ERG, one placed on the cornea
and the other which serves as the reference is commonly placed on the cheek. The ERG is a
potential change that is related to the light induced electrical activity within the retina (webvision,
2012). As the retina deteriorates due to a pathology, the light-induced response of the retina is
reduced and so are the waveforms of the ERG.
3.4 ISCEV standard (2011)
The International Society for Clinical Electrophysiology of Vision (ISCEV) promotes clinical
electrophysiology of vision and defines recording standards to promote cooperation and
communication among the field. This standard was followed as closely as possible for the rat
recordings. The pupils are to be dilated maximally and the pupil size noted. The electrode type
such as the Burian-Allen or the DTL should be used for recording the ERG. The electrode
stability and impedance are to be measured. For recording the rod response the patient is to be
dark-adapted for a minimum of 20 minutes. The stimulus is a dim white flash of 0.01 cd s m-2
with a minimum interval of 2 seconds between flashes. For recording the combined rod-cone
response a white light of 3.0 cd s m-2 is to be used. There should be at least a 10 second interval
between stimuli. Averaging the recorded signals is not required when using the recommended
electrode types. Patient identification, date as well as any events that deviate from the ISCEV
standard are to be reported.
3.5 Current electrode types
The Burian-Allen electrode is a speculum type structure that holds the eye open while a contact
lens with a wire on its periphery floats on the retina. The Burian-Allen is bipolar and reusable
with reference electrodes built in. This type of electrode is size specific and in order to fit the
patient properly, it is available in four sizes. (Figure 4A) The Jet electrode is a contact lens with
an embedded gold foil electrode; it is a mono-polar electrode that comes in one size only. This
contact lens electrode is disposable. The DTL electrode is a silver-nylon thread wire that lies
along the lower lid and sclera or cornea. This type of electrode is not limited by patients eye size
and is disposable. It is held in place by an adhesive located on the white foam pads and is ideal
where user comfort is an issue. (Figure 4B)
A)
B)
Figure 4: A) Photograph of a Burian-Allen electrode. B) Photograph of a DTL electrode.
4. Existing methods for mapping retinal function
The most recent report of an meERG recording in literature was performed by Sundmark (1959)
using a 9 electrode “contact glass.” The Neural Engineering Vision Laboratory (NEVL) at UIC
is investigating the process of electrophysiological mapping of retinal function with the multielectrode CLEAr Lens™. The meERG is comparable to techniques developed for 3D mapping of
the brain and heart from body surface potentials (He & Wu, 1999). Several methods exist that
map retinal function in patients but have several limitations. These methods are briefly discussed
below.
- 10 -
4.1 Perimetry- Humphrey visual field, Goldman perimetry
The Humphrey visual field (HVF) is a test which requires the patient to fixate on a small target.
Spots of light are introduced at defined locations within the visual field and the patient responds
when he or she sees them. The most common variation of the HVF technique is the 24-2 test
which tests the central 24 degrees of the visual field with a 20 degree target. The test is restricted
to the central visual field since visual acuity in the periphery is limited and the test becomes
inaccurate. The Goldman visual field test is a test that measures a patients peripheral vision
manually. A test target is projected at a single location and intensity is increased until the patient
is able to see it. The target is moved to the central visual field from the periphery and feedback
from the patient is recorded. The primary problem of these two tests is that they rely on the
patient’s feedback and are thus not objective.
4.2 Multifocal electroretinogram
The multifocal electroretinogram (mfERG) is a means of applying a pattern stimulus in order to
elicit a response from various areas of the retina simultaneously, developed by Sutter and Tran,
(1992). The recording is done with a standard ERG electrode. The data is analyzed and
correlated to an appropriate area of the retina via a mathematical technique. This test requires the
patient to have good central vision as the test probes 30-40 degrees of the central visual field and
can take a long time (about 8 minutes per eye) to perform. This test is often used to assess the
retinas of patients with retinitis pigmentosa, glaucoma and macular degeneration. A weakness
with the mfERG is that the recordings have uncertain correlation to the biological events that
occur at the retina and the computed waveforms are open to interpretation (Hood et al., 2002).
4.3 Potential advantages of meERG-based approach to mapping retinal
function
The fist account of the measuring ERG potentials at various locations simultaneously was by
Sundmark (1959), followed by Cringle (1986). Recordable differences in ERG amplitudes based
on location of the contact electrode were first noticed by McAllen et al. (1989), and confirmed
by Hennesey and Vaegan (1995) that different electrode types yielded varying recorded
amplitudes. The meERG is essentially means of collecting corneal potentials at several locations
simultaneously in response to a full field light stimuli. The meERG is a noninvasive test that
probes the entire visual field and is likely to be independent of the visual acuity of the patient.
The test can be applied with varying stimuli in order to analyze various cell types or functional
pathways. The meERG results in waveforms that directly reflect the function of the retina. This
test is quick (less than a minute per eye) and yields spatial information about corneal potentials
and has the potential to yield spatial information about the retina with the use of models
(currently being developed) in solving the inverse problem.
4.4 Commercially available ERG stimulus sources
The Ganzfeld stimulus source is a full-field light source which allows for control of background
illumination and the ability to vary stimulus intensity. The Ganzfeld is typically a dome which
fills the visual field and produces a uniform luminance in order to illuminate the retina
homogeneously. Despite several attempts via personal communication with LKC Technologies
Inc., the manufacturer of the MGS-1 Ganzfeld stimulator as well as by Diagnosys LLC, the
manufacturer of the handheld Ganzfeld (ColorBurst) no quantitative information was disclosed
about the spatial variance of their products. This made it difficult to compare our results to the
commercial standard. The relationship between the source and retinal illuminance is believed to
be approximately direct, that is if a Ganzfeld source is used, the resulting light distribution is
nearly homogenous over the whole retina. The homogeneity of the light distribution on the retina
is not influenced by the size of the pupil or the shape of the optical features (Kooijman, 1983).
Retinal illuminance has previously been measured in human as well as rabbit eyes. Retinal
illuminance decreases only slightly toward the periphery when measured in human and rabbit
eyes upon illumination with a Ganzfeld (Kooijman, A. & Witmer, F., 1986). Since the
relationship is considered to be direct, although quite difficult to measure without error the use of
a Ganzfeld source will be necessary to stimulate the retina homogeneously.
Methods and Results by Specific Aim
5. Specific Aim 1 - To optimize a full field, spatially –homogenous
light –flash stimulus source for use with rats.
The distribution of the meERG recorded corneal potentials is affected by the spatial distribution
of the source of the signal, the retina. Since the ultimate goal is to understand the activity of the
retina and reveal areas of abnormal response to stimulus, it is critical to know the distribution of
the stimulus across the retina. Ideally, the stimulus source would produce a uniform, spatiallyhomogenous distribution of light across the entire retina. Since it is difficult to measure the exact
amount of light that hits the retina, it is approximated that it is proportional to that of the light
source. Assuming that the light source is uniform, it is believed that the retina will be illuminated
uniformly as well. For this reason it is critical to use a well-characterized stimulus source.
Instead of purchasing a commercial stimulus source, one was fabricated in the lab in hope that
we would obtain a level of spatial homogeneity that exceeded the current gold-standard
commercial units. Since this data was not accessible from commercial companies, the goal of
the fabrication process was to create a source with uniform luminance.
- 14 -
5.1 Methods
A. Initial design of the stimulus source
An initial design for the stimulus source was created by Dr. Hetling. This consisted of a 14 x 14
x 20 in plywood box with a series of shelves inside and a light source on top and can be seen in
Figure 5A, B. The challenge to meet Specific Aim 1 was to optimize the diffusers and stops
between the flash lamp and the back-lit dome in order to achieve a source, which is the concave
surface of dome, that was of uniform luminance (+/- 5%). A flash head (2140-C Novatron) was
used to produce a flash with an ̴ 0.88 msec duration. A series of four diffusers encased in a
plywood box were installed. The first three diffusers (A, B, C respectively) were made using
sandblasted polymethyl-methacrylate (PMMA) that was sandblasted on both sides. These were
placed in filter slots A and B. Filter slot B contained two sheets of PMMA, the one on top was
sandblasted on both sides while the bottom one was a diffuser sheet cut to size that is typically
used in fluorescent light fixtures. Filter slot C had a single sided sand blasted PMMA sheet and
slot D had the star stop, see Figure 5A. The light was measured from 0 to 70 degrees in 10
degree increments from the normal using a photometer (International light, model IL-1800, with
SED #100 photo diode detector, R#485 radiance barrel and the ZCIE#19555 scotopic filter),
Figure 7, which was calibrated to scotopic candela seconds per meter squared (sc cd s m-2). This
detector was placed on a custom mount that allowed the targeting of +/- 70 degrees of dome
surface. Figure 5(B) is a photograph of the stimulus box with the door open and a light turned on.
In order to further optimize the light source to the desired luminance profile, a “star stop,” a
photography stop in the shape of a star was developed for filter slot D. Figure 6. The process of
adjusting the star stop is represented as a flow chart in Figure 8. Figure 10 demonstrates the
luminance profile as it evolved through the fabrication process.
A)
B)
Figure 5: (A) Schematic of the spatially–homogenous light–flash stimulus source for use in rats with the
photometer detector in place. (B) Photo of the stimulus source for use in rats. Courtesy of Hadi Tajalli.
12 in
Figure 6: Star stop, made out of PMMA and aluminum
tape. When the star stop is added to the light stimulus
box it produces the final luminance profile as seen in
Figure 10.
Figure 7: Schematic of the light diffuser dome
with the photometer detector showing the
method used to measure the luminance at ten
degree increments.
B. Star Stop
The star stop design was based on the following goal, to bring the luminance at the measured
degree intervals as close to the lowest measured value as possible. The luminance was measured
at ten degree intervals starting from zero degrees from the vertical to 70 degrees and averaged,
see Figure 7. The PMMA sheet was subdivided into 6 concentric circles drawn from the center
of the PMMA with the radii illustrated in Figure 8. Opaque aluminum tape was then added
strategically, first perpendicular to the line dawn in each ring and then parallel. The following
flow chart outlines the process.
Figure 8: Schematic ring pattern overlaid on star stop. The overlaid ring pattern was used in the fabrication process
of the star stop. The ring pattern allowed for the division of the PMMA sheet into zones that were later coupled
with the eight degree increments that were measured.
Figure 9: Star stop optimization process. The flow chart describes the procedure used to fabricate the star stop to
produce uniform luminance as measured by the photometer.
5.2 Results
A. Light source calibration
A uniform full-field stimulus is necessary to achieve uniform illuminace of the retina so it was
our goal to create a stimulus source as uniform as possible. Comparing the measured luminance
of the source to a flat line (the ideal luminance profile), a sum of squares was calculated as a
measure of ‘flatness’ and uniformity of luminance; the lower the value the more uniform the
luminance is. The formula for the sum of squares is listed in Table 1. This data is based on five
flashes averaged per degree interval. The sum of squares (the sums of the square differences)
divided by the average of the light distribution before the star stop was 16,414. After the star stop
was applied, the sum of squares was reduced to 7,129. See Table 1 for reference. The addition of
the start stop decreased the variance of the light distribution from 66% to 22%. The table shows
the desired reduction in light based on percentages with the varying degrees and the achieved
light reduction. For clarity, this information was plotted, as described in the next section.
B. Luminance profiles through time
The final luminance profile in Figure 10 was the stopping point due limitations of the overall
design of the stimulus box.. Since the luminance around the periphery of the interior dome must
be the same as the luminance at the top or center, the goal was to increase the amount of
reflecting light to make the two equal. The geometry of the light stimulus box was ultimately the
limiting factor in the luminance profile that could be produced with a single point light source. A
possible solution to this might to be increase the distance between the reflective box walls and
the dome surface, either by using a larger box or a smaller diameter dome. Although the stimulus
source that was created in lab does not meet our goal of uniform luminance (+/- 5%), it is the
best that could be achieved with the given materials.
Degrees
0
10
20
30
40
50
60
70
Average
% change 0 to 70
degrees
Sum of Squares
SS(x)=
Original
Luminance
Profile
sc cd s m-2
2790
2730
2310
1790
1380
1190
1070
945
1775
Ideal
Percent
Change
%
66.12
65.38
59.09
47.2
31.52
20.58
11.68
0
Intermediate
Final
Achieved
Luminance Luminance Percent
profile
Profile
Change
-2
-2
sc cd s m
sc cd s m
%
976
952
65.87
1102
941
65.53
1216
973
57.87
1142
965
46.08
923
872
36.81
811
837
29.66
705
781
27
634
745
21.16
938
883
66
47
22
∑(𝑥 − 𝑥̅ )2
29,135,625
7,358,431
6,295,678
Sum of
Squares/Average
∑(𝑥 − 𝑥̅ )2
𝑥̃
16,414
7,844
7,129
Table 1: Light source calibration data. A comparison between the original luminance profile and the luminance
profile with the addition of the star stop. The goal was to decrease the amount of light at every degree interval such
that the luminance at that point would be equal to that of the lowest measured value. This decrease in luminance is
called the ideal percent change that would be necessary to have uniform luminance. The percent change achieved in
the final luminance profile and the ideal percent change necessary for an optimal full field light-flash stimulus have
very similar values from 0 to 40 degrees. However at 50 degrees and beyond, the limitations of the light stimulus
box design became apparent.
A)
3000
sc cd s m-2
2500
StDev/Average = 0.57
2000
Original Luminance
Profile
1500
1000
Average
500
0
0
10
20
30
40
50
60
70
Degrees from vertical
3000
B)
sc cd s m-2
2500
StDev/Average = 0.29
2000
Intermediate Luminance
profile
1500
1000
Average
500
0
0
10
20
30
40
50
60
70
Degrees from vertical
C)
3000
sc cd s m-2
2500
StDev/Average = 0.10
2000
1500
Final Luminance Profile
1000
Average
500
0
0
10
20
30
40
50
60
70
Degrees from vertical
Figure 10: Luminance profiles. Plotted values are calculated based on (the average of 5 flashes per degree
increment.) The standard deviation divided by the average is reported as an indicator of the flatness of the profile. (A)
Original luminance profile. (B) Intermediate star stop luminance profile. (C) Final star stop luminance profile.
6. Specific Aim 2- To improve the design and function of the rat-sized
meERG contact lens electrode array (CLEAr Lens™), with the goals
of increasing the yield of functional electrodes on each lens and
improving the quality of the recorded signal. This was accomplished
by changing the design of the PMMA base lens and by substantially
improving the way in which the assembled CLEAr Lens™ was
prepared for recording.
6.1 Methods
Prior to an animal recording, the CLEAr Lens™ must be assembled as well as tested to evaluate
the number of working channels. The lens assembly consists of the combination of the PMMA
base lens, an adhesive double sided tape as well as a cable which connects to the amplifier.
Figure 11(B) shows a detailed schematic of the rat base lens as well as a photograph of the
assembled rat CLEAr Lens™. The lens assembly steps are described below.
- 22 -
A)
B)
Figure 11: (A) Photo of rat lens (B) to-scale schematic of rat lens.
A. Lens assembly procedure
The lens assembly procedure began by selecting a cable and making sure that the cable was free
of dust and debris. A lens that was clear of tape residue was selected and placed into an
ultrasonic instrument cleaner for 5 minutes. The lens was then removed and rinsed under
distilled water for several seconds. In order to make sure that the lens was completely dry, it was
allowed to air dry on a paper towel for 10 minutes. Three 1/8 inch balls of dental wax were then
placed equidistant from one another around the inner perimeter of the lens assembly well, see
Figure 12. The concave side of the lens was placed down into the lens assembly well assuring
that the lens was parallel to the assembly table. The cable was placed onto the metal cable
holding shelf with the exposed electrode pad side down and covered with the plastic slip. The red
screw was tightened to hold the plastic slip as well as the cable in place. The x, y, z translation
knobs and rotation lever were used to adjust the lens such that all the holes were aligned with the
electrodes on the cable. Once everything was aligned, the tip of the cable that rests over the lens
was pull back and flipped back onto itself such that a small weight could be used to hold in
down. A piece of pre-formed tape was removed using forceps and positioned onto the lens such
that the holes were all aligned. Using forceps, each precut piece of tape that covered the wells in
the lens were removed. Once all the tape from the wells was removed, the weight holding down
the cable was carefully lifted and the cable was allowed to fall onto the tape attached to the lens.
It was often necessary to use forceps to gently guide the cable back into proper position. Once
the cable was aligned, gentle pressure was applied to ensure that the tape made a tight bond. The
cable was then removed from the metal cable holding shelf by unscrewing the red screw and
lifting the plastic cover. Using forceps, gentle pressure was applied at the side of the lens until
the lens was free from the dental wax. Any residual dental wax was removed using a Kimwipe™
and rubbing alcohol.
Metal weight
that holds
flipped cable
Rotation lever
Assembly well
Screw hole
Shelf to
support cable
Figure 12. Photo of the lens assembly table showing the metal cable holding shelf, the assembly well and the metal
weight. The assembly well, which is a recess to accept the lens is on a rotation plate which rides on an XYZ
translator.
B. Revision of Lens Design
The voids that are present in the base lens separate the electrode pads from the tear film present
on the cornea. These voids need to be filled with a conductive material in order to facilitate a
flow of current. Optimum lens fit is critical in order to achieve a high channel yield and
minimize cross-talk via a thick tear film between the lens and the cornea. Several modifications
were made to the lens design in order to improve on its function.
i.
Analysis of rat eye size vs age
Rat eye curvature measurements were taken twice a week starting at 4 weeks of age in two male
rats in order to determine the age at which the lens best fit the rat eye. The measurements were
performed using the software DinoCapture and ImageJ.
ii.
Measurement Procedure using DinoCapture 2.0 version 1.3.2
The animal was first anesthetized and the eye lubricated by applying a drop of saline. The animal
was then placed into a stereotactic table and its head was secured into position using the bite bar.
A ruler was then placed as close to the eye as possible making sure that it was in the same focal
plane as the horizon of the cornea when the camera viewed the eye from the side. Using the
"Folder" menu, a folder was created with the appropriate name including the rat id number and
the date. This folder needed to be created with DinoCapture in order to save the bitmap images
taken by the microscope such that measurements can performed later on. The "Text" option was
then used to create a label on the images. The magnification was then set to 1 in the top menu. At
this point the position of the ruler was adjusted to make sure it was in the same focal plane as the
horizon of the cornea when the camera views the eye from the side. If these were not in the same
plane, the depth of the focus on the camera would be different and as a result produce a fuzzy
image. By clicking on the Camera icon on the top left corner of the image between 5 and 7
pictures were taken. These pictures were then saved under a subfolder named "Picture" of the
folder created at the beginning. In order to make sure that everything was properly saved, the
"Folder" menu was used again and the folder containing the correct file name selected. A
preview of the images was displayed on the left panel of DinoCapture and one image was
selected for analysis. Using the "Calibration" option on the top right of the menu a new
calibration profile was created with a corresponding name (rat id, date and image number). At
this point the magnification was set to 1 by using the ruler on the image to the known distance of
2 mm that corresponded to the ruler on the screen. By using the “three points arc" on the top
menu, three spots on the circumference of cornea were selected and r, the radius of curvature was
measured, Figure 13. A total of 3 images per animal were used in calculating the radius of the rat
cornea.
Figure 13. Photo of eye and ruler captured using DinoCapture camera. Arc drawn for analysis of eye radius shown
in white with the calculated radius equal to 2.652 mm. Courtesy of Hadi Tajalli.
iii.
Measurement Procedure using ImageJ
The measurement procedure using ImageJ was very similar to that of the procedure with
DinoCapture. The desired jpg image was opened using the ImageJ software. The “Straight” line
tool was selected to mark the edge of the hash marks on the ruler and a straight line was drawn
from the left edge of one hash mark to the left edge of the next. This was done to set the scale
under “Analyze -> Set Scale” in the ImageJ toolbar. In the “Known Distance” box, the distance
of the length of the line drawn was entered in mm. The chosen unit of measure then set to be the
“Unit of Length.” The “Elliptical” option from the top menu was used to create a circle such that
the "w"(width) and "h"(height) of ellipse was equal to diameter of the circle drawn. The "ar"
aspect ratio in this case was one. This drawn circle encompassed the entire cornea.
Measurements were repeated on three images per animal.
iv.
Depth of the lens cup
The depth of the concave part of the lens was modified in order to improve the contact between
the lens and the eye by minimizing the contact between the lens and the eyelids. This was
achieved by sanding the eye side of the base lens from 2.97 mm to a variety of depths. The depth
that is referred to here is pictured in Figure 14. The 2.16 mm lens fit the curvature of the rats eye
the best while the 2.14 mm one was found to be too shallow. The 2.16 mm lens is pictured next
to the original 2.97mm lens in Figure 15.
A)
B)
Figure 14: (A) Schematic of the base lens, (B) Photo of base lens.
v.
Width of the lens voids
The voids in the CLEAr Lens™ were typically filled manually by using a polyamide
microelectrode filler needle (Microfil 28 guage, MF28G67-5) which had a steep learning curve.
The filler needle was slightly smaller than the voids and would break easily, lodging itself in the
void in the process. This rendered the CLEAr Lens™ unfit for use and a new lens had to be
assembled. Similarly, the use of the filler needle introduced small bubbles that would be trapped
in the voids that were difficult to remove. The goal of increasing the width of the voids was to
decrease the time and effort it takes to fill the lenses with saline and to increase channel yield.
The width of the voids was increased from 0.30 µm to 0.40 µm using a hand drill bit. This was
done to all 12 voids in the periphery of the lens (A ring, see Figure 15) and the central void.
C. Lens Filling Procedures
i.
Saline
At the beginning of this study, the CLEAr Lens™ was filled with conductive saline manually by
using a polyamide microelectrode filler needle (WPI™ Microfil 28 guage, MF28G67-5) and
filling 25 voids in order to complete the electrical contact between the corneal surface and the
metal electrodes in the lens cable. This involved inserting the tip of the filler needle into the
bottom of the void and delivering saline via a hand held syringe all the while slowly withdrawing
it from the void. This was a tedious, time consuming process that often yielded less than perfect
results due to the filler needle breaking off in the lens voids and trapping air bubbles that were
difficult to remove. These air bubbles interfered with the electrical connection, while the metal
electrodes were often damaged due to physical scraping by the filler needle. To achieve a higher
yield of working electrodes per lens, a better method of filling the lens holes was needed. In
order to avoid the manual filling procedure, the use of a vacuum chamber was investigated.
Parameters to be optimized included the amount of saline to be place in the lens cup, level of
vacuum applied as well as temperature and duration. After a great deal of optimization, the
following stranded operating procedure was established.
ii.
Saline lens filling procedure
The rat lens was checked for secure attachment to the cable as well as any foreign debris. The
lens was then placed into a Petri dish with the concave side facing up. Using a 1 ml syringe, 3
drops of saline were placed onto the lens. The Petri dish was then placed into the vacuum
chamber making sure that the depressurizing value was in the open position and the pressurizing
valve was in the closed position. The vacuum was then turned on until the pressure gauge
reached 28.5 inHg. When the pressure gauge reached 28.5 inHg, the saline solution was
continuously watched for slight changes in opacity due to freezing of the solution which
typically started after about 60 seconds. Upon slight change in clarity of the saline, the vacuum
pump was shut off and the depressurizing valve was turned to the closed position. If the saline
froze, the procedure failed and needed to be repeated. If the procedure was successful, the lens
remained in the vacuum chamber for a minimum of 5 minutes. The lens cannot be left in the
chamber for a longer period of time due to evaporation of the saline. After 5 minutes had elapsed
the air was slowly introduced back into the vacuum by opening the pressurizing valve. Using a
microscope, the lens was inspected to see if any bubbles were present in the saline or in the lens
voids. If any bubbles were found, the procedure had to be repeated.
iii.
Saline lens cleaning and storage procedure
The lens was first placed into a Petri dish and by using a Kimwipe™, the excess saline was
wiped off. Using a 1 ml syringe, 3 drops of distilled water were placed into the lens and the Petri
dish was placed into the vacuum chamber. The depressurizing valve was set to the open position
while the pressurizing valve was set to the closed position. The vacuum was turned on until the
pressure gauge reached 24 inHg. The pressurizing valve was then opened and the air was
allowed to slowly enter the vacuum chamber. The depressurizing of the lens and the addition of
distilled water was repeated for a total of three time all the while wiping the previously used
distilled water in between the cycles. The lens was then removed from the vacuum chamber and
the distilled water was wiped off using a Kimwipe™ from the lens. The lens was then stored
exposed to air.
iv.
Poly-Ethylene Glycol (PEG)
While the vacuum method had substantially improved the filling procedure as well as the
functional yield of electrodes, it was still time consuming and an imperfect process that required
practice in order to obtain consistent results. The ideal solution would be to fill the voids in the
lens permanently with a clear conductive material that is safe for contact with the cornea. One
of the materials that was considered was a hydrogel called poly-ethylene glycol (PEG). This gel
is easily made conductive by incorporating free ions such as Na+ and Cl- . The PEG fabrication
procedure was optimized by Melanie Kollmer from Richard A. Gemeinhart’s lab.
v.
PEG fabrication and lens filling procedure
Courtesy of Melanie Kollmer, PEG was fabricated by mixing 83 µL of Irgarcure 2959 to 0.817
ml of saline. This mixture was then added to 100 mg of PEGDA (Mw=3400). The rat lens was
then checked for secure attachment to the cable as well as any foreign debris. The lens was then
placed into a Petri dish with the concave side facing up. Using a 1 ml syringe, 3 drops of PEG
were placed onto the lens. The Petri dish was then placed into the vacuum chamber making sure
that the depressurizing value was in the open position and the pressurizing valve was in the
closed position. The vacuum was then turned on until the pressure gauge reached 28.5 inHg.
When the pressure gauge reached 28.5 inHg, the saline solution was continuously watched for
slight changes in opacity due to freezing of the solution which typically started after about 60
seconds. Upon slight change in clarity of the saline, the vacuum pump was shut off and the
depressurizing valve was turned to the closed position. If the PEG froze, the procedure failed and
needed to be repeated. If the procedure was successful, the lens remained in the vacuum chamber
for a minimum of 5 minutes with a black cloth covering the chamber in order to minimize the
amount of UV light entering the chamber. The lens was checked by opening the pressurizing
valve and allowing the air back into the chamber. Using a microscope, the lens was inspected for
bubbles. If none were present, the lens was placed under a 360 nm UV light for 40 minutes to
polymerize the PEG. If bubbles were present, the filling procedure had to be repeated. Once the
PEG polymerized, 4 drops of saline were placed onto the PEG and allowed to sit for 15 minutes
to ensure that the PEG was fully hydrated. Once the PEG was fully hydrated, the excess was
scraped out of the voids such that the remaining PEG was flush with the curved sides of the lens.
vi.
PEG lens storage procedure
The storage of PEG varied based on whether the lens was to be stored short or long term. If the
lens was to be used daily, a drop of saline from a 1 ml syringe placed onto the PEG every day
kept the PEG properly hydrated and functional. The addition of a Petri dish cover was used to
reduce evaporation. If the lens was to be stored long term, using a 1 ml syringe, 5 drops of
distilled water were placed onto the PEG and the excess wiped off with a Kimwipe. The lens was
then stored in an air tight chamber. Prior to the use of a lens filled with PEG that was stored for a
duration of time, 3 drops of saline from a 1 ml syringe were placed onto the PEG and allowed to
rehydrate for 15 minutes.
6.2 Results
A. Analysis of rat eye size vs age
Rat eye measurements that were taken from two male rats were used to develop an
understanding of how rat eye size changes as a function of age. Figure 15 illustrates this
compiled data starting at 24 days of age. According to the data, the rat eye would be an ideal fit
for the lens at 31 days of age since the lens radius of curvature is 2.5 mm and the radius of
curvature of the rats eye at that age is an average of 2.53 mm. Upon testing the lens at this age it
was found that the lens was too large to fit properly. This error was attributed to the improper
alignment of the rat eye during the eye size measurement procedure as well as the marker
placement of the arc calculating software.
Based on manual manipulation which consisted of feeling for a secure fit that kept the rat lens on
the eye without falling off it was determined that the lens fit first became acceptable at 42 days
of age. Based on manual manipulation which consisted of feeling for a secure suction like fit that
kept the rat lens on the eye without falling off it was determined that the lens fit best at 42 days
of age. Rats that were used for meERG recording were 42 and 49 days of age plus or minus a
day for each target eye.
y = 1.2812x0.1948
3.0
2.9
Curvature Radius (mm)
2.8
2.7
Measurement
difference
2.6
2.5
Lens radius
2.4
2.3
2.2
2.1
2.0
20
25
30
35
40
45
50
55
60
65
70
Age (days)
Figure 15: Rat eye radius measurements plotted from the age of 24 to 66 days. Measurements were taken from two
male rats with 3-5 days of rest between measurements to reduce stress from repeated anesthesia. According to the
measurements done in ImageJ and DinoCapture, the rats eye is a perfect fit for the lens at the age of 31 days.
However, upon manual inspection of the lens it was found to best fit at 42 days. Symbols plot measurements from
two animals as well as the error between the measured radius and radius of the eye that fits the 5 mm lens. A line
was fit to the averaged measurements from two rats and is displayed. This trend line was used to calculate the age at
which the lens was to fit.
B. Depth of Lens Cup
The depth of the lens was modified in order to improve the lens seating on the cornea. By
creating a shallower lens, it was no longer necessary to pull back the eye lids of the rat in order
to maintain good electrical contact. The photos in Figure 16 show the original and modified lens,
2.16 mm on the left and the original 2.97 mm on the right.
Figure 16: Two photos of the modified and original depth of lens at 2.16 mm on the left and the original 2.97 mm on
the right. The modification was made using sandpaper and a custom jig to ensure controlled removal of PMMA
material. The shallower lens ultimately proved to better fit the curvature of the rats eye. The voids within the lens
were widened using a hand-held drill bit. The wider voids can be seen in the central and peripheral rings in both
lenses.
C. Width of lens voids
The increase of the width of the wells did not improve either the time it took to fill the voids or
reduce the number of bubbles that were often trapped in the voids during the filling procedure.
To the contrary, once the lens was filled and tipped, the probability of saline dripping out was
increased due to the increase width of the voids. The wider voids can be seen in Figure 15
located in 12 peripheral (A ring) and one central void. Increasing the width of the voids reduced
the likelihood of the filler needle breakage but this was not important once the vacuum filling
method was adopted. No difference in impedance was observed between the different widths of
voids.
D. Lens Filling Procedures
The decrease in filling time and an increase in channel yield reached near perfection (24 or 25
working channels) with the vacuum method when compared to the manual filler syringe
procedure. The channels that were often found to not work were later determined to be flawed
due to an incomplete etching process during cable fabrication. This vacuum filling procedure
was later applied to PEG and allowed for a consistent number of working channels as well as a
faster experimental setup if the experiments were carried out daily. The major drawback to PEG
was the initial filling procedure as well as long term storage. Unless the lens was filled with PEG
flawlessly the first time, the lens and cable assembly had to be taken apart to remove the PEG,
thereby wasting a cable. The initial stages of perfecting the PEG filling technique produced a
number of wasted cables. Long term storage proved to be difficult as certain channels
discontinued working upon rehydration. It appeared as though the PEG separated from the gold
pads on the cable after long term dehydration. Saline lens storage proved to be easier with the
disadvantage of a longer daily setup. Similarly, much of the saline filling procedure depended
strongly on visual feedback and experience and often needed to be repeated after bench top
testing to confirm channel yield. The channel yield proved to be higher for the saline filled lens
due to ability to repeat the filling procedure and test the yield prior to the experiment. On a one
time fill basis, the fill success rate was about the same for both materials with 23 to 25 working
channels. The need to repeat the filling procedure for both materials would ultimately prove to be
a challenge if it was necessary to scale up manufacturing. The vacuum filling technique was
adopted for filling the human CLEAr Lens™ and is now standard lab procedure with 100%
channel yield. The impedance of channel 77 on the CLEAr Lens™ was measured by a lab mate,
Hadi Tajalli and can be seen in Figure 17.
Impedance(k Ohm)
200
150
100
50
0
0
200
400
600
800
1000
1200
Frequency (Hz)
Figure 17: Impedance of channel 77 on the CLEAr Lens™ performed using saline. Courtesy of Hadi Tajalli.
7. Specific Aim 3- To record the meERG signal from normal rats and
to characterize this relatively novel signal with regard to test-retest
and inter animal variability and to quantify spatial differences in
meERG potentials across the rat cornea.
7.1 Methods
Experiments were carried out according to the ACC protocol number 11-154. The rat retina was
illuminated using the spatially homogeneous light stimulus source developed in Aim 2, set to the
- 40 -
strengths listed in Table 2. A aluminum cradle was used to hold the rat steady and position the
eye at the center of the light source with the optical axis of the eye at approximately zero degrees
to the normal of the source. The subject then received approximately full-field illumination. The
recording protocol is developed below. Figure 18 shows a photo of a rat during recording.
Filter size
(in inches)
Flash strength -3.8
(sc cd s m-2)
Flash strength -2.8
(sc cd s m-2)
0.0787
1.144
1.614
1.986
2.812
3.455
3.984
0.01
5.8
20
30
73
116
159
n/a
n/a
n/a
n/a
n/a
n/a
319
Table 2: Light source calibration table with light from filters 0.0787 to 3.894 inches. The power level was increased
from level -3.8 to -2.8 on the flash head power pack and the measurement taken at the largest filter size. The
stimulus strength of 159 sc cd s m-2 was found to be saturating and was the highest stimulus strength used for rat
recordings. The abbreviation n/a indicated that data was not collected at that filter size.
A. Recording Protocol
The animal recording procedure began by weighing the animals and administering the
appropriate amount of anesthetic of Ketamine 100 mg/mL and Xylazine 20 mg/mL under a dim
red light. Once the animal was sedated, corneal anesthetic Proparacane 0.5%, and dilators
Phenyephrine 2.5% and Tropicamide 1% were administered one minute apart. The ground
electrode needle was then placed beneath the skin in the back of the rats neck while the reference
electrode was placed in the cheek. The CLEAr LEns™ was then quickly tested for channel yield
and placed onto the rat eye. The rat was positioned such that the axis of the right eye was
perpendicular to the light stimulus box. Consistency of positioning the animal was based on
visual feedback. MCRack was turned on to recording mode and the number of working
electrodes was confirmed. If certain electrodes discontinued working, the lens was readjusted on
the animal. The smallest filter was placed into the filter slot in the light stimulus box and lowered
to the appropriate height from the rats eye. All wires going into the Faraday cage were turned off
and or unplugged. Alternating filter sizes was done in the dark with a head light set on red light.
Starting with the 0.01 sc cd s m-2, flash strength was increased in the following order; 5.8, 20, 30,
73, 116 and 159 sc cd s m-2 until a total of 7 flash strengths were used. Four flashes at least one
minute apart were recorded for each flash strength. A multi channel systems (MEA-60) amplifier
was used for recording and the signal sampled at 5 kHz. A typical duration for an experiment
was 30 minutes and did not require a booster of anesthetic. Once the recording session was
completed successfully, the rat was returned to its cage. Artificial tears were place onto both eyes
and the rat was monitored until it woke up.
Figure 18: A photograph of an anesthetized rat during a recording with the CLEAr Lens™ in place. Courtesy of
Hadi Tajalli.
B. Analysis Protocol
Data to be used for later analysis was collected using MCRack and later transferred to Excel.
Files corresponding to the appropriate series of flash strengths were grouped together, ordered,
averaged and the a- and b-wave amplitudes evaluated. A-wave amplitudes were determined by
first averaging 50 seconds of the baseline before the flash. Using Excel, the amplitude of the
waveform was taken at t = 8.2 ms after the flash. This amplitude was subtracted from the
baseline to give the final a-wave amplitude. The b-wave amplitude was determined by finding
the maximum value after t = 0 and subtracting the baseline from this value.
7.2 Results
A main goal of the NEVL is to research the potential of the meERG to be used as a tool for
diagnosis and the monitoring of eye diseases that affect the retina. A large number of rat models
of human eye diseases exist today so in order to demonstrate the efficacy and safety of the new
CLEAr Lenstm, rats were used. The final Aim of this work was to characterize the novel meERG
signal by recording from normally-sighted rats in hope that this normative data will provide a
comparative foundation for animal models of retinal dysfunction.
A. Typical meERG response
A typical meERG response recorded with the CLEAr Lens™ filled with saline can be seen in
Figure 19(A). Each meERG waveform is the average of four flashes. Each waveform is offset
vertically and horizontally such that is appears in the correct relative position of the corneal
electrode from which it was recorded. This type of plot will be referred to as a “position plot”
below. The channel yield in this example is relatively high with a few centrally located channels
that have a strong stimulus artifact. It is possible to subtract the artifact but for the purposes of
demonstrating what a typical flash evoked response looks like as well as a 25 channel yield it
was not done for this figure. Figure 19(B) illustrates the channel map, which is the relative
location of the recording electrodes on the lens.
A)
Nasal
Superior
Inferior
B)
Temporal
Figure 19: (A) Position plot (experiment 20111123-23,24,25,26) where responses to four flashes at 73 sc cd s m-2
were averaged with SNR= 103 dB. Anatomical orientation of the lens is noted in the plot; electrode A8 was at the
most superior position. The SNR value was calculated using the following equation 𝑆𝑁𝑅 = 20𝐿𝑜𝑔 10
waveform was offset in order to reflect its actual position on the lens. (B) meERG CLEAr Lens
™
𝑆𝑖𝑔𝑛𝑎𝑙
𝑁𝑜𝑖𝑠𝑒
. Each
electrode map.
In order to illustrate the variability of a-wave and b-wave amplitude values, three animals were
chosen that had a high channel yield with little artifact. The analysis was performed on the same
animals at 6 and 7 weeks of age, and is summarized in Table 3. The mean a-wave and b-wave
amplitudes were calculated by averaging all the electrodes. The standard deviations were then
divided by the mean and multiplied by one hundred in order to comparison between animals. The
symbol N designates the number of working channels that were used to calculate the mean
within a particular experiment. The resulting a- and b-wave means are highly varied from one
experiment to the next, as is typical for conventional ERG recording. The standard deviations of
the a-wave amplitudes are smaller than the standard deviations of the b-wave amplitudes. The
normalized standard deviations vary not only from one animal to the next but also between 6 and
7 weeks of age within the same animal. In order to better understand this trend the ratios of the
standard deviation divided by the mean were plotted in Figure 20.
Experiment date
73 sc cd s m-2
6 weeks
A
SD/Mean
B
SD/Mean
Experiment Date
7 weeks
A
SD/Mean
B
SD/Mean
Rat 1
20111123-A
N=22
Mean
StDev
331
3.2
1.0
1399.7
33.3
2.4
20111129-b
N=22
568.9
3.2
5.6
1768.9
22.3
1.3
Rat 2
20110824-b
N=24
Mean
StDev
243
5.1
2.1
1348.3
188
13.9
20110830-a
N=16
206.9
1.0
5.0
1316.9
19.9
1.5
Rat 3
111122
N=23
Mean
StDev
440
16
3.6
1550.7
18
1.2
111128
N=22
482.5
4.3
9.0
1504.2
14.5
1.0
Table 3: Mean a- and b-wave amplitudes with all electrodes averaged, stimulus = 73 sc cd s m-2. Three animals were
chosen to compare the differences in amplitudes between the animals as well as the differences at week 6 and 7
within one animal. The symbol N designates the number of working channels that were used to calculate the mean
within a particular experiment. Standard deviation divided by the mean values have been multiplied by 100.
B)
Normalized StDev
10
8
6
Rat 1
4
Rat 2
Rat 3
2
15
Normalized StDev
A)
0
10
Rat 1
Rat 2
5
Rat 3
0
6 weeks
7 weeks
6 weeks
7 weeks
Figure 20: (A) The normalized standard deviation of the a-wave amplitudes from Table 3 (B) The normalized
standard deviation of the b-wave amplitudes from Table 3. Three animals were chosen to illustrate the differences in
amplitudes as well as at weeks 6 and 7 within one animal.
The a-wave amplitudes across all 25 electrodes for 22 experiments were averaged together and
their standard deviations plotted in Figure 21 (flash strength 73 sc cd s m-2). The mean was
calculated to be 440, StDev = 122. In order to compare the experiments to one another, each
experiment was normalized by dividing the standard deviation by its mean, Figure 22. The mean
of this ratio was calculated to be 0.04, StDev = 0.02.
700
600
500
µV
400
300
200
100
0
Figure 21: At 73 sc cd s m-2 the a-wave amplitudes of all electrodes for 22 experiments were averaged together and
their standard deviations plotted. The mean was calculated to be 440, StDev = 122. Each bar represents an
experiment.
0.12
StDev/mean
0.1
0.08
0.06
0.04
0.02
0
Figure 22: The mean divided by the standard deviation of Figure 21 plotted. Each bar represents an experiment and
has the same sequence as does Figure 21. Mean = 0.04, StDev = 0.02
B. Inter animal variability
Intra-subject data is not always available for comparison, often requiring a clinician to use a
database of subjects to determine if the result is within a normal range. The acquisition of a
normative data set is critical for the effectiveness of any test. The main goal of the experiments
performed in this study was to begin to document the normal range and understand the
variability of the inter-animal meERG results such that any measurements taken at a future time
will have a normal range to compare to.
i.
A-wave by ring
In order to further understand the basis for the large variances within an experiment, data were
grouped by concentric rings formed by the rat CLEAr Lens™ electrodes. This produced
evaluation areas at equal radial distances from the center of the cornea and allowed for an
increase in sensitivity and in noise reduction in measuring differences between central and
peripheral response amplitudes. This approach in grouping of data is utilized by many imaging
techniques such as the mfERG (ISCEV, 2012) and while it limits the ability to evaluate
asymmetry in the responses, it increases SNR for evaluating differences as a function of radial
eccentricity. With this in mind, the mean a-wave amplitudes in the A, C and M rings were
normalized to the ring B mean and plotted in Figure 23. The following formulas were employed
for calculating the normalized ring averages,
𝐴̅
𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝐴 = 𝐵̅
𝐶̅
𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝐶 = 𝐵̅
𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝑀 =
Equation 1
̅
𝑀
𝐵̅
Since the a-wave amplitudes in ring A have the highest potential for variability due to lens
misalignment issues, in order to have confidence in the calculated mean value by which the
amplitudes would be normalized, the ring B mean was chosen as the common reference value.
The ratios for ring A at 6 and 7 weeks consistently range from 1.0 to 1.4 while the ring C has a
slightly wider distribution ranging from 0.94 to 1.03. The ratios of Ring A, B and C at 6 weeks
of age were calculated and listed in the table next to each figure. The ratios of the central
electrode M are based on the least number of experiments as this centrally located electrode was
not always functioning during the full course of the experiment. Overall, the a-wave ratios for
three rings regardless of age consistently cluster around 1. This is consistent with corneal
potentials that are uniform as a function of radial eccentricity.
1.04
1.02
1.02
1
1
Ratio (A/B)
Ratio (A/B)
1.04
0.98
0.96
0.94
0.92
Mean
0.99
1.00
0.94
SD
0.02
0.04
0.92
N
12
10
0.96
1.04
1.04
1.02
1.02
1
1
0.98
0.96
0.94
0.92
0.9
Weeks 6
7
Mean
0.99
0.97
0.94
SD
0.03
0.01
0.92
N
12
10
0.96
1.04
1.04
1.02
1.02
1
1
0.98
0.96
0.94
0.92
0.9
7 weeks
0.98
0.9
6 weeks
Ratio(M/B)
Ratio(M/B)
7
0.9
6 weeks
Ratio (C/B)
Ratio (C/B)
0.9
Weeks 6
0.98
7 weeks
Weeks 6
7
Mean
0.99
1.01
0.96
SD
0.04
0.05
0.94
N
5
10
0.98
0.92
6 weeks
0.9
7 weeks
Figure 23: A wave amplitudes from rings A, C and M normalized to ring B and plotted based on age of the animal.
Each bar represents an experiment with the table on the right showing the mean and the standard deviation. The
symbol N designates the number of animals that were used to calculate the summery statistics.
ii.
B-wave by ring
Ratios for b-wave amplitudes were normalized to ring B and plotted by ring in Figure 24. The
ratios of the b-wave amplitudes varied as much within a ring as did the a-wave ratios. The central
electrode, M seemed to have less variance for the b-wave amplitude ratios than the a-wave
amplitude ratios. Overall, the variance within the ring was consistent between the a- and b- wave
amplitudes.
1.04
1.02
1.02
1
1
Weeks 6
7
Mean
0.99
1.00
SD
0.01
0.05
N
10
10
Weeks 6
7
Mean
0.99
1.01
0.96
SD
0.08
0.06
0.94
0.94
N
9
10
0.92
0.92
Ratio(A/B)
Ratio(A/B)
1.04
0.98
0.96
0.94
0.92
0.98
0.96
0.94
0.92
0.9
0.9
7 weeks
1.04
1.02
1.02
1
1
Ratio(C/B)
1.04
0.98
0.96
Ratio(M/B)
0.98
0.9
0.9
6 weeks
7 weeks
1.04
1.04
1.02
1.02
1
1
Ratio(M/B)
Ratio(C/B)
6 weeks
0.98
0.96
0.94
7
Mean
1.00
1.00
SD
0.04
0.05
N
7
10
0.96
0.94
0.92
0.92
0.9
0.98
Weeks 6
0.9
6 weeks
7 weeks
Figure 24: B-wave amplitudes from rings A, C and M normalized to ring B from animals at 6 and 7 weeks. Each bar
represents an experiment. The ratios are consistently similar between all animals, clustering around a ratio of 1.
C. Spatial Differences
The most important feature of the meERG measurement is that it provides information about the
spatial distribution of ERG potentials across the cornea. This is the only ERG measurement
technique that measures distinct potentials at different locations on the cornea at the same time.
The attempt to quantify differences in the spatial distribution of corneal potentials for normally
sighted rats might begin to provide information about the health of the retina. In order to achieve
this goal, meERG potentials have been analyzed as a function of electrode position. The a-wave
amplitudes across all rings at 73.4 sc cd s m2 were calculated by recording and averaging four
flashes together per experiment for 18 experiments. Due to the presence of outliers some of the
experiments were not used in this analysis. Ratios of each A ring electrode divided by the mean
of rings C and M multiplied by 100 were calculated and are plotted in Figures 25 and 26. The
following equation defines the ratios:
𝑅𝑎𝑡𝑖𝑜𝐴𝑛 =
𝐴𝑛
𝐶1+𝐶2+𝐶3+𝐶4+𝑀
(
)
5
𝐴𝑛 = 𝐴 𝑟𝑖𝑛𝑔 𝑒𝑙𝑒𝑐𝑡𝑟𝑜𝑑𝑒
𝑥 100
Equation 2
𝐶1−4 = 𝐶 𝑟𝑖𝑛𝑔 𝑒𝑙𝑒𝑐𝑡𝑟𝑜𝑑𝑒𝑠
𝑀 = 𝑀 𝑒𝑙𝑒𝑐𝑡𝑟𝑜𝑑𝑒
This ratio was chosen due to a precedent in our lab developed in a study looking to use the
meERG for glaucoma detection. The use of the a-wave amplitudes over the b-wave amplitudes
were chosen arbitrarily. Values corresponding to the appropriate bin were divided by 216 (18
experiments multiplied by 12 electrodes per experiment) to find the ratios probability. The mean
probability ratio across all 18 experiments was 101.0 with a standard deviation of 4.0. Ratios are
ultimately a means of comparison and a way of standardizing the results.
0.2
P(Ratio)
0.15
0.1
0.05
0
88
90
92
94
96
98 100 102 104 106 108 110 112 114 116 118
Value of Ratio (Peripheral vs. Central)
Figure 25. Probability distribution of a-wave amplitude ratios for 18 experiments at 73.4 sc cd s m-2. The ratios of
each A ring electrodes as defined by equation 2. Values corresponding to the appropriate bin were divided by 216 to
find the ratios probability. Mean value of ratio= 101.0, SD = 4.0.
0.4
P (Ratio)
0.3
0.2
0.1
0
90
92 94 96 98 100 102 104 106 108 110
A-Wave Amplitude Ratio (Peripheral / Central X 100)
Figure 26: For each A-ring electrode position (A1-A12), the average ratio across the 18 experiments was calculated,
resulting in 12 averaged ratios. Mean = 100, SD = 1.6.
D. Test-retest (same animal)
The variability within a data set plays an important role in clinical diagnosis and the ability to
determine whether a condition exists or is changing. Therefore, understanding the repeatability
of results from a single subject is critical. These results help identify the change that is normal
due to growth or noise and differentiate it from a change that might indicate a clinical condition.
In order to characterize this test-retest variability, 8 animals were selected and the same ratios as
those used to calculate spatial differences were used to evaluate the a-wave amplitudes at 6 and 7
weeks of age. A-wave amplitudes were determined from the average of four flashes at 73 sc cd s
m-2. Figure 27 is a series of histograms of the a-wave amplitude ratios at 6 and 7 weeks from 8
different animals. The group of twelve a-wave ratio values obtained in each of the eight animals
at six weeks was compared to the corresponding twelve ratio values obtained at seven weeks
using a paired Student's t-test. In five of the eight animals, the p values were greater than the
criterion value of 0.05, indicating no significant difference between the initial test and the
repeated test one week later. Figure 28 is a histogram with all the a-wave amplitude ratios from 8
rats averaged together at 6 and 7 weeks. When the data for the eight animals were combined, the
resulting p-value was 0.08. While this value is above the typical criterion value for statistical
significance (0.05 > 0.08), a strict interpretation is that there is only an eight percent chance that
the six and seven week measurements are not different, or a 92% chance that they are different.
This change in the distribution of the amplitudes might be due to lens placement or fit relative to
the stimulus source. In order to improve consistency in the future between one experiment and
the next, photographs of lens placement will be taken and the angle between the lens and the
luminance source will be analyzed. The issue of significant structural changes that might have
occurred at the retina was not investigated as this time as it is unlikely to occur over the course of
7 days. Although the origin of the change of the distributions needs to be further investigated it
and has been determined to be beyond the scope of this study, the results nonetheless help set the
3.5
3
2.5
2
1.5
1
0.5
0
p = 0.63
animal 2, 6 weeks
animal 2, 7 weeks
0.83
0.84
0.85
0.86
0.87
0.88
0.89
0.9
0.91
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
1.01
1.02
1.03
1.04
1.05
1.06
1.07
1.08
1.09
1.1
1.11
1.12
1.13
1.14
1.15
1.16
1.17
1.18
1.19
1.2
1.21
1.22
Count
foundation for detection of retinal dysfunction at varying stages of development.
A-wave amplitude ratios
0.83
0.84
0.85
0.86
0.87
0.88
0.89
0.9
0.91
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
1.01
1.02
1.03
1.04
1.05
1.06
1.07
1.08
1.09
1.1
1.11
1.12
1.13
1.14
1.15
1.16
1.17
1.18
1.19
1.2
1.21
1.22
Count
0.83
0.84
0.85
0.86
0.87
0.88
0.89
0.9
0.91
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
1.01
1.02
1.03
1.04
1.05
1.06
1.07
1.08
1.09
1.1
1.11
1.12
1.13
1.14
1.15
1.16
1.17
1.18
1.19
1.2
1.21
1.22
Count
0.83
0.84
0.85
0.86
0.87
0.88
0.89
0.9
0.91
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
1.01
1.02
1.03
1.04
1.05
1.06
1.07
1.08
1.09
1.1
1.11
1.12
1.13
1.14
1.15
1.16
1.17
1.18
1.19
1.2
1.21
1.22
Count
0.83
0.84
0.85
0.86
0.87
0.88
0.89
0.9
0.91
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
1.01
1.02
1.03
1.04
1.05
1.06
1.07
1.08
1.09
1.1
1.11
1.12
1.13
1.14
1.15
1.16
1.17
1.18
1.19
1.2
1.21
1.22
Count
8
6
4
p = 0.0006
2
animal 3, 6 weeks
0
animal 3, 7 weeks
A-wave amplitude ratios
3.5
3
2.5
2
1.5
1
0.5
0
p= 0.02
animal 4, 6 weeks
animal 4, 7 weeks
A-wave amplitude ratios
7
6
5
4
3
2
1
0
p = 0.0006
animal 5, 6 weeks
animal 5, 7 weeks
A-wave amplitude ratios
3.5
3
2.5
2
1.5
1
0.5
0
p=0.60
animal 6, 6 weeks
animal 6, 7 weeks
A-wave amplitude ratios
4
Count
3
p=0.75
2
animal 7, 6 weeks
1
animal 7, 7 weeks
0.83
0.84
0.85
0.86
0.87
0.88
0.89
0.9
0.91
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
1.01
1.02
1.03
1.04
1.05
1.06
1.07
1.08
1.09
1.1
1.11
1.12
1.13
1.14
1.15
1.16
1.17
1.18
1.19
1.2
1.21
1.22
0
A-wave amplitude ratios
3.5
3
Count
2.5
p=0.37
2
1.5
animal 8, 6 weeks
1
animal 8, 7 weeks
0.5
0.83
0.84
0.85
0.86
0.87
0.88
0.89
0.9
0.91
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
1.01
1.02
1.03
1.04
1.05
1.06
1.07
1.08
1.09
1.1
1.11
1.12
1.13
1.14
1.15
1.16
1.17
1.18
1.19
1.2
1.21
1.22
0
A-wave amplitude ratios
5
Count
4
p=0.18
3
2
animal 9, 6 weeks
1
animal 9, 7 weeks
0.83
0.84
0.85
0.86
0.87
0.88
0.89
0.9
0.91
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
1.01
1.02
1.03
1.04
1.05
1.06
1.07
1.08
1.09
1.1
1.11
1.12
1.13
1.14
1.15
1.16
1.17
1.18
1.19
1.2
1.21
1.22
0
A-wave amplitude ratios
Figure 27: A wave amplitude ratios from 8 animals at 6 and 7 weeks of age in response to a flash strength of 73 sc
cd s m-2. The A-wave amplitude ratios were calculated by dividing each peripheral electrode in ring A by the
average of the electrodes in rings C and M. Student’s t-Test was used to determine whether the ratio distributions
were different from one week to the next. In five out of eight animals, the six and seven week distributions were not
significantly different (p < 0.05).
30
25
Count
20
P=0.08
15
6 weeks
10
7 weeks
5
0.83
0.84
0.85
0.86
0.87
0.88
0.89
0.9
0.91
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
1.01
1.02
1.03
1.04
1.05
1.06
1.07
1.08
1.09
1.1
1.11
1.12
1.13
1.14
1.15
1.16
1.17
1.18
1.19
1.2
1.21
1.22
0
A-wave amplitude ratios
Figure 28: Eight distributions of a-wave amplitude ratios from figure 25 combined in one plot. Stimulus strength =
73 sc cd s m-2. The A-wave amplitude ratios were calculated by dividing each peripheral electrode in ring A by the
average of rings C and M. The mean of the 6 week average was found to be 1.04 and 1.01 at seven weeks. The
distribution of a-wave ratios is not different, on average across animals, at 6 weeks than it is at 7 weeks. (P = 0.08)
8. Discussion
The overall goal of this study was to further the development of a novel ERG measurement
technique, the multi-electrode electroretinogram (meERG). This technique is enabled by the
contact lens electrode array, CLEAr Lens™ technology developed in the Hetling (UIC) and
Williams (UW, Madison) labs. The meERG approach is unique in that it measures corneal ERG
potentials at several locations simultaneously, thereby creating a surface potential map of the
cornea. This technique is adaptable to a wide variety of visual stimuli currently used in vision
science and diagnostic ophthalmology. The main application of the meERG technique will be to
detect spatial differences in the response of the retina that are due to eye diseases such as
- 60 -
glaucoma, retinitis pigmentosa, and diabetic retinopathy. This study focused on fabricating a
stimulus source suitable for meERG recording in rats (Specific Aim 1), improving the design and
use of the rat-sized CLEAr Lens™ (Specific Aim 2), and recording and characterizing the first
meERG signals from rats to establish the first normative data set for this new type of ERG
measurement (Specific Aim 3).
8.1 Summary of Results by Specific Aim
A. Summary of results: Specific Aim-1
In order to understand the activity of the retina and reveal areas of abnormal response, the spatial
distribution of the light stimulus source must be well understood. Since the relationship between
the light stimulus source and the activity of the retina is directly linked, fabrication of the
stimulus source was performed in hope that we would obtain a level of spatial homogeneity that
exceeded the current gold-standard commercial units. The work done to fabricate the full-field,
homogeneous stimulus source required a fundamental knowledge of optics and a significant
amount of empirical “trial and error” to reach the design goals. In modifying the stimulus source,
the percent change from zero to 70 degrees from the vertical was decreased from 66.13% to
22.80%. A measure of uniformity, the sum of squares error between measurements and a flat line
(ideal distribution) was also significantly reduced. The geometry of the light stimulus box was
ultimately the limiting factor in the luminance profile that could be produced with an overhead
light source. It was not possible to further increase the amount of light available at 40 to 70
degrees without increasing the amount of light at 0 to 30 degrees. The periphery of the dome
receives less light (which originates directly overhead) and the only way to increase the amount
of light available in the periphery is to add a separate light source to that location. A possible
solution to this in the future might include increasing the distance between the reflective box
walls and the dome surface, either by using a larger box or a smaller diameter dome such that the
amount of light reaching the periphery might be increased. Comparison to commercially
available units was hindered because the companies that produce those units do not publish or
reveal direct measures of uniformity of luminance. However, the final design has proven
satisfactory and robust and is in regular use in the NEVL. This system is used in place of a
commercial system, and does not by itself contribute anything novel to the field of ERG
recording, other than perhaps serving as an example to other laboratories who wish to construct a
low-cost source ($2k for our source, compared to at least $10k for a commercial source).
B. Summary of results: Specific Aim-2
The rat eye corneal curvature was quantified as a function of age using two male rats to
determine the age at which the CLEAr Lens™ would fit best. According to the software ImageJ
and DinoCapture the radius of curvature of the rat eye was 2.5 mm at just over four weeks of age.
Attempts to record the meERG at this age revealed that the eye was too small to properly fit the
lens. This error was attributed to the improper alignment of the rat eye during the measurement
procedure as well as the arc calculating software. The lens was found to best fit at 6 weeks of age
via manual manipulation which consisted of feeling for a secure fit that kept the lens on the eye
without falling off during handling. Other attempts to measure rat corneal curvature as a function
of age could not be found in the literature with the most relevant measurements performed by
Chauhuri, (1983) where enucleated rat eyes were used to determine corneal radii of curvature.
This study focused on measuring the thicknesses and radii of optical compounds as well as
determining the reflective indicies of the lens and tissues with the use of a light beam. Similar
experiments as those performed by Chauhuri were performed in mice in 1984 by Remtulla S. and
Hellett P.E. Neither of these attempts measure the radius of curvature in live animals as a
function of time but rather focused on understanding the anatomy and structure of the rodent eye.
Design revisions to the rat CLEAr Lens™ were made to improve the lens preparation (filling
lens voids to increase channel yield) and to improve lens fit on the rat eye (making the lens cup
shallower). The increase in diameter of the lens voids to improve filling proved to be effective
when hand-filling the lenses with a syringe, but offered no advantage when filling the lens using
the vacuum method. Lens placement and corneal contact were improved by making the lenses
shallower, and all lenses currently in use have been modified in this way.
The primary contribution under this aim was the development of the protocol for using a vacuum
chamber to fill the lenses. This method offered substantial advantages over hand filling in terms
of time required, reduced damage to the CLEAr Lens™ components, and increased channel yield.
The next step in this line of improvements will be to permanently fill the lens holes with a
conductive, transparent, biocompatible material and thus eliminate most of the lens preparation
procedure altogether (lenses still need to be cleaned before and after use). Filling the lens holes
with PEG was explored extensively in collaboration with the Gemeinhart lab (UIC), and while
recording quality and channel yield were comparable to lenses filled with saline, storage proved
to be a problem. To store the PEG in a hydrated state proved unacceptable because of the effects
of the long-term hydrated environment on other lens components (adhesive tape). To store the
PEG in a semi-dehydrated state required it to be re-hydrated prior to use, which proved to be
unreliable in terms of maintaining channel yield. The solution to this problem will require a
different lens assembly adhesive material, or exploration of other candidate conductive materials,
such as conductive silicone. This will be an active area of development for future students in the
NEVL.
C. Summary of results: Specific Aim-3
The main contribution of this work was the recording and analysis of the first significant set of
meERG data ever obtained. The first account of recording corneal potentials at various locations
was by Sundmark in 1959 who used a contact glass fitted with nine electrodes; Sundmark was
able to record potential differences across the retina in human subjects. The most recent
recordings were done by Holland and Herr in rabbit eyes in 1964. The purpose of Holland and
Herrs study was to determine whether electroretinographic changes were present in healthy and
post photocoagulated retina. The development of the meERG as described in this thesis has
proven to be a direct way of recording as many as 25 waveforms simultaneously. A number of
investigators have modeled the ERG source and the resultant corneal potentials, and have
described the potential use of such recorded potentials in the study of retinal function. The most
significant of these is the work by Job et al. 1999, who concluded that different retinal locations
contribute differently to local corneal potentials. Cringle et al. 1989 patented a method of using
simultaneously recorded scleral potentials in diagnosis, but their patent did not describe a method
of analysis of the recorded potentials or finding a relationship between localized retinal damage
and changes in eye surface recordings. An important part of evaluating any new technology or
method with potential diagnostic application is to compare it to existing methods that might
provide similar information. As the meERG technique has the potential to provide information
about spatial differences in retinal function, we here focus on comparing meERG to multi-focal
electroretinography (mfERG), which is the only ERG technique that attempts to generate a
functional map of the retina.
The meERG has several advantages over the mfERG such as its short test time and the ability to
probe the entire retina. The meERG is independent of movement, fixation and positioning errors
such as those present in the mfERG. (ISCEV, 2011) The meERG waveforms directly reflect the
function of the retina, requiring minimal signal processing. Since the meERG uses a Ganzfeld
stimulus, there are less restrictions in the stimulus type which can range from standard scotopic
flashes to paired-flash protocols. Although the potential disadvantages of the meERG over the
mfERG include lower resolution and less sensitivity it is likely that the two techniques may be
complimentary in combining the high spatial resolution of the central retina in the mfERG with
the information on the peripheral function of the meERG.
9. Cited Literature
Cited Literature
Chaudhuri A, Hallett P, Parker J. Aspheric curvatures, refractive indices and chromatic
aberration for the rat eye. Vision Research 1983; 23(12):1351-1363
Cringle SJ, Alder VA, Brown MJ, Yu DY. Effect of sclera recording location on ERG amplitude.
Current Eye Research 1987; 6(9):1109-1114
Cringle SJ. Electroretinogram Apparatus. U.S. Patent # 4,874,237; 1989
Gotch F. The time relations of the photoelectric changes on the eyeball of the frog. J
Physiol. 1903;29:388–416
Granit R. The components of the retinal action potential in mammals and their relation to the
discharge in the optic nerve. J Physiol. 1933;77:207–239
He B & Wu D (1999) Laplacian electrocardiography. Crit Rev Biomed Eng 27(3-5):285-338
Hennessy M, Vaegan. Amplitude scaling relationships of Burian-Allen, gold foil and Dawson,
Trick and Litzkow electrodes. Documenta Ophthalmologica 1995; 89(3):235-248
Holland M. G. & Herr N (1964) The electroretinographic potential field. Localization of retinal
lesions. American Journal of Opthalmology. 57:639-45.
Hood DC, Bach M, Brigell M, Keating D, Kondo M, Lyons JS, Marmor MF, McCulloch DL,
Palmowski-Wolfe AM. ISCEV standard for clinical multifocal electroretinography (mfERG).
Doc Ophthalmo 2012 Feb;124(1):1-13
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Job HM, Keating D, Evans AL, Parks S (1999) Three-dimensional electromagnetic model of the
human eye: advances toward the optimization of electroretinographic signal detection. Med Biol
Eng Comput 37:710-719
Kooijman, A. Light distribution on the retina of a wide-angle theoretical eye, J. Opt. Soc.
Am. 73, 1544-1550 (1983)
Kooijman, A. and Witmer F. Ganzfield light distribution on the retina of human and rabbit eyes:
calculations and in vitro measurements, J. Opt. Soc. Am. A 3, 2116-2120 (1986)
Lamb, T.D., Pugh Jr. E.N. (1992) A quantitative account of the activation steps involved in
phototransduction in amphibian photoreceptors. J. Physiol 449:719–758pmid:1326052.
Masland RH (2001) The fundamental plan of the retina. Nature Neurosci 4:877-886
McAllan A, Sinn J, Aylward GW. The effect of gold foil electrode position on the
electroretinogram in human subjects. Vision Research 1989; 29(9):1085-1087
Purves D, Augustine GJ, Fitzpatrick D, et al., editors. Neuroscience. 2nd edition. Sunderland
(MA): Sinauer Associates; 2001. Anatomical Distribution of Rods and Cones
Remtulla S, Hallett P. A schematic eye for the mouse, and comparisons with the rat. Vision
Research 1985; 25(1):21-31
Sundmark E. The contact glass in human electroretinography. Acta Ophthalmalogica
Supplement 1959; 52:3-40
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Webvision (2012) http://webvision.med.utah.edu/
10. Appendix
10.1 Standard operating procedures
A. Lens Assembly procedure
1. Select a cable and ensure the cable is free of dust and debris.
2. Select a lens that is clear of tape residue as well as saline. Place lens into an ultrasonic
instrument cleaner for 5 minutes.
3. Remove and rinse the lens under distilled water for several seconds and pat dry.
4. Confirm that the wells within the lens are dry and clean.
5. Place three 1/8 in balls of dental wax equidistant from one another around the inner
perimeter of the lens assembly well. Figure 12.
6. Place the lens concave side down into the lens assembly well assuring that the lens is
parallel to the assembly table.
7. Place the cable onto the metal cable holding shelf, exposed electrode pad side down and
cover it with the plastic slip and make sure the red screw is on tight. Figure 12
8. Use the x, y, z translation knobs and rotation lever to adjust the lens such that all the
holes are aligned with the electrodes on the cable.
9. Once everything is aligned, pull back the tip of the cable that rests over the lens and flip it
back onto itself such that a small weight can be placed to hold in down. Figure 12
10. Using forceps, remove a piece of pre-formed tape and position it onto the lens such that
the holes are all aligned.
11. Using forceps remove each precut piece of tape that covers the wells in the lens.
12. Once all the tape from the wells has been removed, carefully remove the weight holding
down the cable and allow the cable to fall onto the tape attached to the lens.
13. It may be necessary to use forceps to gently guide the cable back into its original position.
14. Once the cable is aligned, gently push down on to ensure that the tape bonds to it.
15. It is now safe to remove the cable from the metal cable holding shelf by unscrewing the
red screw and lifting the plastic cover.
16. Using forceps, push at the lens from the side until the lens is free from the dental wax.
17. Remove access dental wax using a Kimwipe and rubbing alcohol.
B. Measurement Procedure using DinoCapture 2.0 version 1.3.2
1. Anesthetize the animal and lubricate the eye by applying one drop of saline.
2. Place the animal in the stereotactic table and secure its head position using the bite bar.
3. Place a ruler as close to the eye as possible making sure that it is in the same focal plane
as the horizon of the cornea when the camera views the eye from the side..
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4. Using the "Folder" menu, create a folder with appropriate name including Rat # and Date.
This folder needs to be created via DinoCapture in order to save the bitmap images taken
by the microscope and measurements can be done later.
5. Use "Text" option to create a label on images, also set the magnification to 1 in the top
menu.
6. Make sure the ruler us in the same focal plane as the horizon of the cornea when the
camera views the eye from the side. If these are not in the same plane the depth of focus
will be different for each and produce a fuzzy image.
7. By clicking on the Camera icon on the top left corner of the image take as many as 5-7
pictures. These pictures will be saved under a subfolder named "Picture" of the folder
created at the beginning.
8. To open the folder use the "Folder" menu again and select the folder containing the
pictures you have taken. Preview of your images will be displayed on the left panel of
DinoCapture and select one for analysis.
9. Use "Calibration" options on the top right of the menu and create a new calibration
profile and name it corresponding with Rat#, Date and Picture#. Make sure
magnification is set to 1 and set (using the ruler on the image) the known distance of 2
mm on the image and finish the calibration. This part is similar to set the scale in Image J
software.
10. Use “three points arc" on the top menu to point three spot on the circumference of cornea
and measure the r (Radius of curvature)
11. Capture 3 images per animal and repeat the measurements on each image.
C. Measurement Procedure using ImageJ
1. Open the jpg image taken by Dino Capture with ImageJ software
2. Select the *Straight* line tool. Choose a hash mark on the ruler and draw a straight line
from the left edge of that hash mark to the left edge of a different hash mark. Next go to
“Analyze -> Set Scale” under the ImageJ toolbar. In the “Known Distance” box, enter
the length the line across the ruler is in mm, and set the “Unit of Length” to mm.
3. Use "Elliptical" option from top menu to create a circle so "w"(width) and "h"(height) of
elliptic would be equal to diameter of the circle you draw. In this case "ar" aspect ratio
will be one. This circle should encompass the cornea.
4. Capture 3 images per animal and repeat the measurements on each image.
D. Saline lens filling procedure
1. Ensure the rat lens is free of foreign debris and is securely attached to the cable.
2.
3.
4.
5.
Place the lens into a Petri dish with concave side facing up.
Using a 1 ml syringe place 3 drops of saline onto the lens.
Place Petri dish into the vacuum chamber.
Close chamber door. Ensure the depressurizing valve is in the open position and the
pressurizing valve is in the closed position. Turn on the vacuum until the vacuum
pressure gauge reaches 28.5 inHg.
6. Once 28.5 inHg is achieved: continuously watch the saline solution for slight changes in
opacity due to freezing which starts around 60 seconds. Upon slight change in clarity of
saline, shut off vacuum pump and turn the depressurizing valve to the closed position.
7. If saline freezes, procedure has failed. Dispose of used saline and restart process from
step #3.
8. Lens must remain in the sealed vacuum chamber for a minimum of 5 minutes. If left in
longer, ensure that the saline does not evaporate.
9. Opening the pressurizing valve and allowing the air back into the vacuum chamber.
10. Using a microscope; inspect the lens. If bubbles are present in the saline or in lens wells it
will be necessary to repeat the procedure.
E. Saline lens cleaning and storage procedure
1. Place the lens in a petri dish and using a Kimwipe wipe off the excess saline.
2. Using a 1ml syringe, place 3 drops of distilled water into the lens and place in vacuum.
3. Close chamber door. Ensure the depressurizing valve is in the open position and the
pressurizing valve is in the closed position. Turn on the vacuum until the vacuum
pressure gauge reaches 26 inHg.
4. Open the pressurizing valve and allow the air back into the vacuum chamber.
5. Repeat steps #2 through #4 for a total of three times all the while wiping the previously
used distilled water from the lens using a kimwipe.
6. Remove the lens from the vacuum chamber and wipe off all of the distilled water from
the lens.
7. Store the lens exposed to air.
F. PEG lens filling procedure
1.
2.
3.
4.
Ensure the rat lens is free of foreign debris and is securely attached to the cable.
Place the lens into a Petri dish with concave side facing up.
Using a 1 ml syringe place 3 drops of PEG onto the lens.
Place petri dish into the vacuum chamber.
5. Close chamber door. Ensure the depressurizing valve is in the open position and the
pressurizing valve is in the closed position. Turn on the vacuum until the vacuum
pressure gauge reaches 28.5 in Hg.
6. Once 28.5 in Hg is achieved: continuously watch the PEG for slight changes in opacity
due to freezing which starts around 60 seconds. Upon slight change in clarity, shut off
vacuum pump and turn depressurizing valve to the closed position.
7. If PEG freezes procedure has failed, restart process from step #3.
8. Lens must remain in the sealed vacuum chamber for a minimum of 5 minutes. If left in
longer, ensure that the PEG does not evaporate. During this time cover the vacuum
chamber with a black cloth to minimize the amount of UV light entering the chamber.
9. Check lens by opening the pressurizing valve and allowing the air back into the vacuum
chamber.
10. Using a microscope; inspect the lens. If bubbles are present in the PEG or in lens wells it
will be necessary to repeat the procedure.
11. Once there are no more bubbles, place the lens under a 360 nm UV light. Place the light
as close to the lens as possible by resting the light on the edges of the petri dish.
12. Allow the PEG to polymerize for 40 minutes.
13. Once the PEG has polymerized, turn off the UV light and place 4 drops of saline into the
well with the PEG.
14. Cover the lens with the cover to the petri dish and allow to sit for 15 minutes to ensure
that the PEG is fully hydrated.
15. Once the PEG is fully hydrated, scrape the excess out of the well. The goal is to cut the
PEG such that it is flush with the curved sides of the lens.
G. PEG fabrication procedure
1. To make the PEGDA hydrogel add 83µL of Irgarcure 2959 to 0.817 ml of saline.
2. Add the mixture to 100 mg of PEGDA (Mw=3400).
Courtesy of Melanie Kollmer.
H. PEG lens storage procedure
1. If the lens will be used daily, simply place one drop of saline from a 1 ml syringe onto the
PEG every day to make sure it stays hydrated.
2. Place a small petri dish cover to reduce evaporation.
3. If the lens needs to be stored long term, using a 1 ml syringe, place 5 drops of distilled
water onto the PEG filled lens and wipe off the excess with a Kimwipe.
4. Store the lens in an air tight chamber.
5. Prior to using a lens that has been stored long term, place 3 drops of saline from a 1ml
syringe and allow the PEG to fully rehydrate for 15 minutes.
I. Recording Protocol
1. Animals were anesthetized under a dim red light using Ketamine and Xylazine.
2. Corneal anesthetics of Proparacane, Phenyephrine and Tropicamide were administered
one minute apart.
3. The ground electrode needle was place in the back of the rats neck while the reference
electrode was placed in the cheek.
4. The CLEAr LEnstm was tested for channel yield and placed onto the rats eye. The rat was
positioned such that the axis of the right eye was perpendicular to the light stimulus box.
Consistency of positioning the animal was based on visual feedback.
5. MCRack was turned on to recording mode and the number of working electrodes was
confirmed. If certain electrodes discontinued working, the lens was readjusted on the
animal.
6. The smallest filter was placed into the filter slot in the light stimulus box and lowered to
the appropriate height from the rats eye.
7. All wires going into the Faraday cage were turned off and or unplugged.
8. Alternating filter sizes was done in the dark with a head light set on red light.
9. Starting with the 0.01 sc cd s m-2, flash strength was increased in the following order; 5.8,
20, 30, 73, 116 and 159 sc cd s m-2 until a total of 7 flash strengths were used.
10. Four flashes at least one minute apart were recorded for each flash strength.
11. An ALA amplifier was used for recording and the signal sampled at 5kHz.
12. A typical duration for an experiments was on average 30 minutes and did not require an
anesthetic booster.
13. Once the recording session was completed successfully, the rat was returned to its cage.
Eye drops were place onto both eyes and the rat was monitored until it woke up.
J. Analysis Protocol
1. Data was collected using MCRack and transferred to excel.
2. Files corresponding to the appropriate series of flash strengths were grouped together,
ordered, averaged and the a and b-wave amplitudes measured.
3. A-wave amplitudes were determined by first averaging 50 seconds of the baseline before
the flash.
4. Using excel, the amplitude of the waveform was taken at t=8.2 ms after the flash. This
amplitude was subtracted from the baseline to give the final a-wave amplitude.
5. The b-wave amplitude was determine by finding the max value after t=0 and subtracting
the baseline from this value.
VITA
NAME:
Yelena Krakova
EDUCATION:
B.S., Biological Sciences, University of Illinois at Chicago, 2005
M.S., Bioengineering, University of Illinois at Chicago 2012