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Spatial anomalies of visual processing in retinitis pigmentosa and a potential treatment

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Spatial anomalies of visual processing in retinitis pigmentosa and a potential treatment

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Content PhD Dissertation
Spatial Anomalies of Visual Processing in
Retinitis Pigmentosa and a Potential
Treatment
Wan-Qing Yu
NEUROSCIENCE GRADUATE PROGRAM
Committee
Greg Field (Chair)
Norberto Grzywacz
Eun-Jin Lee
Judith Hirsch
May 2015
Contents
1 Research Background 1
2 Functional Changes of Retinal Ganglion Cells in S334ter-line3 Rats 3
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3 The Effect of TIMP-1 on the Cone Mosaic 26
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4 The Effect of AAA on the Cone Mosaic 53
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
i
CONTENTS ii
5 Future Directions 77
References 78
Acknowlegements 97
Chapter 1
Research Background
Retinitis pigmentosa (RP) is the leading cause of inherited retinal blindness world-
wide, having an incidence of 1 per 4000 births each year (Hartong et al., 2006).
Currently, no treatment is available for RP. About 45 genes are identified to cause
nonsyndromic RP (Daiger and Bowne, 2007). The onset of RP starts with the death
of rods, and it is followed by a slow progressive phase of cone loss. It typically con-
tainsthreephases(JonesandMarc,2005): photoreceptorstress, photoreceptordeath
and neuronal remodeling. The length of each phase varies with genetic mutations.
For some models, cones take some time to die out and before they do so, their mo-
saic undergoes massive reorganization. The S334ter-line-3 rat we use is a transgenic
model developed to express a rhodopsin mutation which is similar to that found in
human RP patients. In this RP model, the death of rods led to the migration of
cones, which rearranged themselves into a regular mosaic of rings (Ji et al., 2012;
Lee et al., 2011; Zhu et al., 2012). Similar mosaic has been observed in other animal
model of RP and also human patients (Carroll et al., 2004; Choi et al., 2006; Duncan
et al., 2007a; Joeres et al., 2008; Rossi et al., 2011). Normally, mosaic of photorecep-
tors exhibits spatial regularity and homogeneity (Wässle and Riemann, 1978). This
feature is important for retina to uniformly sample the visual space (French et al.,
1977; Manning and Brainard, 2009). The rearrangement of the cone mosaics in RP
1
CHAPTER 1. RESEARCH BACKGROUND 2
retinas severely disrupt the normal spatial organization. Consequently, it impairs
the spatial visual functions. Cones are essential for our day vision. Thus, one of the
significances of this project is to seek a treatment to restore the cone mosaic back to
normal before they die.
In order for any kind of the therapeutic method on photoreceptor layer to be
effective, it is required that the inner retinal circuitry is still functional normally.
Therefore, the investigation on functional changes of retinal ganglion cells after the
loss of rods in S334ter-line-3 rats is presented first. We found that despite the spatial
distortion of the receptive field of RGCs, fundamental functional properties were still
maintained. This indicated that the inner retinal circuitry was largely intact. It
provide the premise on the photoreceptor therapy. In the following chapter, TIMP-
1 was used to manipulate the cone mosaic back to homogeneous pattern without
killing the cones. This provides a potential therapy to RP retina. In the future,
retinal function should be assessed in the drug treated retinas to determine whether
the redistributed cones restore the spatial receptive fields of RGCs.
Chapter 2
FunctionalChangesofRetinalGanglionCells
in S334ter-line3 Rats
2.1. Introduction
Retinitis pigmentosa (RP) is the leading cause of inherited retinal blindness, affect-
ing millions of people in the world. The onset of RP starts with the death of rods,
and it is followed by secondary degeneration of cones. Although no effective rescue
of photoreceptors is available, recent progress in gene therapy (Acland et al., 2001;
Bennett et al., 2012) and stem cell transplantation (MacLaren et al., 2006; Tucker
et al., 2011) provides hope to restore light sensitivity for RP patients. However, a
common premise of these treatments is that the inner retinal circuitry is intact and
retinal ganglion cells (RGCs) are functioning normally. Therefore, it is essential to
evaluate how the inner retina react to photoreceptor degenerating.
Considerable effort has been made to identify the structural remodeling in the
inner retina following the degeneration of photoreceptors (Fariss et al., 2000; Jones
and Marc, 2005; Puthussery and Taylor, 2010). Anatomical evidences from human
RP patients (Humayun et al., 1999; Santos et al., 1997; Stone et al., 1992) suggest
3
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 4
that severity of remodeling and neuronal loss in the inner retina correlated with cone
loss. Similarly, in RP animal models (Gargini et al., 2007; Jones et al., 2011; Ray
et al., 2010; Strettoi et al., 2003), neuronal remodeling follows a clear temporal (i.e.
from rod bipolar to cone bipolar) and spatial pattern (i.e. from outer to inner retina)
which associated with progressive degeneration of photoreceptors.
Although morphological modifications of inner neurons may not be significant,
functional changes could happen at the early stage of retinal degeneration. Record-
ings from rd mouse (Margolis et al., 2008; Stasheff, 2008) revealed elevated sponta-
neous activity and 10 Hz rhythmic spiking in the RGCs. And later studies suggest
that the oscillatory activity originated from membrane oscillations in bipolar and
amacrine cells (Borowska et al., 2011; Choi et al., 2014; Menzler and Zeck, 2011). In
P23H rats, shrinkage of receptive field size was detected before complete loss of rod
(Sekirnjak et al., 2011). In these studies, RGC responses were collectively analyzed
for either ON and OFF groups or adding a transient and sustained separation in
OFF group. However, in mammalian retinas, there are about 15 20 types of RGCs
(Masland, 2001; Wässle, 2004) which transmit light signal in parallel pathways. It
is possible that the progression of disease affects differently in distinct RGC types,
like what has been shown in a mouse model of Glaucoma (Della Santina et al.,
2013). In this study, we would like to address the issue that whether degeneration
of photoreceptors can cause differential functional alterations in different RGC types.
The animal model we used is a transgenic model developed to express a rhodopsin
mutation similar as that found in human RP patients. Previous in our laboratory,
we showed that in this model, the death of rods did not induce immediate death of
cones, instead it led to the migration of cones, which rearranged themselves into a
regular mosaic of rings (Ji et al., 2012; Lee et al., 2011; Zhu et al., 2012). Similar
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 5
mosaic has been observed in other animal model of RP (Diego et al., 2013) and
also human patients (Carroll et al., 2004; Choi et al., 2006; Duncan et al., 2007a;
Joeres et al., 2008; Rossi et al., 2011). A homogeneous and regular cone mosaic is
important for visual sampling. Thus, it is also interesting to find out the functional
consequences of this dramatic remodeling of cone mosaic. In this study, we recorded
simultaneously hundreds of RGCs from both wild type and RP rats using high den-
sity multielectrode arrays. We estimated both spatial and temporal receptive fields
in a high resolution. Then we performed a semi-supervised classification on RGCs
on both retinas to ascertain whether distinct functional types can be found in RP
retinas and whether they were similar as those found in wild type retinas. After
identifying major RGC types, we then compared shape of spatial receptive fields,
dynamic of temporal integration and spontaneous activity across types to figure out
whether consistent changes exist in different functional types.
2.2. Materials and Methods
Animals and Tissue Preparation
The third line of albino Sprague-Dawley rats homozygous for the truncated murine
opsin gene (created a stop codon at Serine residue 334; S334ter-line-3) was obtained
from Dr. Matthew LaVail (University of California, San Francisco, CA). Heterozy-
gous S334ter-line-3 rats are obtained through mating homozygous S334ter-3 female
rats and Long Evans male rats (Charles River). For control, Long Evans rats are
used. All rats were housed under cyclic 12/12-hour light/dark conditions with free
access to food and water. This model shall be referred to as the Retinitis Pigmentosa
(RP) model in the rest of the article. Animals were treated in accordance with the
regulations of the Veterinary Authority of University of Southern California and with
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 6
the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Animals were deeply anesthetized by intra-peritoneal injection of ketamine (100
mg/kg; KETASET, Fort Dodge, IA) and xylazine (20 mg/kg; X-Ject SA, Butler,
Dublin, OH) and the eyes were enucleated. After enucleation, the anterior part of
the eye and vitreous was removed. Then, the eye cup was placed in oxygenated
Ames’ solution at room temperature. The mid-periphery of the retina was dissected
and isolated from the retinal pigment epithelium. Then, it was transferred to MEA
chamber, and positioned in the center of the array with the retinal ganglion cell
layer down. The whole procedure was done under the infrared illumination. For
recordings, the retina was kept at 35 C and was perfused with Ames’ solution
bubbled with 95% O
2
and 5% CO
2
.
Electrophysiology and Visual stimulation
Extracellular recordings are performed using a high-density MEA. It consists of 519
electrodes, in which the inter-electrode distance is 30 m, covering about 0.5 mm
2
(Field et al., 2010). Light stimulus is projected to photoreceptors through transpar-
ent MEA. Binary white noise with either 20 x 20 m or 60 x 60 m unit is used to
estimate the receptive fields. Square-wave drifting gratings in 8 directions are used
to classify direction-selective ganglion cells. Contrast sensitivity was measured using
light pulses on gray background ranging from -100% to 100% contrast.
Functional classification on RGCs
RGCs classification was first done by hand. The output of the hand classification
was fed into a semi-supervised classification. In this way, the boundary of the cluster
was determined objectively across preparations. In the hand classification, a cluster
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 7
boundary was firstly drawn manually in a 2D space, which consist of two principal
components of either time courses or autocorrelation function. Response properties
of full field pulses and drifting gratings were then inspected within early defined clus-
ter. Any outliers were removed from the cluster. This procedure was repeated until
no cluster can be drawn from the feature spaces. The semi-supervised classification
was fully described in another paper (Ravi et al., 2013). The authors identified op-
timal feature space for DS, 3 ON and 5 OFF RGC types and fit Gaussian mixture
models with or without initial seeds. In order to track the identity of functional
types, identical feature spaces and classification sequence were kept in this study
with minor modifications. The modifications followed three rules. First, a type was
skipped when no or few cells (n < 4) in this type can be identified during the hand
classification; Second, additional types were added at the end of the original clas-
sification described in the paper; Third, an alternative feature space was searched
whenever the contamination rate was higher than 20% using the original space. And
the substitute space should be as close as the original one. The potential modifica-
tions can be justified by two factors. First, a higher density array was used in this
study, which could preferably record from different populations of RGCs; Second,
retinas in a disease condition were also analyzed, which could brought in uncertain-
ties in the classification.
RGC receptive field characterization
The spatial and temporal receptive field was estimated using spike-triggered average
(STA) with white noise stimulation (Chichilnisky, 2001). High-spatial (20 20
m) and low-temporal (refresh at 6-8 frames) white noise was used to estimate fine
spatial receptive field. In order to quantify the size and shape of spatial receptive
field, theestimatedreceptivefieldwasfirstbinarizedusingthresholdas4.5SD.Those
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 8
pixels where the sensitivity was higher than the threshold was called the effective
receptive field. The size of the receptive field was thus proportional to the number of
pixels above the threshold. The convexity index was used to quantified the receptive
field shape. For a given RGC, a convex hull was drawn for the effective receptive
field. And the convexity index was defined as the ratio between the size of effective
receptive field and the convex hull covered the receptive field. In order to quantify
the temporal receptive field, the time courses were fitted with a difference of two
low-pass filters.
2.3. Results
Photoreceptor remodeling leads to distorted RGC receptive fields
In S334ter-line3 RP rats, four major structural changes have exhibited in the
outer retina at postnatal day 60 (P60). First, reduction in thickness of outer segment
layerwassignificant(Fig.2.1AandC).Second, relativetoagematchedWTrats, cone
outersegments(COSs)wereshorteranddistorted(Fig.2.1BandD).Third, COSslay
flat along the outer plexiform layer. Finally, cone photoreceptors migrated away from
a regular mosaic arrangement to form ring-like patterns across the retina (Fig.2.1E
and F). These observations were consistent with previous studies(Ji et al., 2012).
Asconesmigrate,itisexpectedthatRGCswouldnotrespondtolightinthewhole
visual field. It is unclear how the receptive field (RF) properties change accordingly.
One possible outcome is that RFs would exhibit irregular shape. To determine the
consequences on retinal signaling of these structural changes in the outer retina, P60
RP and healthy rat retinas were recorded on a large-scale MEA while presenting vi-
sual stimuli (Anishchenko et al., 2010; Litke et al., 2004). In particular, checkerboard
noise stimuli were presented to estimate the spatiotemporal RFs of recorded RGCs
by computing the spike-triggered average (STA) stimulus (Chichilnisky, 2001). As
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 9
Figure 2.1: Cone remodeling in RP retinas.
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 10
expected, RFs of individual RGCs from RP retinas exhibited larger departures from
a Gaussian shape compared to WT retinas (Fig.2.2A and B). RFs from RP reti-
nas did exhibit some spatial structure; they tended to form arcs, partial rings, and
sometimes complete rings. This structure is reminiscent of that observe in the cone
mosaic. Indeed, when the receptive fields of individual RFs were summed together,
a binarized map of spatial sensitivity across the recorded piece of retina revealed
the same ring-like pattern observed across the cone outer segments (Fig.2.2D). In
contrast, these maps from healthy WT retinas were more homogeneous and did not
exhibit large gaps in light sensitivity over space (Fig.2.2C).
The regions in the RP retinas with little to no light sensitivity were not caused by
a failure to record from RGCs in those regions. There was no significant decrease in
the number of identifiable RGCs in RP compared to WT retinas ( WT: 290:8 27:7,
RP: 349:8 17:8, p = 0.9351). Estimates of RGC soma locations from the electrical
images of the recorded cells (see Materials and Methods) indicated that RGC density
was unchanged in areas with low visual sensitivity. Thus, the remodeling in the
location of COS was reflected in the spatial structure of RFs in individual RGCs and
across the population of RGCs. However, despite this reorganization in the outer
retina, RGCs continued to respond to light.
Functionally distinct RGC types remain after loss of ONL
Do functionally distinct RGC types persist after rod death and cone migration, or do
thesechangesintheouterretinapropagatetoobscurethedistinctresponseproperties
of different RGC types? To answer this question, a semi-supervised classification of
RGCs based on their light response properties method was applied to applied on
both WT and RP retinas (see Materials and Methods). Consistent with previous
studies, RGCs in WT retinas could be classified into distinct types (Fig.2.3A). Each
identified type exhibited a mosaic-like arrangement of RFs (Fig.2.3C). This indicates
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 11
A
B
WT RP
Figure 2.2: Distorted receptive fields in RP retinas. A. Receptive field examples
for both WT and RP. First row: ON cells, Second row: OFF cells. Left four: WT, Right
four: RP. B. Binary response maps of WT and RP retinas. White spot stands for the
location where existence of light response from at least one RGC. Red curve: MEA array
edge
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 12
Figure 2.3: Functional classification and matching. Functional classification for
ON small transient type in WT (A) and RP (B) retinas. Classified cells were labeled in
dark red. The spatial receptive fields (RFs) tiled the space in WT (C) retina but not in
the RP retinas (D). The histogram of soma location of classified type indicates that the
classified type form a regular mosaic in both WT (E) and RP retinas (F). Insets: MEA
array contour with estimated soma location. Dark red curve: a random distribution
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 13
that each type corresponded to a morphologically distinct type because they also
exhibit a mosaic-like arrangement. It also indicates that each RGC type identified
by this classification approach could not be further subclassified.
Despite extensive remodeling in the outer retina, this classification approach also
revealed functionally distinct RGC types in RP retinas at P60 (Fig.2.3B). Typically,
thesuccessofafunctionalclassificationofRGCswouldbecheckedbytestingwhether
a putative type exhibited a mosaic-like arrangement of RFs, with neighboring RFs
overlapping at approximately the 1-sigma contour of a Gaussian fit (Devries and
Baylor, 1997). However, just as cone migration in RP retinas disrupted the spa-
tial structure of individual RFs (Fig.2.2), it also disrupted the coordinated (mosaic-
like) sampling performed by neighboring RFs (Fig.2.3D). This precluded determining
whether these putative RGC types exhibited a mosaic-like arrangement of RFs. In-
stead, regular spacing was checked by estimating the approximate location of each
RGC soma by the electrical signal produced by each RGC across the MEA (Fig.2.3E
and F insets, see Materials and Methods). The nearest-neighbor distributions for
RGC locations revealed that RGCs classified into types in RP retinas were regularly
spaced, similar to the spacing observed in WT retina (Fig.2.3E and F), suggesting
that they corresponded to morphologically distinct types and could not be further
subclassified.
Remarkably, parameter spaces that were used to classify RGC types in WT reti-
nas, also frequently identified distinct RGC types in RP retinas (Fig.2.4). While the
total number of types and number of cells in each type varied across preparations,
there was also substantial consistency in the functional classification of RGCs be-
tween WT and RP retinas (Table 2.1). Overall, seven major types emerged in WT
(Figs.2.4A and C) and RP (Figs.2.4B and D) retinas in addition to direction selec-
tive RGCs. To test whether the seven types that were most consistently identified
could be matched one-to-one, the temporal RFs were compared across distinct RGC
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 14
Figure 2.4: Distorted receptive fields in RP retinas. A. Receptive field examples
for both WT and RP. First row: ON cells, Second row: OFF cells. Left four: WT, Right
four: RP. B. Binary response maps of WT and RP retinas. White spot stands for the
location where existence of light response from at least one RGC. Red curve: MEA array
edge
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 15
Retina DS ON Sus ON ST ON LT OFF Sus OFF ST OFF LT OFF Sluggish
WT1 44 4 4 1 6 9 8 13
WT2 47 10 7 10 7 7 7 11
WT3 55 12 14 19 15 12 17 5
WT4 64 13 12 19 12 15 14 None
RP1 63 20 23 16 15 7 22 15
RP2 69 24 None 16 31 2 24 None
RP3 50 16 10 6 13 14 15 10
RP4 60 12 11 19 13 13 15 22
RP5 35 5 15 23 16 13 11 14
Table 2.1: Number of cells identified for each functional type in 4 WT and 5 RP retinas.
types and between WT and RP retinas (Fig.2.4). The temporal RFs were estimated
from the STAs of the recorded RGCs and reflect the average temporal dynamics of
visual stimuli within the spatial RF that preceded spiking (see Materials and Meth-
ods). Temporal RFs were compared across WT and RP conditions because they are
potentially be more robust to changes in the spatial arrangement of cones. Indeed,
while quantitative changes in temporal RFs were observed between WT and RP reti-
nas (see Fig.2.6), the relative kinetics of the three ON and four OFF type RGCs were
qualitatively preserved (Fig. 2.5). This suggests that individual RGC types could
be matched 1-to-1 between healthy and RP retinas.
Non-discriminantmodificationofspatialreceptivefieldbycone
migration
The shape of RGC receptive field in RP retinas deviated from Gaussian as cones mi-
grated (Fig.2.2A). The irregular RFs were observed in all functional types (Fig.2.5A).
In order to determine whether the cone migration affect RF shape uniformly across
different types. RF shapes were compared within each type. The shape of the recep-
tive field was quantified using convexity index (see Methods and Materials), which
was defined by the ratio of receptive field size and the size of its convex hull. The
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 16
Figure 2.5: Spatial receptive field properties. A. RF examples for three functional
types. B. Distribution of convexity index of RF. Correlations between RF displacement
and convexity index for WT (C) and RP (D) retinas. E. A reduction in size of RF in
all RGC functional types. F. A linear correlation between the reduction of RF size and
original RF size.
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 17
larger the index, the more convex the receptive field shape was. In WT retinas, since
the receptive fields were Gaussian, their convexity index were close to 1 (Fig.2.5B
blue shade). The distribution and the peak was similar for all the functional types
examined. In RP retinas, the distribution of the convexity index was much broader,
and with peaks at a lower value (Fig.2.5B red shade). Notably, the distribution of
the index was similar among types, too. Thus, cone migration affected the receptive
field shape indiscriminately across functional types.
As cones migrate, some migrated out to form the rings, while some stayed near
the neighborhood. Thus, for those RGC cells sitting under the holes, their receptive
fields would be more likely displaced and distorted. On the contrary, those sitting
further away from the center of the holes would be more likely to maintained Gaus-
sian receptive fields. In order to test this hypothesis, we examined the relationship
between the receptive field displacement and shape. The receptive field displacement
was the distance from its center to RGC cell location. A negative correlation was
revealed between convexity index and displacement in RP retinas (Fig.2.5D, r
2
=
0.25, = -139.3, p < 0.00001), but not in WT ones (Fig.2.5C, r
2
= -0.002, =
-35.5, p = 0.476). It indicated that the shape of receptive field was mostly related
with the relative location of the RGC to cone ring structure.
The size of a RGC receptive field was largely determined by its size of dendritic
field and types of bipolar cells connected. The more photoreceptorsśignal a RGC
collectes, the larger its RF is. The size of RGC receptive field varied with functional
types in WT retina (Fig.2.5A). OFF LT had the largest receptive field, while OFF
sluggish had the smallest (Fig. 2.5B). In RP retinas, although RF sizes decreased
significantly in all the types, the order was maintained (Fig.2.5E). Moreover, the
shrinkage of the receptive field was proportional to the receptive field size (Fig.
2.5F,R
2
= 0.98). This was consistent with the previous observation that the typical
distance between two cones was smaller in RP retinas (Ji et al., 2015).
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 18
RGC temporal dynamics was slower in RP retinas
The RGCs process visual signals using a combination of temporal channels, i.e. fast
and slow, transient and sustained. To determine how the temporal dynamics was
affected by loss of rods and migration of cones, the time courses were compared
within each type.
Overall, all the time courses of RP cells were slower than WT ones (Fig.2.6A).
In order to compare the time courses quantitatively, the time courses were fit with
a difference of low-pass filters (see Materials and Methods) and then three features
were extracted from these fitted time courses: time to peak, time from trough to
peak and degree of transiency (Fig.2.6A top-right panel). First, the time to peak
was a measurement of response latency. The time to peak of time courses in RP
retinas was delayed on average about 35.6 ms than in WT ones, with the ON ST
delayed the most and OFF ST the least (Fig.2.6B). Next, the time to zerocrossing
was the time to peak responses given a step stimulus. All the RGCs had longer time
to zerocrossing (Fig.2.6C). OFF ST was the least affected again. Finally, the degree
of transiency changed dramatically for OFF ST, OFF Sus and ON Sus, but not for
the other 4 types (Fig.2.6D). In particular, OFF ST became more sustained while
others All these results indicated that the major change in time courses was that
the temporal dynamics was slower in RP retinas than WT ones. But the amount of
changes varied with RGC types.
Changes of properties of DS cells
Directional selective RGCs were also one of the major types we identified in both
WT and RP retinas. Because of the spatial-temporal inseparability of their recep-
tive field, it is not suitable to compare the properties of receptive field of DS cells
directly. Instead, the directional selective tuning of individual cells and populational
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 19
Figure 2.6: Temporal dynamics was slower in RP retinas. A. Comparison of time
courses in all 7 functional types between WT and RP retinas. B. Changes in response
latency (indicated as a in up-right corner panel). C. Changes in time to zerocrossing
(indicated as b in up-right corner panel). D. Changes in degree of transiency (indicated
as DoT in up-right corner, both S1 and S2 were the integration under the curve)
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 20
Figure 2.7: Temporal dynamics was slower in RP retinas.
distribution of preferred direction were examined and compared.
Similar number of DS cells were identified in WT and RP retinas (WT: 52:54:5,
RP: 56:8 7:5, p = 0.64). The tuning curve of each DS cell was measured using
square-wave drifting gratings in 8 directions and was fitted by von Mises function
(Fig.2.7A). Both the mean and variance of the tuning curve width was larger in RP
retinasthanWTones(Fig.2.7B,Mann-Whitney,p<0.00001; Ftest,p=0.036). The
symmetry of the tuning curve was quantified by circular skewness. The distribution
of the skewness was broader, which indicated that more DS cells in RP retinas had
a skewed tuning curve (Fig.2.7C).
In WT rats, the preferred motion aligned roughly 4 cardinal axis (Fig.2.7D), as
reported in the rabbit and mouse. In RP retinas, although individual cells had clear
directional preference, the distribution of preferred directions did not form clean 4
clusters (Fig.2.7E). However, Raoś spacing test showed that in RP retinas, the distri-
bution was not uniform, either. But it is more uniform than in WT ones (Fig.2.7F).
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 21
Thus, in RP retinas, the preferred direction still maintained some clustering.
Spontaneous activity was elevated in all types but one
Previous studies noted elevated spontaneous activity in all or part of the RGCs
in photoreceptor degenerating model (Margolis et al., 2008; Sekirnjak et al., 2011;
Stasheff, 2008; Stasheff et al., 2011). But it was not quite certain whether different
functional types had different changes in spontaneous activity.
Figure 2.8: Spontaneous activity for major types of RGCs
In our RP models, the changes of spontaneous activity varied with functional
types. For ON sustained cells, the average spontaneous firing rates in RP retinas
was higher than WT ones (Fig. 2.8A raster plot. WT: 2:19 1:10 Hz (n = 18),
RP: 15:30 0:48 Hz (n = 77), p < 0.00001). However, for OFF Sustained type had
a reduced spontaneous activity (Fig.2.8B raster plot), from 41:89 1:78 Hz (n =
39) to 20:29 0:69 Hz (n = 88) (Fig.2.8D, p < 0.00001). For the remaining cell
types, they all showed an increased spontaneous activity, but with various degree
(Fig.2.8C). Indeed, bursts were a significant feature in RP cells. Power spectrum
analysis suggest a weak rhythmic firing 7Hz in RGC cells (Fig.2.8A and B, right
panel).
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 22
2.4. Discussion
Cone migration reshapes the receptive fields of RGCs
ThehealthofconesiscompromisedimmediatelyafterthedeathofrodsinRPdisease
which has been verified among different animal models (Gargini et al., 2007; Jones
et al., 2003; Linberg et al., 2005). Previous studies on the functional alterations
of RGCs following the photoreceptor degenerations thus focus on changes in firing
properties as well as the synaptic mechanism underlying them. However, it was
first shown in the S334ter-line3 rat model that the death of rods can trigger spatial
rearrangement of cones (Ji et al., 2012; Lee et al., 2011; Zhu et al., 2012), which
was also reported in P23H-1 (Diego et al., 2013). Moreover, despite the extensive
structural remodeling, cones survive at least four months after the complete loss of
rods. These observations promote the current work to take an effort on receptive
field properties in RGCs.
We chose heterozygous RP rats around P60 when only few rods remained in
the retinas (Martinez-Navarrete et al., 2011; Zhu et al., 2012). At this stage, ONL
already collapsed into a thin layer and rings of cones were distributed across the
whole retina with about 100-200 um in diameter (Fig.2.1). Consistent with previous
studies on other RP models (Margolis et al., 2008; Sekirnjak et al., 2011; Stasheff,
2008), our recordings suggested that the majority of RGCs responded to light vividly.
Interestingly, we observed that a large number of RGCs exhibit receptive fields with
irregular shapes (Fig.2.2A). These shapes comprised partial or whole rings. And not
surprising, if we stitched individual receptive fields together, we obtained a response
map which resembled the cone mosaic (Fig.2.2B). These results implied that cones
probably maintained their synaptic connections during the migration.
The reshaping of RGC receptive fields was not been reported before. In an earlier
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 23
recording on P23H-1 rats, the authors did not report any changes in receptive field
shape. There are three possible reasons. Firstly, it is probably that we used a
higher resolution white noise to probe the details of receptive field (20 um vs 44
or 55 um). Also, the spatial distribution of cone rings was not uniform. In our
recordings, we targeted at mid-periphery of dorsal retina where the number of rings
was abundant. The information of cone mosaic in P23H-1 rats was not available at
the time of recording. The authors might targeted at a region with low density of
cone rings. Moreover, in our model, cone density stays normal for a long period,
which suggest the cone migration. In P23H-1 rats, the anatomical evidence suggest
that the formation of rings might be also due to the death of cones. Therefore, it
is more probably that RGCs would lose partial or the whole receptive fields rather
than reshape them.
Preservation of diverse functional channels
The visual information is processed in parallel pathways in the retina. It is imple-
mented by distinct synaptic connections among five major types of neuronal cells.
As a result, RGCs at the end of each pathway display particular functional prop-
erties to light stimuli. Previous studies showed that structural disturbances and
neuronal remodeling in the inner retina followed the death of photoreceptors. At the
stage which we examined, no death of neurons in the inner retina has occurred ??.
However, functional alterations can be detected before any structural modification.
Thus, it is of particular interest to check whether the functional channels are still
preserved in our model.
Earlier recordings on retinal degeneration model often classified RGCs into four
types according to the polarities and transiency of their light responses. However, in
rat retinas, about 10-15 morphological types of RGCs can be classified (Sun et al.,
2001). Recently, a semi-supervised classification relying heavily on temporal infor-
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 24
mation of RGC receptive field was developed which can identify up to 12 functional
types of RGCs in rat retinas (Ravi et al., 2013). We adapted the method for our
recordings and identified 7 types of RGCs besides DS cells in most of the wild-type
retinas. Among the 7 non-DS cells, six of them were identifiable in the original pa-
per. In the RP retinas, the similar functional types can also be classified Fig.2.4,
although the overall dynamic was slower. This suggest the synaptic connectivity of
remaining cone pathways was largely intact in the RP retina.
Functional alterations differ across RGC types
Previous studies in other RP models showed similar functional changes exist among
different groups of RGC cells, such as increased spontaneous activity (Margolis et al.,
2008; Stasheff, 2008) and shrinkage of receptive field (Sekirnjak et al., 2011). How-
ever, in a recent recording from a mouse model of Glaucoma, the authors demon-
stratedthedifferentialfunctionaldegenerationamongRGCcells(DellaSantinaetal.,
2013). Since we performed a more thoroughly RGC classification, it provides an op-
portunity to investigate the issue in details.
Firstly, changes in receptive field size and shape were consistent across the func-
tional types (Fig.2.5). The size of all RGC receptive fields was decreased. The
reduction of the size was linearly correlated with the size. It can be explained by
photoreceptor remodeling. As rods die, cones tend to migrate closer to each other
(Ji et al., 2015). This in turn reduced the near-neighbor distance between cones.
Even under the assumption that cones maintained the synaptic connections while
moving, the light sensitive region would be shrinked for each RGC. And the more
cones a RGC connects, the larger reduction in the size, vice versa. Secondly, the
temporal dynamics was slower among all types in RP retinas. However, the degree
of changes depended on functional types (Fig.2.6). The slower dynamics might be
due to either the compromising of cone health(J et al., 2012) or aberrants in synaptic
CHAPTER 2. FUNCTIONAL CHANGES OF RGCS 25
transmissions (Puthussery et al., 2009). Thirdly, spontaneous activity was elevated
in all the RGCs but OFF sustained type (Fig.2.8). OFF-sustained type showed a
significant reduction in their firing rate which was inconsistent with other RP mod-
els. Moreover, we did not observe 10 Hz rhythmic firing which was reported in rd1
and rd10.
2.5. Conclusion
We have shown that photoreceptor degeneration in S334ter-line3 rats caused follow-
ing changes in RGCs: 1. dramatic distortion of spatial receptive field; 2. slower
temporal dynamics; 3. broader distribution of preferred direction in DS cells and 4.
elevated spontaneous activity in all functional types but not sustained OFF types.
However, despite these functional changes, the distinction between functional types
was still preserved and similar functional types of RGCs could be identified. Overall,
it suggest that the inner circuitry of the retina was largely intact in RP retinas after
the loss of rods.
Chapter 3
The Effect of TIMP-1 on the Cone Mosaic
in the Retina of the Rat Model of Retinitis
Pigmentosa
1
3.1. Introduction
The outer nuclear layer (ONL) of the vertebrate retina contains a tightly packed,
uniform array of rods and cones, which is essential to ensure that the visual world
is regularly sampled with no empty visual space. The density of rods constrains
visual sensitivity and the spacing of cones determines resolution and thus acuity of
vision (Williams and Coletta, 1987a). Past studies have described that regular and
homogenous spacing of photoreceptors, as seen in some mammalian species and ze-
brafish (Bruhn and Cepko, 1996a; Kram et al., 2009; Larison and Bremiller, 1990;
Lin et al., 2004; Raymond et al., 1995; Wikler and Rakic, 1991, 1994), are important
for sampling the visual space efficiently (French et al., 1977; Manning and Brainard,
2009). However, cones in the S334ter-line-3 rat model of Retinitis Pigmentosa were
recently shown both to survive for a longer period of time after the early rod deaths
1
The research in this chapter has already published in IOVS (Ji et al., 2015). ARVO is the
copyright holder.
26
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 27
and to remodel in their mosaic pattern into orderly arrays of rings (Ji et al., 2012;
Lee et al., 2011; Zhu et al., 2012). Similar dark patches (i.e. holes) are noted in
several human eye diseases caused by retinal dystrophy, inherited retinal degenera-
tion, and photo-pigment genetic perturbations in M-cones (Carroll et al., 2004; Choi
et al., 2006; Duncan et al., 2007a; Rossi et al., 2011). The center of these rings lack
photoreceptors, indicating local loss of visual function. Consequently, knowledge on
modulating and rearranging photoreceptors from the ring patterns into more regular
and homogenous distribution would help improve conditions in these patients.
In the past studies, it has been reported that the balance in the level of en-
zymes that mediate the degradation of the extracellular matrix (ECM) is important
for modulation of migration of neurons, including photoreceptors (Akahane et al.,
2004; German et al., 2008; Hansson et al., 2011). In mammals, these enzymes are
the Metalloproteinase (MMP-degrades ECM) (Matrisian, 1992) and its natural in-
hibitor, Tissue Inhibitor of Metalloproteinase (TIMP) (Murphy et al., 1991), and
together, they modulate neural organization by remodeling and organizing of ECM
in normal and pathological retinas (Cornelius et al., 1995; Yamada et al., 2001). In
particular, a previous study showed that TIMP-1 applied to co-cultured rat retinal
neurons with human retinal epithelial cells led to modulation of photoreceptor mi-
gration (German et al., 2008). Also, opposite from some other members of the TIMP
families, TIMP-1 does not inhibit endothelial cell migration. Among members of the
MMP and TIMP families, MMP-9 and its inhibitor, TIMP-1, are predominantly ex-
pressed in the interphotoreceptor matrix (IPM) (Ahuja et al., 2006). This indicates
that TIMP-1 may play a role in modulating turnover of IPM, which is important
for various photoreceptor functions and maintenance (Chaitin and Wortham, 1994;
Feeney, 1973a,b; Hageman et al., 1991; Hollyfield et al., 1988; Lazarus and Hageman,
1992; Yao et al., 1990, 1994).
In human and animal models with various ocular diseases including retinal degen-
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 28
eration, the level of TIMP-1 is significantly upregulated (Kim et al., 2014; Matsuo
et al., 1997; Zeiss et al., 1998). Positive correlation between TIMP-1 expression and
tumor growth in several cell lines indicate that TIMP-1 may also play a key role as
a survival factor (Bertaux et al., 1991; Hayakawa et al., 1992; Kikuchi et al., 1997;
Soloway et al., 1996; Yoshiji et al., 1998). It was proposed that TIMP-1 may protect
ECM-bound growth factors critical for cell survival (Yamada et al., 2001).
Inthepresentstudy, weinvestigatedifexogenousapplicationoftheTIMP-1could
affect the mosaic of cones in S334ter-line-3 rat retinas. Because we studied the effects
of TIMP-1 on the mosaic of cones, we needed statistical tools to compare the spatial
distribution of these cells in different conditions (Raven, 2003). One of the most
commonly used statistical measures is the areas of Vornoi domains regions of space
obtainablebyenclosingeachcellinthemosaicinspaceclosesttoitselfthananyother
cells. Another statistical analysis focused on the nearest-neighbor distance (NND),
the distance to the closest neuron for every cell (Wässle and Riemann, 1978). Using
these analyses, we report for the first time that the use of TIMP-1 brings statistically
significant changes to the cone mosaic in S334ter-line-3 allowing it to become similar
to that in normal retinas in their homogeneity. Ultimately, deeper understanding
of the action of TIMP-1 could help future therapeutics against various eye diseases,
where cone mosaic remodeling would benefit.
3.2. Materials and Methods
Animals
The third line of albino Sprague-Dawley rats homozygous for the truncated murine
opsin gene (created a stop codon at Serine residue 334; S334ter-line-3) was obtained
fromDr. MatthewLaVail(UniversityofCalifornia, SanFrancisco, CA).Homozygous
S334ter-3 male rats are mated with homozygous S334ter-3 female rats to produce
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 29
offspringfortheS334ter-3transgenethatareusedthroughoutthisstudy. Forcontrol,
age-matchedSpragueDawleyrats(Harlan, Indianapolis, IN)wereused. Allratswere
housed under cyclic 12/12-hour light/dark conditions with free access to food and
water. Both sexes of normal (control) and S334ter-line-3 rats were used. This model
shall be referred to as the Retinitis Pigmentosa (RP) model in the rest of the article.
Animals were treated in accordance with the regulations of the Veterinary Authority
of University of Southern California and with the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research.
Administration of Tissue Inhibitor of Metalloproteinase 1
TIMP-1(Sigma, Saint Louis, MI) was prepared in sterile-filtered phosphate-buffered
saline (PBS), adjusted to pH 7.4 and sterile-filtered before administration. TIMP-1
was administered by intravitreal injection with a fine glass microelectrode through
the sclera at the level of the temporal peripheral retina. For preliminary testing, 4
L of several different final concentrations of the TIMP-1 (10, 25 and 50 g/mL)
were applied on normal and RP rats at postnatal day (P)20, P30, P45 and P60.
Survival periods of 1-3 hours, 3, 5 days and 1-6 weeks were tested. Both 25 and 50
g/mL gave similar end results in terms of the degree of change in the mosaics of
M-opsin-immunostained reactive cones (termed M-cones), thus 4 L of 25 g/mL
was used for the rest of the experiments. It was also determined that the optimal
stage for the injection of TIMP-1 was P45, the age when cones are arranged in rings
across the entire retina12. As for survival periods, 1 hour, 2 weeks and 6 weeks were
used as they best described the progress of cone mosaic changes with application of
TIMP-1. Sham injections, for controls, consisted of 4 L of the same sterile-filtered
PBS used to prepare the TIMP-1. For each animal, one eye was used to inject TIMP-
1 while the other was used to inject saline for comparison. Surgeries on rats were
performed under anesthesia induced by intra-peritoneal injection of ketamine (100
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 30
mg/kg; KETASET, Fort Dodge, IA) and xylazine (20 mg/kg; X-Ject SA, Butler,
Dublin, OH). The entire injection procedure required only a few minutes, allowing
us to finish before the animals recovered from anesthesia.
Tissue Preparation
Animals at P45 (1 hour survival period), P59 (2 weeks survival period) and P87 (6
weeks survival period) were used (n = 15 for each stage). Animals were deeply anes-
thetized by intra-peritoneal injection of pentobarbital (40 mg/kg body weight) and
theeyeswereenucleated. Animalswerethenkilledwithanoverdoseofpentobarbital.
The anterior segment and crystalline lens were removed and the eyecups were fixed
in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 30 minutes to 1
hour at 4

C. Following fixation, the retinas were carefully isolated from the eyecups
and were transferred to 30% sucrose in PB for 24 hour at 4

C. For storage, all
retinas were then frozen in liquid nitrogen, and stored at -70

C, thawed, and rinsed
in 0.01 M phosphate buffered saline (PBS; pH 7.4). For cryostat sections, eyecups
were embedded in OCT embedding medium (Tissue-Tek, Elkhart, IN), then quickly
frozen in liquid nitrogen and subsequently sectioned along the vertical meridian on
a cryostat at a thickness of 20 m.
Immunohistochemistry
Forimmunohistochemistry, 20-m-thickcryostatsectionswereincubatedin10%nor-
malgoatserum(NGS,JacksonImmunoResearchLaboratories, Inc., WestGrove, PA)
or normal donkey serum (NDS, Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA) for 1 hour at room temperature. Sections were then incubated overnight
withrabbitpolyclonalantibodydirectedagainstglialfibrillaryacidicprotein(GFAP;
Sigma, Saint Louis, MI). This antiserum was diluted in PBS containing 0.5% Triton
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 31
X-100 at 4

C. Retinas were washed in PBS for 45 minutes (3 15 min) and af-
terwards incubated for 2 hour at room temperature in carboxymethylindocyanine-3
(Cy3)-conjugated affinity-purified donkey anti-rabbit IgG (Jackson ImmunoResearch
Laboratories, Inc., West Grove, PA). Next, the sections were washed for 30 minutes
with 0.1M PB and coverslipped with Vectashield mounting medium (Vector Labs,
Burlingame, CA). For whole-mount immuno staining, the same immunohistochem-
ical procedures described above were used. However, incubation times with the
primary antibodies,were longer (two nights with rabbit polyclonal antibody directed
against middle-wavelength-sensitive opsin (M-opsin) (Zhu et al., 2012), mouse mon-
oclonal antibody directed against glutamine synthetase (GS; Chemicon, Temecula,
CA) and so were those with the secondary antibodies (one night either with Cy3-
conjugated donkey anti-rabbit IgG or with Alexa 488 donkey anti-mouse IgG).
Fordouble-labelstudies, wholemountswereincubatedfortwonightsinamixture
of anti-M-opsin and anti-GS markers. Incubation with these antibodies used 0.5%
Triton X-100 in 0.1 M PBS at 4

C. After this incubation, whole mounts were rinsed
for 30 minutes with 0.1 M PBS. Afterwards, we incubated them with Cy3-conjugated
donkey anti-rabbit IgG and Alexa 488 donkey anti-mouse over night at 4

C. Whole
mounts were then washed again for 30 minutes with 0.1 M PB and cover-slipped with
Vectashield mounting medium. Sections and whole mounts were then analyzed using
a Zeiss LSM 510, (Zeiss, NY) confocal microscope. Immunofluorescence images were
processed with the Zeiss LSM-PC software. Finally, the brightness and contrast of
the images were adjusted using Adobe Photoshop 7.0 (Adobe Systems, Mountain
View, CA). All Photoshop adjustments were carried out equally across sections.
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 32
Terminal deoxynucleotidyl transferase dUTP nick end labeling
(TUNEL) Staining
CelldeathwasvisualizedbyamodifiedTUNELtechnique, accordingtothemanufac-
turer’s instructions (In Situ Cell Detection kit, Boehringer Mannheim, Mannheim).
The P45, P59, and P87 cryostat sections of normal retinas from the control and
TIMP-1 groups were incubated with Proteinase K (10 g/ml in 10 mM Tris/HCl,
pH 7.4-8.0) for 10 minutes at 37

C. The sections were incubated with TUNEL
reaction mixture (terminal deoxynucleotidyl transferase plus nucleotide mixture in
reaction buffer) for 60 min at 37

C. The sections were then washed again for 30 min
with 0.1 M PB and cover-slipped with Vectashield mounting medium.
Construction of Nuclei-positions map
Confocal micrographs of the retinas (n = 3-5 animals for each group) were taken
at the focal level of the nuclei of M-cones, covering 1 1mm
2
areas at the mid-
peripheral region of the superior wing of the retina. The micrographs were used to
composecollagesusingPhotoshop. EachnucleusoftheimmunolabeledM-coneswere
visualized using the zoom tool (Fig. 3.6) and each nucleus was marked with a white
dot using the paint tool in Photoshop. The circular dots were slightly lesser in size of
the actual nuclei and were kept even throughout the entire working space. This way,
in cases when two nuclei are close to one another, the two dots marking them neither
touched each other nor overlapped. The resulting "Nuclei-positions map" allowed
easy identification of the position of each M-cones in the micrographed retinal area.
Also, using these images, the density of M-cones (total number/ 1 1m
2
, n = 3-5
animals for each group) was measured.
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 33
Statistical analysis
The previously described nuclei-positions maps were used for the NND and Voronoi
analyses. For the Voronoi analysis, the Voronoi domain for each cell was generated
and the areas of each polygon were calculated and plotted in a histogram. For the
NND analysis, the distance to the nearest neighboring cell was measured for each
dot (Wässle and Riemann, 1978). The distributions were plotted in a histogram. In
turn, for the Voronoi analysis, the Voronoi domain for each cell was generated and
the areas of each polygon were calculated and plotted in a histogram. To remove the
artifacts induced by the edge, we did not include cells around the boundaries.
These NND histograms were then compared to simulation distributions generated
from a random-positions model. This model was programmed to yield expected
distributions for mosaics that were random in the spacing of cells. The model took
into account the constraint in spacing induced by the cone-nucleus size ( 5m). The
curves generated by the model were overlaid on the NND histograms for comparison.
We also extracted statistics from the distributions for analysis. The skewness of the
Voronoi distribution was also determined. The formula used for quantifying skewness
was:
g
1
=
1
n
P
n
i=1
(x
i
x)
3
(
1
n
P
n
i=1
(x
i
x)
2
)
3=2
(3.1)
All the statistics were expressed as mean standard errors of the mean. One-
way unbalanced ANOVA and post-hoc Tukey’s least significant difference procedure
(LSDtest)wereusedtoexaminethedifferencesamongthegroupofmeans. Thetests
were performed and graphs were generated by MATLAB version 8.2.0 (The Math-
Works Inc., Natick, MA). A difference between the means of separate experimental
conditions was considered statistically significant at alpha level of 0.05.
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 34
3.3. Results
Absence of glial activation and M-opsin cone cell death with
TIMP-1
First, the safety of TIMP-1 in concentration and volume used for intraocular injec-
tions in this study (25 g/mL, 4 L) was tested. To check if TIMP-1 was toxic to
retinal cells, normal retinas from the control and the TIMP-1-treated groups were
immunostained with GFAP, a marker for glial activation associated with retinal de-
generation (DiLoreto et al., 1995; Tanihara et al., 1997). The controls showed no
significant upregulation of GFAP expression at 1 hour (data not shown), 2 weeks
(Fig.3.1A), and 6 weeks (data not shown). The GFAP expression is seen predom-
inantly in the nerve fiber layer (NFL). Similar results were observed among the
TIMP-1 groups, i.e., no significant upregulation of GFAP at 1 hour (Fig.3.1B), 2
weeks (Fig.3.1C), and at 6 weeks (Fig.3.1D). Moreover, we did not observe Tunel
positive cells in all groups (data not shown). In summary, TIMP-1 did not cause
glial activation and cell death in both normal and RP retinas.
In addition, the number of M-cones were measured within the 1 1mm
2
areas
at the mid-peripheral region of the superior wing of the retinas. Retinas of all four
conditions showed decreasing mean M-cone densities with age and increasing survival
period (Fig.3.1E, p < 0.000001, two-way ANOVA). This is a common observation
that arises with the aging of animal and the subsequent retinal growth (Harman
et al., 2003; Ji et al., 2012; McCall et al., 1987). However, no statistically significant
differences were observed in the number of M-cones between the control and the
TIMP-1 groups for both normal and RP retinas (p = 0.5576, two-way ANOVA).
The greatest visible difference in the mean M-cone density occurred in RP retinas 6
weeks after TIMP-1 application (p < 0.05).
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 35
Figure 3.1: Confocal micrographs taken from cryostat sections of normal retinas pro-
cessed for GFAP immunoreactivity shown for the 2-weeks control (A), and the 1 -hour
(B), 2-weeks (C), and 6-weeks TIMP-1 groups. The drug caused no significant upreg-
ulation of GFAP expression. The summary graphs illustrated for mean cone density (E)
measured from the 11 mm
2
sampling areas (in the superior mid-peripheral region) of
all normal control, TIMP-1 treated normal, RP control and TIMP-1 RP retina groups (n
= 3-5 animals per group). Data are presented as mean standard error. ONL, outer-
nuclear layer; OPL, outer-plexiform layer; INL, Inner-nuclear layer; IPL, Inner-plexiform
layer; GCL, Ganglion-cell layer; NFL, Nerve-fiber Layer. Scale bar = 50 m.
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 36
DisturbanceofthemosaicofM-conesinRPretinaswithTIMP-
1
To examine if exogenous application of TIMP-1 can modulate the M-cone mosaic in
vivo, this drug was administrated intraocularly into RP rat eyes. The M-cones were
labeled in the whole-mount retinas in all groups. The RP retinas of the controls
(Figs.3.2A-C) and the TIMP-1-treated groups (Figs. 3.2G-I) immunostained with
M-opsin showed fairly intact cone morphologies. For mosaic quantification, we used
the nuclei-positions map (Figs.3.2D-F, J-L). In these figures, the geometry of their
mosaic can be seen clearly. The control RP retinas showed nuclei forming the rim of
the rings and the cones´ processes pointing towards the center of the regions devoid
of cell bodies (Figs.3.2A-C). Furthermore, the size of these rings increased with age
(Figs.3.2D-F), which was consistent with our previous observations (Ji et al., 2012).
Such M-cones mosaic showed remarkable change with TIMP-1. The rings lost first
their sharpness and eventually disappeared (Figs.3.2J-L). Even after only 1 hour,
the rings became less defined and smaller compared to the control group (Fig.3.2J).
At 2 weeks, the rings disappeared and cones redistributed themselves homogenously
(Fig.3.2K). Such striking change continued even at 6 weeks (Fig.3.2L).
Voronoi analysis on RP retinas was performed in order to quantify changes in
homogeneity of the mosaic and the gradual disappearance of rings. Examples of the
resulting Voronoi tessellation are shown in insets besides the histograms (Figs.3.3A-
F). In the RP control retinas, the majority of Voronoi domains were small as M-
cones are clustered around the rings. Furthermore, a few large Voronoi-domain
areas were observed. These larger areas resulted from the regions with few or no
cones in the rings. Hence, the histograms from the data had longer tails, resulting
in highly skewed distributions (Fig.3.3A-C, J). The insets in Figs.3.3A-C illustrate
the alternation between small and large Voronoi domains in the RP retinas. This
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 37
Figure 3.2: Confocal micrographs taken from whole-mount RP retinas processed for M-
opsin immunoreactivity (A-C, G-I) and nuclei-positions maps (D-F, J-L). In these maps,
each dot represents a nucleus of a M-cone as obtained from the micrographs. The
micrographs for control groups show P45 RP (A), P59 RP (B), P87 RP (C) retinas 1
hour, 2 weeks, and 6 weeks post-saline application respectively. Rings are observed in
the mosaics of RP-controls (A-F). The micrographs for TIMP-1 groups show P45 RP
TIMP-1 (G), P59 RP TIMP-1 (H), P87 RP TIMP-1 (I) retinas 1 hour, 2 weeks, and
6 weeks post-application of the drug respectively. TIMP-1 loosens rings and increases
the homogeneity of the mosaic of M-cones (G-L). 1hour, hour; wk, week; RP, retinitis
Pigmentosa; TIMP-1, Tissue inhibitor of metalloproteinase 1. Scale bars = 500 m.
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 38
Figure 3.3: Histograms generated from the Voronoi analysis on the 1 1mm
2
sampling
areas from all RP controls (A-C), TIMP-1 treated RP (D-F), and normal controls (G-I)
(n = 3-5 animals per group). Results are shown with survival times of 1 hour, 2 weeks
and 6 weeks. Examples ( 170 m x 170 m) of the resulting Voronoi domains are
shown for each group. The summary graphs for the mean sknewness values obtained
from the Voronoi domain distribution curves are plotted for each groups (J). Also graph
for the mean coefficient of clustering measures in all groups are illustrated (K). Data are
presented as mean standard error. The symbol ? represents p < 0.05 or better.
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 39
alternation was not random but showed a specific pattern in that small Voronoi
domains were surrounded by other small Voronoi domains whereas large Voronoi
domains were surrounded by other large Voronoi domains. This was illustrated by
calculating the coefficient of clustering (CC), the ratio between the local and the
global coefficient of variance of Voronoi domain size. This coefficient is important
because the existence of a cluster of large Voronoi domains indicates the existence
of holes and thus, the inhomogeneity of the mosaic. Typically, RP retinas revealed
high CC (Fig.3.3K). In TIMP-1 treated RP groups, the rings gradually disappeared
andconesredistributedthemselveshomogeneously. Withincreasingsurvivalperiods,
the cones spread out to occupy areas inside rings and large Voronoi domains became
smaller, and less skewed (Figs.3.3D-F, J).
Voronoi analysis on normal control retinas (Fig.3.3G-I) was performed in order
to compare the homogeneity of the mosaic between TIMP-1 treated RP groups and
normal control groups. Examples of the resulting Voronoi tessellation are shown in
insets besides the histograms (Figs.3.3G-I). ). In the normal control retinas, the
distribution of Veronoi domains was close to Gaussian thus less skewed (Fig.3.3G-I,
J). In order to compare the distribution of VD in among three groups, RP control,
RP TIMP-1, and normal control, we examined both skewness of the distributions
and CC. The skewness of distributions were significantly different from RP control,
and TIMP-1-treated RP and normal control retinas (p < 0.0001, two-way ANOVA).
Post-hocanalysisshowedsignificantlylowersknewnessvalueinnormalcontrolgroups
and RP TIMP-1 groups compared to RP controls at both 2 weeks and 6 weeks (post-
hoc test, alpha = 0.05). This indicated that Voronoi domains with extremely larger
size is reduced and cones in RP retinas became more homogeneous with TIMP-1
after 2 weeks. Furthermore, homogeneity of cone mosaic is restore closely to normal
control groups after 2 weeks. This was also confirmed by measurement of CC. Our
results showed statistically significant differences in CC in among groups (Fig.3.3K,
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 40
p = 0.0001, two-way ANOVA). The M-cones in TIMP-1 treated RP retinas were
still highly clustered at 1 hour, but significantly reduced close to normal level after 2
weeks (post-hoc test, = 0:05). In summary, TIMP-1 induced mosaics of M-cones
in RP retinas to gain homogeneity and become close to normal.
TIMP-1 injection induces irregularity of M-opsin cones in RP
retinas
We examined if the homogenous M-cone mosaics in TIMP-1 treated RP retinas
are also regular as in normal mammalian retinas (Ji et al., 2012; Lee et al., 2011).
Two critical hallmarks for a normal cone mosaic are homogeneity and regularity.
Homogeneity means that the spatial statistics of cones are similar in different regions.
In turn, regularity means that the distance from a cone to its neighbors is similar
for different cones. In Figure3.3, we showed that TIMP-1 induced mosaics of M-
cones in RP retinas to gain homogeneity. Next, we performed Nearest-neighbor-
distance regularity index (NND-RI) to determine the regularity. Thus, we measure
regularity by the regularity index (RI, (Cook, 1995; Wässle and Riemann, 1978)).
It is the ratio of the mean to the standard deviation of the distances from each cell
to its nearest-neighbor. In addition, we plotted a distribution of the NND for the
random-position model with the same density and with minimum distance of 5m at
each time point (solid lines). The histograms for the normal control groups showed
near-Gaussian distributions that did not conform well with the predictions from the
random-positionsmodel(Fig.3.4A,B).ThemeanNNDsinthenormalcontrolretinas
at 2 weeks and 6 weeks were 10.29 0.08 m and 10.88 0.07 m, respectively.
These distributions were distinct from the random positions model. Subsequently,
the mosaics showed high regularity with RI value of 3.94 0.03 and 4.22 0.26
at 2 weeks and 6 weeks, respectively. However, the NND distribution changed with
TIMP-1 treated RP groups. The distributions from the TIMP-1 treated RP retinas
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 41
Figure 3.4: Distribution of distances between nearest-neighbor M-cones within the 1
1mm
2
sampling areas from normal control (A,B), TIMP-1 treated RP (C,D), and TIMP-
1 treated normal groups (E, F) (n = 3-5 animals per group) at 2 weeks and 6 weeks after
treatments. The histograms are overlaid with distributions generated from the random-
positions model (solid line in each histogram). With the application of TIMP-1, the NND
distributions became closer to the simulated random distribution. The summary graphs
for mean NND (G), and the mean RI (H) for all groups are illustrated. Data are presented
as mean standard error. The symbol ? represents p < 0.05 or better.
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 42
showed smaller mean NNDs of 8.95 0.04 m and 9.15 0.31 m at 2weeks and
6weeks, respectively (Figs.3.4C, D). The RI values for TIMP-1 2weeks was 3.31
0.12 m and 6weeks was 3.08 0.14 m.
In order to understand if lower RI values of M-cone mosaic in TIMP-1 treated
RP retinas were direct consequence of TIMP-1 treatment or if it were an indepen-
dent of TIMP-1 effect, we examined the regularity also in normal retinas treated
with TIMP-1(Figs.3.4E-H). To address this question, we applied TIMP-1 to nor-
mal retina that has both homogeneity and regularity. The M-cones were labeled in
the whole-mount retinas in all groups (control groups: Figs.3.5A, B, C; TIMP-1:
Figs.3.5G, H, I). The images of marked nuclei of M-cones help visualize the ge-
ometry of their mosaics (Figs.3.5D-F, J-L). The M-cones in control groups showed
regular and homogenous distribution patterns (Figs.3.5A, B, C) that were similar
to that seen in the normal mammalian retinas (Ji et al., 2012; Lee et al., 2011).
The nuclei-positions map emphasizes this similarity in M-cone patterns (Figs.3.5D,
E, F; Fig.3.6). However, the mosaic of M-cones showed some changes with TIMP-1
(Figs.3.5G-L; Fig.3.6). First, the orientation of array of the outer segments were
disturbed in some regions (Figs.3.5G, H, I, squares). Rather than showing steady
orientation as in control groups, variable orientations were sometimes observed in
retinas with TIMP-1 (Figs.3.5G, H, I, squares). More importantly, TIMP-1 led to
change in the arrangement of some cell bodies after 2 weeks that seem to show lose
in regularity (Figs.3.5K, L, ellipses; although not much after 1 hour, Fig.3.5J).
TheNNDanalysisonTIMP-1treatednormalretinasshowedthatthedistribution
became more skewed and broader compared to normal controls with significantly
less mean NND of 9.93 0.21 m by 6 weeks (Fig.3.4E and F). The mean RI also
declined compared to normal controls with value of 3.19 0.16 m. In addition,
the NND distribution showed better fit to the random distribution (solid lines). We
then compared the mean NND (Fig.3.4G) and RI (Fig.3.4H) for normal control, RP
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 43
Figure 3.5: Confocal micrographs taken at the level of the OS with low power objectives
from the whole-mount normal retinas processed for M-opsin immunoreactivity (A-C, G-
I). Here, global features of the OS are presented as they are strongly labeled and due
to their signals becoming too intense when nuclei (although successfully labeled) were
focused. Nuclei-positions maps and each dot therein represent the location of soma of
M-cones in the above corresponding panels. The micrographs for control groups show
P45 N (A), P59 N (B), P87 N (C) retinas 1 hour, 2 weeks, and 6 weeks post-saline
application, respectively. The retinal cone mosaics seem regular and homogenous (D, E,
F). The micrographs for TIMP-1 groups show P45 N (G), P59 N (H), P87 N (I) retinas
1 hour, 2 weeks, and 6 weeks post-application of TIMP-1, respectively. The orientation
of the array of M-cone outer segments are disturbed in their orientation with TIMP-1
(squares; G, H, I). The nuclei-positions maps (J, K, L) reveal that, not so much after 1
hour, but by 2 weeks and 6 weeks, M-cone mosaic seem less regular (examples enclosed
in ellipses). hour, hour; wk, week; N, normal; RP, retinitis Pigmentosa; TIMP-1, Tissue
inhibitor of metalloproteinase 1. Scale bars = 500 m.
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 44
Figure 3.6: Confocal micrographs taken with high-power objective at a focal plane of
the nuclei in the whole-mount normal (A, B) and RP (C,D) retinas processed for M-opsin
immunoreactivity 2 weeks after saline and TIMP-1 treatment. Nuclei were successfully
immunolabeled in all retinas. Where the disruption of orientation occurred as viewed at
a focal plane of the OS in the normal retina with TIMP-1 treatment is enclosed with an
ellipse (B). It’s illustrated that Nuclei positions maps (A-D) were constructed by marking
the location of cell bodies using white dots. Scale bars, 50 m, of panel A and B is
indicated in panel A. Scale bar, 50 m, of panel C and D is indicated in panel D.
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 45
and normal retinas with TIMP-1 treatment. The two-way ANOVA analysis showed
significant differences in both mean NNDs and RIs among the different groups of
retinas (Fig.3.4G mean NND, p = 0.0001; Fig.3.4H RI, p = 0.0005), but not between
different stages (2 weeks and 6 weeks) after intraocular treatment. Compared to the
normal control retinas, the TIMP-1 treated normal retinas showed statistically lower
mean NND and RI at 6 weeks. (Fig.3.4G and H, post-hoc test, = 0.05). However,
the mean NND in TIMP-1 treated normal retinas were still significantly higher than
in TIMP-1 treated RP retinas (Fig.3.4G. post-hoc test, = 0.05). Consistent with
this observation, the mean RI in TIMP-1 treated normal retinas were lower than
normal controls; however, not significantly different from that of the TIMP-1 treated
RPs (Fig.3.4H. post-hoc test, = 0.05). These indicated that M-cone mosaic in
TIMP-1 treated RP retinas did not reach the degree of regularity seen in normal
retinal mosaics. In addition, TIMP-1 led to loss of local spatial regularity in the
mosaics of M-cones in normal rat retinas. In summary, the loss of regularity in
TIMP-1 treated RP retinas may largely be caused by TIMP-1.
Remodeling of Müller cell processes in RP retinas with TIMP-
1
In this paper, we focused on TIMP-1 since it is one of the regulators of the ECM,
thus being important for cellular migration. Another retinal process contributing to
the migration of neurons is the Müller glial cell. We thus decided to test whether
Müller cell processes in RP retinas were also affected by TIMP-1. Therefore, we
immunostained RP-control and TIMP-1-treated retinas with M-opsin and glutamine
synthase (GS), a marker for Müller cells (Haverkamp and Wässle, 2000; Lee et al.,
2002). Consistent with our previous work (Lee et al., 2011), the RP-control retina
showed remodeled processes of the Müller cells filling the insides of each ring of
M-cones after 1 hour (data not shown), 2 weeks (Fig.3.7A) and 6 weeks (data not
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 46
Figure 3.7: Confocal micrographs taken from RP whole mounts of control and TIMP-1
groups processed for GS (green) and M-opsin (red) immunoreactivities. Double exposure
of control retina at 2 weeks (A) and its higher-power micrograph (B) show rings of M-
cones around remodeled Müller-cell processes in characteristic broccoli-like shape. Just
1 hour after application of TIMP-1, M-cones and Müller-cell processes begin losing their
broccoli-like shapes (C). A higher-power micrograph shows this loss more clearly (D).
After 2 weeks, the mosaic of M-cones and Müller-cell processes is almost homogeneous
(E). However, a higher-magnification reveals some tendency for some groups of M-cones
to migrate closer to each other, showing that the mosaic is becoming less regular (F).
Scale bars = 100 m.
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 47
shown). A high-magnification view of a ring marked by the inset rectangle revealed
these remodeled processes more closely (Fig.3.7B). RP retinas at 1 hour after ap-
plication of TIMP-1 showed disturbance of rings as they became smaller and less
distinct (Fig.3.7C). A higher-power micrograph revealed that the Müller cells pro-
cesses were filling inside the center of the shrinking rings (Fig.3.7D). RP retinas at
2 weeks (Figs.3.7E, F) and 6 weeks (data not shown) post application of TIMP-1
showed homogeneously distributed M-cones and Müller-cell processes. In summary,
these results indicated that the Müller-cell processes in RP retinas are also remodeled
with cone mosaic significantly upon application of TIMP-1.
3.4. Discussion
TIMP-1 does not cause cell death
Why does TIMP-1 treatment cause such dramatic effects in RP retinas? The results
reveal that this drug is not acting through retinal damage. To start, neither saline
nor TIMP-1 introduce reduction in the cone density (Fig.3.1). Moreover, the glial
activation associated with TIMP-1 (DiLoreto et al., 1995; Tanihara et al., 1997) is
also not detected in normal retinas (Fig.3.1). And lack of significant TUNEL positive
staining indicates no sign of cell deaths in these retinas (results not shown). Thus,
the reduction of the mean cone density that we observe with greater survival time
is not explained by cell deaths but by the growth of the total retinal area with age
(Fig.3.1). In addition, the density is the number of cells divided by area. Hence, any
density changes must be due to area variations. Furthermore, we also demonstrated
previously that the mean retinal areas from P30 to P180 increased significantly in
normal and RP retinas (Ji et al., 2012). Therefore, the retinas were shown to grow
with age. Such growth results in the declining density of different types of retinal
cells (Harman et al., 2003; Ji et al., 2012; McCall et al., 1987). In particular, greater
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 48
retinal expansion in the peripheral retinal regions compared to the central region
(Mastronarde and Thibeault, 1984; Reichenbach et al., 1993) may have made our
mid-peripheral regional density results more significant.
Mosaics of M-cones can be manipulated by TIMP-1 treatment
In the present study, two mosaic properties were studied statistically ï£¡ homogene-
ity and regularity. Both properties are important as they are the basis of even
sampling of visual world, which provides visual acuity (French et al., 1977; Manning
and Brainard, 2009). One of the main results of the current study is that TIMP-1
causes change in the mosaic of cone photoreceptors in RP retina to become more
homogenous. Homogeneity is a measurement of the spatial statistical properties of
the mosaic and are as constant as possible over large portions of the retina. When a
mosaic exhibits rings, the mosaic is not homogeneous, because the statistics in their
rims are different from those in the areas with little or no cones (center of rings).
Therefore, we are looking for an analysis that will provide: (1) the degree of global
homogeneity, and (2) existence of holes. Classical tools such as quadrat analysis
would provide only the former. In turn, with largest-empty-space analysis, only in-
formation about existence of holes is provided. In contrast, the Voronoi-domain)
analysis, though not typically used as an homogeneity test, can detect the global
homogeneity and existence of holes (Figs.3.2B, E). Thus, to emphasize ring-induced
inhomogeneity, we measured the distribution of areas of Voronoi domains. These
domains are large inside the rings and small in their rims. Such rings become visibly
disturbed and less distinct only after 1 hour of TIMP-1 treatment (Fig.3.2G, J).
By 2 weeks, the rings are no longer obvious as cells cover the space homogeneously
(Figs.3.2H, K). The Voronoi domain analysis results statistically confirmed such ob-
servation. The skewness of the small Voronoi domain areas in RP retinas declined
significantly as M-cones start migrate to fill inside the empty rings with TIMP-1
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 49
treatment (Fig.3.3D-F, J). As the cells move away from the crowded rim of rings,
the mean coefficient of clustering decrease significantly over time. All these changes
that TIMP-1 brings to the retina make the mosaic properties closer to what is ob-
served in the normal retinas (Fig.3.3G-I, J, K). Another important result from our
study is that the regularity of the mosaic is lost with TIMP-1 treatment. We think
of regularity as an even or uniform arrangement at small spatial scales, i.e., relatively
local. One can measure regularity in many ways, but in this paper, we used the sim-
plest definition, namely, the similarity of distances between nearest neighbors. The
results from the NND analysis showed that TIMP-1 induced mosaic to become closer
to a random distribution with significantly less NND and RI compared to the nor-
mal retinas (Fig.3.4A-D, G, H). Thus, although clear improvement of homogeneity
is achieved, the mosaic became irregular.
Ultimately, the aim of drug treatment therapy is to improve both homogeneity
and regularity. However, with TIMP-1 treatment, we see a clear improvement of
homogeneitywithoutaccompanyingrestorationofregularity. Thus,inordertobetter
understand if such irregularity is a direct consequence of TIMP-1 treatment or it is
independent of TIMP-1 effect, we applied the treatment to normal retinas that have
homogenous and regular mosaic. As results, we observed M-cone mosaic significantly
lose its regularity at 6 weeks and become close to a random distribution. Thus, the
lose of regularity may largely be caused by TIMP-1. Even if TIMP-1 fails to promote
regularity, the effects of this drug on homogeneity appear to be so dramatic that we
may still consider TIMP-1 as a potential therapeutic tool. TIMP-1 would improve
sampling of the visual field simply by causing homogeneity.
A possible reason for dystrophic retinas to show more dramatic change in the
mosaic pattern with TIMP-1 may be that there is more space for cones to migrate
after the rods die (Zhu et al., 2012). In our previous study, death of rods induces slow
rearrangement of cones into regular mosaics of rings. Although the number of cones
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 50
remains similar in normal and dystrophic retinas even at an older age, rods in RP die
in "hot spots" that increase progressively as circular waves, leaving behind "rod-less"
zones (Ji et al., 2012; Zhu et al., 2012). Our work also clearly demonstrated that
Mï£¡ller-cell processes remodel to occupy these zones, interact with the cones, and
induce cone migration to the edges of the holes of rods (Ji et al., 2012; Lee et al.,
2011). Therefore, dramatic change in the mosaic with TIMP-1 may result in more
space for cones to migrate.
What are the possible mechanisms underlying modulation of
mosaics of M-cones with TIMP-1?
ThesimplesthypothesisisthatTIMP-1actsthroughtheextracellularmatrix(ECM).
For cones to migrate during the change in the mosaic, interactions between the cells
and the ECM are necessary (Raines, 2000; Streuli and Akhtar, 2009). Cell migration
depends on the stiffness of the ECM (Lu et al., 2011). As the ECM comprises mostly
collagen, the mammalian enzymes that modulate its functions are the MMPs and
their inhibitors, the TIMPs (Alexander and Werb, 1989; Padgett et al., 1997). For
MMP and TIMP to play an essential role in the organization of the ECM (Cornelius
et al., 1995; Yamada et al., 2001), the balance in the level of these enzymes is crucial.
Such balance is disrupted in pathological retinas, and often, the level of TIMP-1 is
significantly upregulated with ocular disease (Kim et al., 2014; Matsuo et al., 1997;
Zeiss et al., 1998) TIMP-3 mRNA was also over expressed in human Retinitis Pig-
mentosa and Sorsbyï£¡s fundus dystrophy conditions and was localized to structures
including photoreceptor inner segments and Bruch’s membrane (Fariss et al., 1998;
Jomary et al., 1995; Jones et al., 1994). Thus, a reasonable hypothesis to explain our
results is that exogenous application of TIMP-1 disturbed the enzymatic balance and
the mechanical properties of the ECM even further (Matrisian, 1992). There is also
evidence that TIMP-1 can affect the migration of cells by modulating focal adhesions
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 51
composed of integrins and various cytoplasmic proteins in the ECM, which cells use
as aids during their migration (Akahane et al., 2004). Thus, if controlled release of
growth factors from the ECM is necessary to maintain the M-cones in rings, the fall
in the release of growth factors sequestered in the ECM due to addition of TIMP-1
would lead to fewer M-cones in rings.
Finally, itiswellknownthatglialcellsarealsoregulatedbysignalsfromtheECM
(Xie et al., 2014). The above-mentioned integrins are found expressed in glia and
modulate their migration and organization (Xie et al., 2014). Our hypothesis is that
Müller glial cells are involved in the migration of cones by complex interactions with
surroundingECM.Müllercellprocessesareshownincloseassociationwithmigrating
cones (Fig.3.7) (Lee et al., 2011). In a previous study (Lee et al., 2011) , we showed
effects of DL--aminoadipic acid (AAA) on cone mosaics, which were similar to those
observed in our current study with TIMP-1. AAA is a gliotoxin, which disrupts
Müller-cell metabolism (Jablonski and Iannaccone, 2000; Karlsen et al., 1982a; Rich
et al., 1995a; West et al., 2008). Thus, TIMP-1 may have affected the interaction
between cones and Müller cells through ECM. Further support for this hypothesis
comes from Müller cells playing a role in the regulation of expression and production
of TIMP and MMP through a feedback system involving the ECM (Limb et al.,
2002a; Miyata et al., 2012a). Our results are consistent with this hypothesis, since
Müller-processes remodel with cones in TIMP-1 treated RP retina (Fig.3.7).
3.5. Conclusion
We have shown that exogenous application of TIMP-1 can significantly modulate the
mosaicsandrestoreitshomogeneityintheRPretinas. Theresultingmosaicalsoloses
regularity and become close to random distribution both in the RP and in normal
retinas. All these changes occur without being toxic to retinal cells. Our findings
CHAPTER 3. THE EFFECT OF TIMP-1 ON THE CONE MOSAIC 52
have clear therapeutic implication as they suggest that treatment with TIMP-1 could
improve sampling of visual field by improving homogeneity. In the future, we will
assess the efficacy of TIMP-1 when administered prior to the appearance of rings
and later stages of disease.
Chapter 4
Remodeling of the Cone Mosaic by Unfetter-
ing the Cones from the Müller Cells in a Rat
Model of Retinitis Pigmentosa
4.1. Introduction
Retinitis pigmentosa (RP) is characterized by an initial loss of rods, followed by a
slow, progressive loss of cones. Because cones are essential for high-acuity vision,
their loss leads to a reduction in the quality of everyday life, and eventually, to
vision loss (Punzo et al., 2009; Williams and Coletta, 1987b). The outer nuclear
layer (ONL) of the vertebrate retina contains a tightly packed, uniform array of rods
and cones, which is essential to ensure that the visual world is sampled with no
empty visual space. The density of rods constrains visual sensitivity and the spacing
of cones determines resolution and visual acuity (Williams and Coletta, 1987b). Past
studies have described that regular and homogenous spacing of photoreceptors, as
seen in some mammalian species and zebrafish (Bruhn and Cepko, 1996b; Kram
et al., 2009; Larison and Bremiller, 1990; Lin et al., 2004; Raymond et al., 1995;
Wikler and Rakic, 1991, 1994), is important for sampling the visual space efficiently
53
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 54
(French et al., 1977; Manning and Brainard, 2009). However, cones in the S334ter-
line-3 rat model of RP were recently shown both to survive for a longer period of
time after the early rod deaths and to remodel in their mosaic pattern into orderly
arrays of rings (Ji et al., 2012; Lee et al., 2011; Zhu et al., 2012). Similar dark
patches or holes are noted in several human eye diseases caused by retinal dystrophy,
inherited retinal degeneration, and photo-pigment genetic perturbations in M-opsin
cones (Carroll et al., 2004; Choi et al., 2006; Duncan et al., 2007b; Rossi et al., 2011).
The center of these rings lack photoreceptors, indicating local loss of visual function.
Consequently, knowledge on modulating and rearranging photoreceptors from the
ring patterns into a more homogenous distribution may improve visual perception in
these patients.
We discovered that the rearrangement of cones in rings is modulated by Müller
cells (Lee et al., 2011) and demonstrated that treatment with DL--aminoadipic
acid (AAA), a compound that transiently disrupts the metabolism of Müller cells
(Jablonski and Iannaccone, 2000; Karlsen et al., 1982b; Rich et al., 1995b; West
et al., 2008), induces rearrangement of cones (Lee et al., 2011) . Previously, AAA
has been used to study the treatment of RP and diabetic maculopathy (Bringmann
et al., 2006; Reichenbach et al., 2007). Furthermore, AAA is known to enhance the
number of donor photoreceptors integrated into the recipient photoreceptors after
transplantation (Johnson et al., 2010; Takeda et al., 2008). However, AAA can be
toxic to glial cells and the toxic effect of the drug is dosage and time dependent
(Rich et al., 1995b). In addition, the different routes of administration of the drug
including intravitreal, subretinal, and subcutaneous injection, show differences in
toxicity. The intravitreal injection has the least toxic effect (Pedersen and Karlsen,
1979; Rich et al., 1995b). In this study, we performed intravitreal injections of AAA
and determined the appropriate dosage of AAA to cause the homogenous spreading
of cones in the S334ter-line-3 rat retina.
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 55
In the S334ter-line-3 rat retina, we found specialized adherens junction associ-
ated protein, ZO-1, in the network of rings of cones (Lee et al., 2011). ZO-1 is
normally present between the photoreceptor inner segments and the apical processes
of Müller cells (Campbell et al., 2006; Paffenholz et al., 1999; Pearson et al., 2010;
Tserentsoodol et al., 1998). Therefore, we hypothesized that AAA promotes remod-
eling of the cone mosaic by unfettering the Müller cells from the cones through the
modulation of ZO-1.
4.2. Materials and Methods
Animals
The third line of albino Sprague-Dawley rats homozygous for the truncated murine
opsin gene (created a stop codon at Serine residue 334; S334ter-line-3) was obtained
fromDr. MatthewLaVail(UniversityofCalifornia, SanFrancisco, CA).Homozygous
S334ter-3 male rats are mated with homozygous S334ter-3 female rats to produce
offspring for the S334ter-3 transgene that are used throughout this study. This
model shall be referred to as the RP model in the rest of the article. RP rats were
sacrificed at post-natal (P) days 30, 31, 32, 33, 37, 44, 50, 51, and 72 (N = 9 for
each stage). Controls were age-matched Sprague Dawley rats (N = 9 for each stage;
Harlan, Indianapolis, IN).Allratswerehousedundercyclic12:12hour(hr)light/dark
conditions with free access to food and water. Both sexes of control and RP rats
were used. Animals were treated in accordance with the regulations of the Veterinary
Authority of University of Southern California and with the ARVO Statement for
the Use of Animals in Ophthalmic and Vision Research.
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 56
Administration of Alpha-aminoadipic Acid (AAA)
DL--Aminoadipic acid (AAA, Sigma) was prepared in phosphate-buffered saline
(PBS), adjusted to pH 7.5 and sterile-filtered before administration. AAA was ad-
ministeredbyintravitrealinjectionwithafine-glassmicroelectrodethroughthesclera
at the level of the temporal peripheral retina. The high concentration of AAA is
known to affect the photoreceptors (Jablonski and Iannaccone 2000; West et al.
2008). For preliminary testing, 4 L of several different final concentrations of the
AAA (5, 10, 25 and 50g/mL) were applied on RP rats at P30. Survival periods
of 1-3 days, 1, 2, and 6 weeks were tested. Both 25 and 50g/ml gave similar end
results in terms of the degree of change in the number of M-opsin immunoreactive
cones (termed M-opsin cones for simplicity) and mosaics of M-opsin cones. The
5g/ml of AAA did not affect the mosaic of cones in RP retina. However, 10g/ml
of AAA gave similar end results in terms of the degree of change in the mosaic of
cones as in the 25g/ml or 50g/ml of AAA but did not affect the cell number of
cones after 3 days of injection. Thus, results from 50g/ml (Fig. 4.1) and 10g/ml
(Figs. 2-5) of AAA were reported. It was also decided that the optimal stage for
the injection of AAA was P30, the age when cones were arranged in rings across
the entire retina. As for survival periods, 3 days, 2 weeks and 6 weeks were used
as they best reflected the progress of cone mosaic changes with application of AAA.
Sham injections, for control, consisted of 4l sterile saline. For each animal, one eye
was used to inject AAA and the other eye was used to inject saline for comparison.
Surgeries on rats were performed under anesthesia induced by intra-peritoneal injec-
tion of ketamine (100 mg/kg; KETASET, Fort Dodge, IA) and xylazine (20 mg/kg;
X-Ject SA, Butler, Dublin, OH). The entire injection procedure required only a few
minutes, allowing us to finish before the animals recovered from anesthesia.
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 57
Preparation and Administration of siRNA
The siRNA technology to produce a knockdown of target proteins in the eye is well
established (Pearson et al., 2010). Targeting siRNA against ZO-1 was generated us-
ing the pre-design silencer select ZO-1 siRNA (Gene Name: tight junction protein 1;
Gene Aliases: ZO-1; Locus ID: 292994, Species: Rat, si RNA ID: s146925, Invitro-
gen, UK). The siRNA was resuspended and diluted to the appropriate concentration
in sterile buffer containing lipofectamine using RNAase-free plasticware. Controls
have a 4l injection of Silencer Select Negative Control siRNA (Catalog# 4390843,
Invitrogen, UK). ZO-1 siRNAs were applied by intravitreal injection to the temporal
peripheral retina at P50. Various concentrations of ZO-1 siRNA (15 and 25M) were
administered and monitored after 24hr and 48hr. ZO-1 siRNA 25M was the most
efficient in terms of the degree of change in the mosaics of M-opsin-cones, thus 4L
of 25M was used for the ZO-1 related experiments.
Tissue Preparation
Animalsweredeeplyanesthetizedbyintra-peritonealinjectionofEuthasol(40mg/kg
body weight) and the eyes were enucleated. Animals were then killed by an overdose
of Euthasol. Their eyesánterior segments were then removed and the eyecups were
fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4,
for 1.5-2 hr. Following fixation, the retinas were carefully dissected and transferred
to 30% sucrose in PB for 24 hr at 4

C. For storage, all retinas were then frozen
in liquid nitrogen, and stored at -70

C, thawed, and rinsed in 0.01 M phosphate
buffered saline (PBS; pH 7.4). For cryostat sections, eyecups were embedded in
OCT embedding medium (Tissue-Tek, Elkhart, IN), then quickly frozen in liquid
nitrogen and subsequently sectioned along the vertical meridian on a cryostat at a
thickness of 20 m.
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 58
Immunohistochemistry
For fluorescence immunocytochemistry, 20m thick cryostat sections were incubated
in 10% normal goat serum (NGS) or 10% normal donkey serum (NDS) and 1%
Triton X-100 in PBS for 1 hr at room temperature. Sections were then incubated
overnight with a rabbit polyclonal antibody directed against: mouse green opsin (M-
opsin, dilution1:1000)(Zhuetal.,2003)mousemonoclonalantibodydirectedagainst
glutamine synthetase (GS, Chemicon; dilution 1:100), and zonula occludens (ZO-1,
Invitrogen; 1:500). Each antiserum was diluted with PBS containing 0.5% Triton
X-100 at 4

C. Retinas were washed in PBS for 45 min (3 15 min). Afterwards,
we incubated the retinas for 2 hr in carboxymethylindocyanine (Cy3)-conjugated
affinity-purified, donkey anti-rabbit IgG (Jackson Immuno Labs, West Grove, PA,
USA; dilution 1:500); Alexa 488 anti-mouse (Molecular Probes, Eugene, dilution
1:300) or Cy5-conjugated, donkey anti-mouse IgG (Jackson Immuno Labs; dilution
1:300) at room temperature. The sections were washed for 30 min with 0.1M PBS
andcoverslippedwithVectashieldmountingmedium(VectorLabs,Burlingame,CA).
For whole-mount immunohistochemical staining, the same procedure was used. For
M-opsin and GS, the primary antibody incubation was for 2 days and the secondary
antibody incubation was for 1 day. FITC conjugated mouse monoclonal antibody
directed against ZO-1 was incubated for 1 day. For double and triple labeling, sec-
tions and whole mounts were incubated in a mixture of following antibodies: GS and
ZO-1; ZO-1, GS, and M-opsin. Sections and whole-mounts were then analyzed using
a Zeiss LSM 510, (Zeiss, NY) confocal microscope. Immunofluorescence images were
processed with Zeiss LSM-PC software. The brightness and contrast of the images
were adjusted using Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA).
For presentation, all Photoshop manipulations (brightness and contrast only) were
carried out equally across sections.
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 59
Statistical analysis
The density of M-opsin cones was counted in three retinal whole-mount preparations
from each group. Confocal micrographs of the retinas were taken at the focal level
of the nuclei of M-opsin cones, covering 1 1 mm
2
areas at the central region (1mm
away from optic disc) of the superior part of the retina. At these locations we made
serial optical sections using a confocal microscope. By following each M-opsin cone
throughout the sections, we ensured that every M-opsin cone in the selected region
was counted.
For the Voronoi analysis, the Voronoi domain for each cell was generated and the
areas of each polygon were calculated and plotted in a histogram. To remove the
artifacts induced by the edge effect, we did not include cells around the boundaries.
The skewness of the Voronoi distribution was also determined. The formula used for
quantifying skewness was:
g
1
=
1
n
P
n
i=1
(x
i
x)
3
(
1
n
P
n
i=1
(x
i
x)
2
)
3=2
(4.1)
All the statistics were expressed as mean standard errors of the mean. One-
way unbalanced ANOVA and post-hoc Tukey’s least significant difference procedure
(LSDtest)wereusedtoexaminethedifferencesamongthegroupofmeans. Thetests
were performed and graphs were generated by MATLAB version 8.2.0 (The Math-
Works Inc., Natick, MA). A difference between the means of separate experimental
conditions was considered statistically significant at alpha level of 0.05.
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 60
4.3. Results
Spatial Pattern of Cones in S334ter-line-3 Rat Retinas after
AAA Treatment
In order to redistribute the cones homogenously and minimize toxicity to cones in
RP with AAA, we injected AAA with various doses. First, we examined the cone
mosaic in higher concentration of AAA (50g/ml). The illustrated whole-mounts
were taken from the central part of the superior (1-mm away from the optic disc)
P33 normal, P33 RP control, and P33 AAA-treated RP retinas (Fig. 4.1). In P33
normal retina, M-opsin cones were distributed homogeneously throughout the retinas
(Fig. 4.1A). In P33 RP control retina, multiple rings of cones were observed (Fig.
4.1B). The effect of AAA (50g/ml) was monitored 3 days (3D) after the injections
in P30 RP retinas. In P33 AAA-treated RP retinas, M-opsin cones were distributed
homogeneously throughout the retina (Fig. 4.1C). These results indicate that AAA
induces a homogenous mosaic of cones.
Next, we set out to investigate whether cones die in the RP retinas treated with
50g/ml of AAA. The cell number of the M-opsin cones within the 11 mm
2
retinal
areas (see methods) was evaluated. In normal retina, we counted their segments
but in RP retinas; however, due to loss of some cone outer segments (COS) in P33
RP control, we counted their cell bodies. Loss of COS was previously detected in
rd1/rd1 mice (Lin et al., 2009) and S334ter-line-3 rats (Hombrebueno et al., 2010;
Li et al., 2010). In P33 normal and P33 RP control retinas, the mean density of
cells was 4; 921 96 cells/mm
2
and 5; 616 213 cells/mm
2
, respectively. In contrast,
in P33 RP AAA-treated retinas, the M-opsin cone cell density was 3; 539 117
cells/mm
2
. The density was statistically significantly different from the densities of
M-opsin cones from P33 normal and P33 RP control retinas (p = 0.0011, one-way
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 61
Figure 4.1: Confocal micrographs of whole-mounts processed for M-opsin immunohisto-
chemical staining in saline-treated normal (A), saline-treated RP (B), and AAA-treated
(C) RP eyes. Saline and AAA (50g/ml) are injected at P30. AAA-treated RP retinas
show disruption of M-opsin rings after 3 days of injection. The summary graph illustrates
mean cone density (D) measured from the 1 1 mm
2
sampling areas (for details, see
methods) of saline-treated normal (Normal), saline-treated RP (RP) and AAA-treated
RP (RP AAA 3D) retina (n = 3-5 animals per group). The density of the AAA- treated
RP retina was statistically significantly different from the densities of M-opsin cones from
Normal and RP retinas 3 days after injection. Data are presented as mean standard
error. The symbol ? indicates p < 0.05. AAA, DL--aminoadipic acid; P, postnatal; D,
day; N, normal, RP, retinitis pigmentosa, Scale bar = 100 m.
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 62
ANOVA, post-hoc LSD test) (Fig. 4.1D). Our results show that 50g/ml of AAA
can redistribute cones homogenously in RP retina but kills cones after 3 days of AAA
treatment.
In order to minimize the cone death after AAA injection, we used 10g/ml of
AAA. The concentration of AAA was compared to saline and was monitored at
3 days (3D), 2 weeks (2WK), and 6 weeks (6WK) after the injections at P30 RP
(Fig. 4.2). The illustrated whole-mounts were taken from the central part of the
superior (1-mm away from the optic disc) P72 normal, P72 RP control, P33 AAA-,
P44 AAA-, and P72 AAA-treated RP retinas (Fig. 4.2). In P33 normal (data not
shown), P44 normal (data not shown), and P72 normal retinas (Fig. 4.2A), M-opsin
cones were distributed homogeneously throughout the retinas. In P33 RP (data not
shown), P44 RP (data not shown), and P72 RP control retinas (Fig. 4.2B), rings
of cones were observed. In P33 AAA-treated RP retinas, M-opsin cones were dis-
tributed homogeneously throughout the retina (Fig. 4.2C). This result indicates that
3-day duration was long enough for AAA to change the mosaic of cones dramatically
throughout the retina. This change in the mosaic of cones was also observed in 2
weeks (Fig. 4.2D) and persisted until at least 6 weeks (Fig. 4.2E).
In addition, we counted the number of M-opsin cones within 1 1 mm
2
areas
of the same region. In P72 normal and P72 RP control retinas, the mean density
of cells was 5; 315 146 cells/mm
2
and 5; 283 289 cells/mm
2
, respectively. In
P33 AAA-, P44 AAA-, and P72 AAA-treated RP retinas, the mean density of cells
was 5; 333 315 cells/mm
2
, 2; 796 148 cells/mm
2
, and 2; 513 429 cells/mm
2
,
respectively. The density of M-opsin cones in P33 AAA-treated RP retina showed
no significant difference from normal and RP control retinas. In contrast, the density
of M-opsin cones in P44 AAA- and P72 AAA-treated RP retinas showed statistically
significant difference from the first 3 groups (p < 0.0001, one-way ANOVA, post-hoc
LSD test) (Fig. 4.2F). Our results indicate that 10g/ml of AAA can redistribute
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 63
Figure 4.2: Confocal micrographs of whole-mounts processed for M-opsin immunohis-
tochemical staining in saline-treated P72 normal (A), saline-treated P72 RP (B), AAA-
treated P33 RP (C), AAA-treated P44 RP (D), and AAA-treated P72 RP (E) retinas.
Saline and AAA (10g/ml) are injected at P30. AAA-treated RP retinas show disruption
of M-opsin cone rings after 3 days of injection and the disruption of cone rings persist
until 6 weeks. The summary graphs illustrates mean cone density (F) measured from
the 1 1 mm
2
sampling areas of saline-treated normal (N Saline 6WK), saline-treated
RP (RP Saline 6WK) and AAA-treated RP (RP AAA 3D, 2WK and 6WK) retinas (n =
3-5 animals per group). The density of M-opsin cones in AAA-treated RP retina after 3
days showed no significant difference from normal and RP control retinas. In contrast,
the density of M-opsin cones in AAA-treated RP retinas after 2 and 6 weeks showed
statistically significant difference from the first 3 groups. Data are presented as mean
standard error. The symbol ? indicates p < 0.05. P, postnatal; N, normal, RP, retinitis
pigmentosa; WK, week, Scale bar = 100 m.
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 64
cones homogeneously without killing cones in early stage of the treatment.
AAA induces the spatial homogeneity of cone mosaics in RP
HomogeneityofthemosaicandtheexistenceofconeringswasassessedusingVoronoi
analysis (Ji et al., 2015). Voronoi diagram was generated based on the cone mosaic.
An example of Voronoi tessellation is shown in the inset besides the histogram for
each group (Figs. 4.3A-D). The insets in Figure 4.3A illustrate the alternation be-
tween small and large Voronoi domains in the P72 RP control retinas. This alter-
nation was apparently not random, but showed a clustering pattern in that small
domains were surrounded by other small domains, whereas large domains were sur-
rounded by other large domains (Fig. 4.3A). We could quantify the correlation
between the sizes of neighbor domains by calculating the coefficient of clustering
(CC). The CC was the ratio between the global coefficient of variation and the aver-
age local coefficient of variation in Voronoi-domain sizes (Ji et al., 2015). Thus, the
CC highlights that large domains only occur in holes, whereas small domains only
occur in the rims of the rings. Thus, P72, RP control retinas exhibited high CC,
confirming that the spatial alternation between small and large Voronoi domains
was not random. In contrast, in AAA-treated RP groups, the rings disappeared
and cones redistributed themselves homogeneously. The cones spread out to occupy
areas inside the rings and large Voronoi domains became smaller and less skewed
(Figs. 4.3B-E). In order to compare the distribution of Voronoi domains among the
four groups, RP control, P33 AAA-, P44 AAA-, and P72 AAA-treated RP retinas,
we examined both the skewness of the distributions and the CC. The skewness of
the distributions was significantly different from RP control and AAA-treated RP
retinas (p = 0.0055, one-way ANOVA). Post-hoc analysis showed significantly lower
skewness value in RP AAA groups compared to the RP controls at both 3 days and
2 weeks (post-hoc test, = 0.05). This indicated that the Voronoi domains with
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 65
Figure 4.3: Histograms generated from the Voronoi analysis on the 1 1 mm
2
sampling
areas from saline-treated (A) and AAA-treated RP (B-D) retinas. Results are shown
with survival times of 3 D, 2 weeks, and 6 weeks. Examples ( 170m 170m) of
the resulting Voronoi domains are shown for each group. The summary graphs for the
mean skewness values obtained from the Voronoi domain distribution curves are plotted
for each groups (E). Also, the graphs for the mean coefficient of clustering measured in
all groups are illustrated (F). Data are presented as mean standard error. The symbol
? indicates p < 0.05. AAA, DL--aminoadipic acid (AAA); D days; WK, week; RP,
retinitis pigmentosa. Scale bar = 100 m.
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 66
extremely large size are reduced and cones in RP retinas became more homogeneous
with AAA after 3 days. Our results showed statistically significant differences in CC
between RP control and AAA-treated RP retinas (Fig. 4.3F, p = 0.0011, one-way
ANOVA). In summary, AAA induced mosaics of M-opsin cones in RP retinas to gain
homogeneity.
Modulation of ZO-1 Expression with AAA in RP Retina
In our previous study, we found that the interactions between cones and Müller-cell
processes were essential for the maintenance of rings in the RP retina (Lee et al.,
2011). In addition, we found ZO-1 still existed in the network of rings of cones (Lee
et al., 2011). Therefore, we hypothesized that AAA may promote remodeling of the
cone mosaic by unfettering the Müller cells from the cones through the modulation
of ZO-1. Thus, we first explored the behavior of ZO-1 in RP after AAA treatment.
Whole-mounts were labeled with antibodies to M-opsin, GS, a marker of Müller-
cell, and ZO-1. Figure 4.4 shows an example of P50 normal, P50 RP control, and
P50 AAA-treated RP (treated for 3 days) retina whole-mounts processed for these
antibodies. In this figure, the focal plane of the outer limiting membrane (OLM)
was examined. The normal retina treated with saline showed labeled M-opsin cones
segments throughout the photoreceptor array (Figs. 4.4A, D, red). GS immunore-
activity displayed the normal spatially homogeneous mesh network of Müller-cell
processes (Figs. 4.4B, D, green). In the OLM region, ZO-1 was expressed and colo-
calized with GS (Figs. 4.4C, D, blue). Triple labeling of M-opsin, GS, and ZO-1
confirmed that photoreceptor inner segments and the apical processes of Müller cells
had a close association with ZO-1 (Figs. 4.4D). In RP control retinas, we again
observed an array of rings of cones (Fig. 4.4E, H). Furthermore, we observed the
processes of Müller cells formed broccoli-like shapes (Fig. 4.4F, H) and ZO-1 formed
a network of rings (Fig. 4.4G, H). The labeling showed that the ZO-1 also underwent
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 67
Figure 4.4: Confocal micrographs taken from whole-mounts of saline-treated P50 normal
(A-D), saline-treated P50 RP (E-H), and AAA-treated P50 RP (I-L) retinas processed
for M-opsin (red), GS (green), and ZO-1 (blue) show immunohistochemical staining
patterns. Saline and AAA (10g/ml) are injected at P47. The micrographs show P50
N (A-D) retinas 3 days post-saline application. Triple immunohistochemical labeling of
M-opsin (A), GS (B), and ZO-1 (C) shows that ZO-1 is closely associated between cone
inner segments and the apical processes of Müller cells in P50 normal retinas (D). The
micrographs for P50 RP (E-H) retinas show 3 days post-saline application. Triple labeling
of M-opsin (E), GS (F), and ZO-1 (G) shows that the ZO-1 is closely associated with
segments of photoreceptors and processes of Müller cells in ring (H). The micrographs for
AAA-treated P50 RP retinas show 3 days post-application of the drug. Triple labeling of
M-opsin (I), GS (J), and ZO-1 (K) shows that the ZO-1 is no longer expressed between
cones and Müller cells with AAA treatment (L). In addition, Müller cell processes are
homogeneously distributed (J). AAA, DL--aminoadipic acid (AAA); N normal; D days;
RP, retinitis pigmentosa; GS, glutamine synthetase; ZO-1, zonula occludens 1. Scale bar
= 20 m.
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 68
remodeling to allow the processes of Müller cells and cones to establish contact (Fig.
4.4H). In contrast, RP eyes injected with AAA no longer showed rings of cones (Fig.
4.4I) or broccoli-like processes of Müller cells (Fig. 4.4J). Müller-cell processes were
homogeneously distributed with cones (Fig. 4.4L). Furthermore, we also observed
disappearance of ZO-1 expression after 3 days of AAA treatment (Fig. 4.4K). The
disappearance of ZO-1 coincided with the rearrangement of the cones (Figs. 4.4K,
L).
Next, we examined the expression of ZO-1 in vertical sections of RP retinas post-
application of AAA. We observed the effects of ZO-1 after 3 (P33) days, and 2 (P44)
and 4 (P58) weeks of AAA injection at P30. Vertical sections immunolabeled for GS
(Figs. 4.5A, D, G, J, M) and ZO-1 (Figs. 4.5B, E, H, K, N) are shown in Figure
4.5. In normal retinas, Müller-cell processes were present throughout the retina (Fig.
4.5A) and ZO-l immunostaining appeared at the OLM (Fig. 4.5B). In the OLM re-
gion, ZO-1 was expressed and colocalized with GS (Figs. 4.5C arrow). ZO-1 formed
a continuous line at the OLM. In contrast, the RP retina showed discontinuous and
fragmented immunostaining for ZO-1 at the OLM (Figs. 4.5E). Double immunola-
beling of GS (Fig. 4.5D) and ZO-1 (Fig. 4.5E) showed that the ZO-1 expression was
weak and fragmented but still colocalized with GS at the outer part of the retina
(Fig. 4.5F). Interestingly, AAA treatment did not affect the overall expression of GS
(Fig. 4.5G) but suppressed ZO-1 expression after 3 days of treatment (Fig. 4.5H,
I). This disappearance (Fig. 4.5H, I) coincided with the rearrangement of the cones
(Fig. 4.2C). We observed reappearance of ZO-1 (Fig. 4.5K) without affecting the
GS expression (Fig. 4.5J) 2 weeks after the AAA treatment (Fig. 4.5L). This result
persisted through 4 weeks of post-application of AAA (Figs. 4.5M-O). Therefore, we
suggest that disappearance of ZO-1 allows movement of the cones, and the spatial
fixation of the cones occurs following reappearance of ZO-1.
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 69
Figure 4.5: Confocal micrographs of vertical saline-treated P50 normal (A-C), saline-
treated P50 RP (D-F), AAA-treated P33 RP, AAA-treated P44 RP, and AAA-treated
P58 RP retinal sections processed for GS (A, D, G, J, M) and ZO-1 (B, E, H, K,
N) immunohistochemical staining patterns. Double exposure shows that the ZO-1 is
expressed and co-localized with GS at the OLM (C - arrows). Double labeling of GS
(D) and ZO-1 (E) in P50 RP retina shows that the ZO-1 expression is weaker and
more fragmented but still co-localized with GS at the OLM (F). In AAA-treated P33 RP
retina (G-I), GS immunohistochemical staining still appears in Müller cells but ZO-1 is
no longer expressed in outer part of the retina (I). In AAA-treated P44 RP retina, ZO-1
reappeared after 2 weeks of AAA treatment in RP retinas (K, L). In AAA-treated P58
RP retina, GS (M) and ZO-1 (N) consist with 2 weeks of AAA treatment (O). AAA,
DL--aminoadipic acid (AAA); N normal; D days; WK weeks; RP, retinitis pigmentosa;
GS, glutamine synthetase; ZO-1, zonula occludens 1; OLM, outer limiting membrane;
ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner
plexiform layer, GL, ganglion cell layer. Scale bar = 50 m.
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 70
ZO-1 Plays a Critical Role in Shaping the Ring Mosaic in RP
Figure 4.6: Confocal micrographs of whole-mounts processed for M-opsin immunohis-
tochemical staining in non-targeted siRNA for 24 hr (A), ZO-1 siRNA treated for 24 hr
(25M, B), and ZO-1 siRNA treated for 48 hr (25M, C) at P50. ZO-1 siRNA treated
RP retinas show disruption of M-opsin cone rings in both 24 and 48 hrs. RP, retinitis
pigmentosa; ZO-1 zonula occludens 1. Scale bar = 100 m
Figure 4.7: Whole-mounts for M-opsin (red) and ZO-1 (green) immunohistochemical
staining in P50 RP retinas treated with non-targeted siRNA (A-D) and ZO-1 siRNA
(25M, E-H) for 24 hrs. In control retina, ZO-1 is closely associated with segments
of photoreceptors (C, D). In ZO-1 siRNA treated retina, cones are re-occupying the
space homogeneously (E, G) and ZO-1 is suppressed (F, G). D and H are higher-power
micrographs of C, G, respectively. RP, retinitis pigmentosa; ZO-1, zonula occludens 1.
Scale bar= 50m.
To confirm if the modulation of ZO-1 was essential to promote the cone mosaic
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 71
rearrangement, we suppressed ZO-1 expression using ZO-1 siRNA. If use of ZO-1
siRNA leads to the same remodeling of the cone mosaic, it suggest that ZO-1 plays
a critical role in shaping the ring mosaic in RP. The effect of ZO-1 siRNA was
compared to non-targeting siRNA (Fig. 4.6A) and monitored at 24hrs (Fig. 4.6B)
and 48hrs (Fig. 4.6C) after the treatment in P50 RP retinas. The data related to
non-targeting siRNA injection were similar to 24hrs (Fig. 4.6A) and 48hrs (data
not shown). RP eyes injected with non-targeting siRNA showed the aforementioned
arrays of M-opsin cone rings throughout the retina (Fig. 4.6A). In contrast, RP
eyes treated with ZO-1 siRNA showed no rings after 24hrs and 48hrs (Figs. 4.6B,
C). These effects of ZO-1siRNA indicate that ZO-1 between cones and Müller-cell
processes are necessary for the maintenance of rings in the RP retina.
Figure 4.7 showed double immunological labeling of M-opsin and ZO-1 in whole-
mount retinas. In RP retinas treated with non-targeting siRNA for 24hrs, we again
observed an array of rings of cones (Fig. 4.7A). Furthermore, we observed ZO-1 in
a network of rings (Fig. 4.7B). Merged image of M-opsin and ZO-1 showed that the
segments of cones were closely associated with ZO-1 (Figs. 4.7C, D). In ZO-1-siRNA-
treated retina, cones filled the retina homogeneously (Figs. 4.7E, G, H) and ZO-1
was suppressed (Figs. 4.7F, G, H). Thus, inhibition of ZO-1 promoted remodeling
of the cone mosaic by unfettering the Müller cells from the cones.
4.4. Discussion
Mosaics of M-opsin cones can be manipulated by AAA treat-
ment in RP retina
To improve sampling of the visual field by rearranging cones while minimizing cone
toxicity with AAA treatment, we completed a dose response paradigm of intravitreal
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 72
injections of DL--AAA. Within 3 days of AAA applications, we observed a signifi-
cant spatial homogeneity induction of the cone mosaic, which may involve changes in
the integrity of the outer limiting membrane (OLM) (Ishikawa and Mine 1983). This
suggestion is supported by previous studies showing gross retinal changes within the
first 3 days after AAA injection (Chen et al., 1999; Rich et al., 1995b). RP retinas
treated with a higher dose of AAA (50g/ml) showed significant reduction in cone
density after 3 days of AAA injection compared to RP retinas. RP retinas treated
with lower doses of AAA (10g/l) did not show significant changes in the number
of cones in 3 days post-injection (Fig. 4.2). However, there were fewer cones around
2 weeks following AAA injection. This result is consistent with the previous report
showing significant apoptosis in the photoreceptor populations after AAA (Pedersen
and Karlsen, 1979; Qiu et al., 2005). Furthermore, the toxic effects of the drug are
dosage and time-dependent (Rich et al., 1995b). Thus, AAA fails to retain the cone
density even in the lower concentration of the drug that we used. Nevertheless, the
effects of this drug on the distribution of cone mosaic are dramatic. Homogeneity is a
global measurement that quantifies if the spatial distribution of a mosaic is uniform
or not. When a mosaic exhibits rings, the mosaic is not homogeneous, because the
statistics in their rims are different from those in the areas with fewer or no cones
(center of rings). In this study, we used the Voronoi-domain analysis on lower dose
of AAA-treated retina to detect the global homogeneity and existence of holes (Ji
et al., 2015). The Voronoi-domains were larger inside the rings and smaller in the
rims in RP retina (Fig. 4.3A). Such rings became visibly disturbed after 3 days of
AAA treatment (Fig. 4.3B). At 2 weeks and 6 weeks, M-opsin cone cells covered the
space homogeneously (Fig. 4.3C, D). The Voronoi domain analysis confirmed such
observation statistically. The skewness of the Voronoi domain distribution in RP
retinas declined significantly as M-opsin cones started their migration to fill inside
the empty rings with AAA treatment (Fig. 4.3E). Moreover, as the cells moved away
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 73
from the crowded rim of rings, the mean coefficient of clustering decreased signifi-
cantly over time (Fig. 4.3F). However, with AAA treatment, we could not prevent
the cones from dying (Figs. 4.1 and 4.2). Thus, AAA would improve sampling of
the visual field by leading to homogeneity. But at this stage, AAA treatment is
unlikely to be of therapeutic value. It would be of considerable interest to identify
alternative reagents (Ji et al., 2015) or combining with trophic factors that can pro-
vide protection to the residual cones in RP retina (LaVail et al., 1992). We hope to
analyze such potential side effects of AAA with specific neurotrophic factors in the
future. For example, ciliary neurotrophic factor (CNTF) can rescue photoreceptors
in many degenerative retinas (Cayouette and Gravel, 1997; LaVail et al., 1998) and
thus, combining AAA and CNTF may minimize AAA induced toxicity to cones and
Müller cells. This combination may not only preserve cones, but also maintain a
spatially homogeneous mosaic in the later stages of RP. Alternatively, combining
CNTF and brain derived neurotrophic factor for systematic intravitreal injections
after AAA treatment might be an alternative. The combination of these two factors
has demonstrated powerful neuroprotection of photoreceptors in retinal degenerative
retinas (Caffé et al., 2001; Chen and Weber, 2001).
How does AAA cause cones to re-occupy the space homoge-
nously?
Our current study demonstrated that AAA treatment triggered the cone mosaic re-
modeling through ZO-1 (Figs. 4.4-4.7). ZO-1 is present between the inner segments
of rods and cones and the apical processes of Müller cells to form OLM (Pearson
et al., 2010). Pharmacological interference (e.g., AAA) or genetic disruption of the
ZO-1 leads to significant impairment in OLM integrity (van de Pavert et al., 2004;
van Rossum et al., 2006). In the RP retinas, ZO-1 still existed after the remodeling of
cones (Lee et al., 2011). In this study, we observed disappearance of ZO-1 expression
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 74
after 3 days of AAA treatment (Figs. 4.4, 4.5). However, it reappeared 2 weeks after
the AAA treatment (Fig. 4.5). This disappearance coincided with the movement
of the cones (Fig. 4.2C). Therefore, we suggest that suppressed expression of ZO-1
allows the migration of cones and the reappearance of ZO-1 fixed the spatial distri-
bution of cones. Further support for this hypothesis comes from reversible effects of
AAA on ZO-1 and the integrity of OLM (West et al., 2008). These findings suggest
that the ZO-1 represents at least one critical component for cone rearrangement in
RP retina.
Alternatively, AAA may have affected the interaction between cones and Müller
cells through the extracellular matrix (ECM). Müller cells were involved in the mi-
gration of cones by complex interactions with surrounding ECM. In a new study (Ji
et al., 2015), we showed effects of Tissue Inhibitor of Metalloproteinase 1 (TIMP-1)
on cone mosaics, which were similar to those observed in our current study with
AAA. Furthermore, Müller cells played a role in the regulation of expression and
production of TIMP and Matrix Metalloproteinase (MMP) through a feedback sys-
tem involving the ECM (Limb et al., 2002b; Miyata et al., 2012b). Our results were
consistent with this hypothesis, since Müller-processes remodeled with cones in AAA
treated RP retina (Fig. 4.4).
Expression of glutamine synthetase in AAA-treated RP retina
AAAistoxictotheMüllercells(Richetal.,1995b)andcausesselectivereversiblecy-
totoxicityinMüllercellsandbrainglialcellsbothinvivo(Karlsenetal.,1982b;Olney
etal.,1971;Richetal.,1995b)andinvitro(GarthwaiteandRegan,1980;Hucketal.,
1984). Hallmarks usually associated with disruption of Müller cells
´
function include
decreased glutamine synthetase (GS) activity and cellular retinaldehyde-binding pro-
tein immunoreactivity, and increased glial fibrillary acidic protein (Karlsen et al.,
1982b; Linser and Moscona, 1981; Reichelt et al., 1997). However, in this study, we
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 75
did not observe dramatic changes in expression of GS (Figs. 4.4, 4.5). This may
due to the fact that the current concentration of AAA was not high enough to cause
observable changes in expression of GS in Müller cells. Furthermore, Müller cells
appear to recover in the first week after AAA injection (West et al., 2008).
Alternate strategy to reshape the spatial organization of cones
In our recent study (Ji et al., 2015), we have shown that exogenous application of
TIMP-1 can significantly modulate the mosaics and restore its homogeneity without
damaging the cones in the RP retinas. In addition, we suggested that treatment with
TIMP-1 could improve sampling of visual field by improving homogeneity. Here, we
present an alternative strategy to reshape the spatial organization of cones. In ZO-1-
siRNA-treated retinas, cones spread out in the retina homogeneously (Fig. 4.6) after
ZO-1 was suppressed (Fig. 4.7). These results suggested that inhibition of ZO-1 with
ZO-1 siRNA was consistent with the primary mechanism (freeing the Müller cells
from the cones through the modulation of ZO-1) behind the remodeling of the ring
mosaic to homogeneity. Thus, ZO-1 siRNA strategy can be potentially important as
a pharmacologic target to specific human eye diseases caused by retinal dystrophy,
inherited retinal degeneration, and photo-pigment genetic perturbations in M-opsin
cones that shows similar dark patches (i.e. holes) (Carroll et al., 2004; Choi et al.,
2006; Duncan et al., 2007b; Rossi et al., 2011). However, ZO-1 expression needs to
be carefully monitored in order to translate its potential use into a pharmacologic
tool of retinal dystrophy. There are several reasons: (1) higher doses of ZO-1 siRNA
have greater effects on reducing ZO-1 expression, but they may cause significant
photoreceptor death 48 hrs after application (Pearson et al., 2010). (2) ZO-1 also
exists at the tight junctions of the retinal pigment epithelium (Daniele et al., 2007)
and at the composition of tight or adherens junctions around gap-junctional plaques
at the outer plexiform layer (OPL) (Puller et al., 2009). Hence, an intravitreal
CHAPTER 4. THE EFFECT OF AAA ON THE CONE MOSAIC 76
injection of ZO-1 siRNA may also impact these RPE junctions and OPL, causing
side effects on the retina. Alternatively, one can target or explore other adherens
junction associated proteins (e.g., b-catenin) with siRNAs that may have an impact
on the tight junction between cones and Müller cells.
Chapter 5
Future Directions
In Chapter2, I’ve shown that with the extensive photoreceptor remodeling, the most
dramaticfunctionalchangesofRGCswerespatialproperties. InChapter3andChap-
ter4, I’ve shown that both TIMP-1 and AAA could restore the cone mosaic in RP
retinas back to a homogeneous distribution similar as in the normal retinas. It is
interesting to find out whether the treated cones still retain the normal function.
Further more, how the manipulation of cone mosaic could affect the spatial proper-
ties of RGCs. Is it possible that the treatment on cone mosaic could bring receptive
field of RGCs back to normal? To answer these questions, a series of experiments
can be designed and implemented. We could perform ERG recording to measure the
cone function. For RGCs, we could use the similar design and analysis shown in 2.
77
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Acknowledgements
The research included in this dissertation could not have been performed if not for
the assistance, patience, and support of many people. I would like to express my
gratitude first and foremost to my thesis advisor Dr. Norberto Grzywacz for his help
andsupportduringmyPhDstudies. Ibenefitalotnotonlyfromourresearch-related
discussion but also from the general discussion on scientific thinking. I would also
like to thank Dr. Eun-Jin Lee for her support on my thesis projects. She brought me
to understand the importance of disease research. I would additionally like to thank
Dr. Greg Field for providing me the opportunity to use the advanced equipment and
guide me closely along the path.
In addition, thank you for all of my guidance and dissertation committee: Dr.
Judith Hirsch, Dr. James Weiland and Dr. Bart Kosko. Thank you for all the
lab mates and friends: Dr. Xiwu Cao, Dr. Joaquin Rapela, Dr. Junkman Lee,
Dr. Yerina Ji, Dr. Nadav Ivza, Dr. Arvind Iyer, Yun Sung Eom, Sneha Ravi and
Xiaoyang Yao. Also, thanks Denise Steiner for the administrative support.
Last but not least, I would like to extend my deepest gratitude to my parents,
my husband and my parents-in-law. Without your love, support and understanding,
I could never accomplished this much.
97

Abstract (if available)

Abstract Retinitis pigmentosa (RP) is the leading cause of inherited retinal blindness worldwide, having an incidence of 1 per 4000 births each year. Currently, no treatment is available for RP. The onset of RP starts with the death of rods, and it is followed by a slow progressive phase of cone loss. The S334ter-line-3 rat we use is a transgenic model developed to express a rhodopsin mutation which is similar to that found in human RP patients. In this RP model, the death of rods led to the migration of cones, which rearranged themselves into a regular mosaic of rings. In this dissertation, I first showed that while many RGC functional properties are strongly impacted by rod death, functional distinctions between these RGC types remain (Chapter 2). Then, I showed that impact of intravitreous injection of TIMP-1 (Chapter 3) and AAA (Chapter 4) on cone mosaic, followed by elucidating possible mechanism of the manipulation (Chapter 4). Lastly, I briefly described the possible future directions (Chapter 5).

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Creator Yu, Wan-Qing (author)

Core Title Spatial anomalies of visual processing in retinitis pigmentosa and a potential treatment

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 05/05/2017

Defense Date 12/04/2014

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag AAA,OAI-PMH Harvest,retina,retinal ganglion cell,retinitis pigmentosa,TIMP-1

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Advisor Field, Greg (committee chair), Grzywacz, Norberto M. (committee member), Hirsch, Judith (committee member), Lee, Eun-Jin (committee member)

Creator Email wanqingy@usc.edu,wanqingyu@gmail.com

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