Experimental Eye Research 82 (2006) 597–607 www.elsevier.com/locate/yexer
a,
b
d
c,1 a
b,c,
d
a
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA b
Neuroscience Program and Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA c
Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA d
Department of Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, Iowa City, IA 52242, USA
Received 28 April 2005; accepted in revised form 24 August 2005
Available online 5 October 2005
Abstract
We have functionally and morphologically characterized the retina and optic nerve after neural progenitor cell transplants to healthy rat eyes and eyes damaged by acute elevation of intraocular pressure (IOP). Green fluorescent protein-expressing adult rat hippocampal progenitor cells
(AHPCs) were transplanted by intravitreal injection into healthy eyes and eyes damaged with acute ocular hypertension. Pupil light reflexes (PLR) and electroretinograms (ERGs) were recorded preoperatively and postoperatively. Eyes were subsequently prepared for immunohistochemical analysis and confocal imaging.
Transplanted AHPCs were found in 8 of 15 (53%) acute ischemic eyes 62 days after surgery and 5 of 10 (50%) healthy eyes 32 days after grafting. Analysis of PLR and ERG function in acute ischemic eyes revealed no statistically significant difference compared to controls after transplantation for all observed functional parameters. Transplant into healthy rat eyes revealed no PLR or ERG amplitude deficits between transplanted and non-transplanted (control) eyes. Morphological and immunohistochemical analysis revealed that transplanted AHPCs survived and differentiated in both normal and injured retinal environments. Morphological integration occurred primarily within the inner retinal layers of the acute ischemic eyes. AHPCs were found to express neuronal and glial markers following transplantation.
Transplanted AHPCs have the ability to integrate and differentiate in ischemia damaged retinas. PLR and ERG analysis revealed no significant difference in functional outcome in transplant recipient eyes.
q
2005 Elsevier Ltd. All rights reserved.
Keywords: electroretinogram; pupillometry; optic nerve; stem cells; retinal ischemia
1. Introduction
The death of retinal neurons is a hallmark feature of glaucoma, acute ocular hypertension, diabetic retinopathy and other ocular diseases characterized by ischemic insults.
Traditionally, retina and optic nerve damage has been considered irreversible in humans and animals due to the lack of the regenerative capacity of the mammalian central
* Corresponding author. Address: Neuroscience Program and Department of
Genetics, Development and Cell Biology, Iowa State University, Ames, IA
50011, USA. Tel.:
C
1 515 294 3112; fax:
C
1 515 294 8457.
E-mail addresses: sgrozdan@iastate.edu (S.D. Grozdanic), dssakagu@ iastate.edu (D.S. Sakaguchi).
1
Present Address: Department of Biomedical Sciences, Ontario Veterinary
College, University of Guelph, Guelph, Ontario, Canada N1G2W1.
0014-4835/$ - see front matter q
2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.exer.2005.08.020
nervous system (CNS). The retina, as a part of the CNS, is the target of many degenerative diseases with blindness as a final common outcome. Retinal regeneration has been a field of interest for more than 50 years (
Stone, 1950
). Recent studies have shown that some regenerative activity and the potential re-establishment of visual processing can be achieved by different transplantation techniques (
Girman et al., 2003; Lund et al., 2001; Sagdullaev et al., 2003; Woch et al., 2001 ).
However, some transplantation procedures are primarily based on the availability and use of fetal retinal tissue, which is associated with major ethical and practical issues. Alternative strategies are needed in order to provide an unlimited supply of transplantation substrate.
Recent discoveries in the field of neural stem/progenitor cell biology offer new hope for treatment of incurable chronic neurodegenerative diseases (
Kim et al., 2002; Ourednik et al.,
2002
) and also potentially provides an alternative to the use of fetal tissue (
Cameron and McKay, 1998
). It is important to note

598 S.D. Grozdanic et al. / Experimental Eye Research 82 (2006) 597–607 that studies examining the transplantation of ‘neural stem cells’, are likely grafting mixed populations of cells, some of which may be ‘true’ neural stem cells, but that also contain cells that are more differentiated and therefore these cells are best-termed neural progenitor cells.
Neural stem or progenitor cells are primordial cells postulated to give rise to the array of more specialized cells of the CNS (
Cameron and McKay, 1998 ). They are defined by
their ability to differentiate into cells of all CNS lineages
(neurons, oligodendroglia and astroglia), to give rise to new progenitors with similar potential, and to populate developing and/or degenerating CNS regions (
Gage et al., 1995
). These cells have been isolated from adult, developing and embryonic brain and in vitro studies have revealed that they possess the ability to adopt a variety of cellular fates (
Gage et al., 1995;
Shatos et al., 2001; Svendsen et al., 1999
).
Studies in fish and amphibians have shown that new retinal cells are continually formed from retinal progenitor cells during the life of the animal (
Fernald, 1990 ).
Furthermore, probably the most remarkable examples of the potential of stem cells is their ability to regenerate the
CNS and is based on the studies of retina and optic nerve
regeneration in lower vertebrates ( Raymond and Hitchcock,
1997; Reh and Nagy, 1987 ). While identification of
naturally occurring retinal progenitors in adult mammalian eyes has given hope for the existence of regenerative
mechanisms in humans ( Coles et al., 2004; Tropepe et al.,
2000 ) so far there is no evidence of spontaneous retinal
regeneration in injured and/or diseased mammalian eyes.
However, the application of stem cell based therapies might offer the possibility to replace lost neurons or at least protect disease affected neurons in pathological eye conditions. While multiple studies have demonstrated the capability of neural progenitors to survive and morphologically differentiate into neuron-like cells in ischemic (
Guo et al., 2003; Kurimoto et al., 2001 ) and traumatized (needle-
injured) retinas (
Chacko et al., 2000; Nishida et al., 2000 ),
there are only two published reports, which have demonstrated some preservation of visual function after neural
progenitor transplantation in laser-damaged eyes ( Zhang et al., 2004 ) and eyes with retinal degeneration ( Klassen et al., 2004 ). In order to validate the efficacy of stem cell
based therapy it is essential to determine: whether neural progenitors are capable of survival in an environment which is continuously exposed to potential neurotoxic factors such as retinal ischemia and whether transplantation of neural progenitors can provide protection and recovery of compromised retinal neurons.
The principal purpose of this study was to determine whether transplanted green fluorescent protein-expressing adult rat hippocampal progenitor cells (AHPCs) could survive, integrate, differentiate and protect (or recover) function in acute ischemic eyes. Furthermore, we were interested to observe whether transplantation of neural progenitors would interfere with function and morphology of healthy rat eyes.
2. Materials and methods
2.1. Animals
All animal studies were conducted in accordance with the
ARVO Statement for Use of Animals in Ophthalmic and
Vision Research, and procedures were approved by the Iowa
State University Committee on Animal Care. Adult Brown
Norway rats ( n
Z
31) were used in the study.
2.2. Culturing procedures
Adult rat hippocampal progenitor cells (AHPCs) were obtained from F.H. Gage (Laboratory of Genetics, The Salk
Institute). The AHPCs were originally isolated from the brains of adult Fischer 344 rats as reported by
Palmer et al. (1997)
The AHPCs were maintained in polyornithine/laminin-coated tissue culture flasks (T75; Fisher Scientific, Pittsburgh, PA) in at 800 RPM for 4–5 min.
A previously published procedure to generate an ischemia-
reperfusion insult in rats was used ( Grozdanic et al., 2003a,b ).
Briefly, adult Brown Norway rats ( n
Z
21) were anesthetized and the anterior chamber was cannulated with a 25-gauge needle connected to a reservoir containing 0.9% NaCl. The intraocular pressure in experimental eyes was controlled by the height of the reservoir to maintain a pressure of 110 mmHg for
60 min. To prevent potential infection, antibiotic ointment
(neomycin
C polymyxin B
C bacitracin; Bausch & Lomb
Pharmaceuticals Inc; Tampa, FL) was applied topically after the procedure. Ten days after ischemia-reperfusion insult
15 rats received AHPC transplant, while six rats received transplant of non-viable cells (repeatedly frozen and thawed
AHPCs which were exposed to UV illumination for 10 min).
.
Dulbecco’s Modified Eagle’s Medium/Ham’s F12 (DMEM/
F12, 1:1 Gibco BRL, Gaithersburg, MD) supplemented with
N2 (1%; Gibco-BRL), 20 ng/ml basic fibroblast growth factor
(bFGF; Promega Corp., Madison, WI) and
L
-glutamine
(2.5 mM; Gibco BRL). To harvest the AHPCs for in vitro analysis, they were detached from the flasks using ATV solution (Gibco BRL), collected and pelleted by centrifugation
2.3. Transplantation procedure
AHPCs were transplanted by injection using a bevelled glass micropipette attached, via a saline (0.9% NaCl) filled polyethylene tube, to a 20 m l Hamilton syringe. Animals received intraocular injections of AHPCs through the superiorlateral aspect of the pars plana. Two microliter of cell suspension ( w
50,000 cells/ m l) were slowly injected into the vitreal chamber of the eyes. An aliquot of cells used for each transplant session were subsequently plated into sterile culture dishes and visualized using fluorescence microscopy to verify
GFP expression and the viability of the transplanted cells.
2.4. Acute ischemia model in rats

2.5. Healthy rats for histological analysis.
2.6. Functional monitoring
2.6.1. Computerized pupillometry as we previously described (
2004
). Briefly, rats were anesthetized and a light plane of anesthesia was maintained with 1% halothane, 30% NO and
70% O
2
S.D. Grozdanic et al. / Experimental Eye Research 82 (2006) 597–607
Sixty-two days after ischemia-reperfusion insult rats were euthanized and tissue was collected for histological analysis.
Ten adult Brown Norway rats received intravitreal transplant of the AHPCs in the right eye. The left eye was non-injected and served as a control. Thirty-two days after transplantation, rats were euthanized and tissue was collected
The pupil light reflex was evaluated with a custom-made computerized pupillometer (University of Iowa, Iowa City, IA)
Grozdanic et al., 2002, 2003a,
to avoid suppression of the pupil light reflex response as detected with the use of higher doses of anesthetic. The computerized pupillometer was attached to two infrared sensitive CCTV video cameras for simultaneous visual monitoring of both pupils. However, one channel computerized pupillometer was used to record the movement of the pupil from the control (non-operated) eye, while the stimulus light was alternated between the control and operated eye.
paraformaldehyde in 0.1
2.8. Antibodies
M
PO
4
599 buffer. Eyes were removed, post-fixed and then cryoprotected in 30% sucrose in 0.1
M
PO
4 buffer. Tissue was embedded, (Tissue-Tek OCT compound,
VWR International, West Chester, PA) frozen and sectioned coronally at 20–40 m m thickness using a cryostat (American
Optical, Buffalo, NY). Sections were thaw mounted onto
Superfrost microscope slides (Fisher Scientific) and stored at
K
20 8 C until processed.
Specific primary antibodies (see Section 2.8) were used to identify proteins associated with differentiated cell phenotypes.
For antibody staining, tissue sections were washed in phosphate buffered saline (PBS; 137 mM NaCl, 2.68 mM
KCl, 8.1 mM Na
2
HPO
4
, 1.47 mM KH
2
PO
4
) and incubated in blocking solution (5% goat serum; 0.4% bovine serum albumin, BSA; Sigma, and 0.2% Triton X-100, Fisher
Scientific, in PBS). Primary antibodies were diluted in blocking solution and preparations were incubated overnight at 4 8 C in a humid chamber. On the following day the preparations were rinsed with PBS and incubated with an appropriate secondary antibody for 90 min at room temperature. Slides were then rinsed and cover-slipped using
Vectashield Fluorescence (Vector Laboratories, Burlingame,
CA) mounting media.
2.6.2. Electroretinography
To quantify damage to the retina due to chronic elevation of the IOP, a simultaneous recording of electroretinogram from both eyes (control and operated) was performed with two
Ag–AgCl electrodes as previously reported (
Grozdanic et al.,
2002, 2003a, 2004 ). The luminance of the Ganfeld dome
surface was measured with a J17 LumaColor TM photometer equipped with a J1803 luminance head (Tektronix, Willsonville, OR). Measured luminance in our system was 1600
G
200 cd/m
2
. A flash ERG routine was delivered at a 0.2 Hz frequency (10 averaged signals per recording session, sensitivity 100 m V/division, low-cut frequency 0.5 Hz, highcut frequency 10 kHz, analysis time 500 msec). Oscillatory potentials were recorded by delivering light stimuli at a 0.2 Hz frequency (10 averaged signals per recording session, sensitivity 100 m V/division, low-cut frequency 50 Hz, highcut frequency 500 Hz, analysis time 100 msec). Isolated cone responses were recorded from previously light adapted eyes by delivering stimuli at 20 Hz (50 averaged signals per recording session, sensitivity 50 m V/division, low-cut frequency 0.5 Hz, high-cut frequency 10 kHz, analysis time 500 msec).
2.7. Histological and immunohistochemical examination
Immunohistochemical examination was performed as described previously ( after performing functional recordings at the appropriate survival periods, the rats were deeply anesthetized with halothane and
Van Hoffelen et al., 2003
perfused transcardially
). Briefly,
with 4%
Antibodies and concentrations used in this analysis were as follows: anti-microtubule associated protein (MAP) 2ab
(mouse IgG; 1:500; Sigma) was used as a marker of inner retina neurons (ganglion cells and the inner plexiform layer)
(
Caceres et al., 1986
); anti-neurofilament antibody (mouse
IgG; 1:200; Developmental Studies Hybridoma Bank, Iowa
City, IA) was used as a marker of retinal ganglion cell axons and the optic nerve; anti-class III beta tubulin (TUJ1) (mouse
IgG; 1:200; Chemicon International, Temecula, CA) was used as an immature neuronal marker; anti-Calretinin (rabbit;
1:3,000; Chemicon) was used to identify this calcium binding protein which has been used as a marker of a subclass of
horizontal cells, amacrine cells, and ganglion cells ( Massey and Mills, 1999; Volgyi et al., 1997
); anti-synaptic vesicle protein 2 (SV2) (mouse IgG; 1:100; Developmental Studies
Hybridoma Bank) was used to identify the synaptic layers in
the retina ( Buckley and Kelly, 1985 ). Anti-glial fibrillary acidic
protein (GFAP)(mouse IgG; 1:200; ICN Immunobiologicals,
Costa Mesa, CA) was used as a marker of astrocytes and
reactive Mu¨ller glia of the retina ( Debus et al., 1983 ) and anti-
RIP (mouse IgG; 1:100; Developmental Studies Hybridoma
Bank) was used as a marker for oligodendrocytes ( Friedman et al., 1989 ).
After labelling with primary antibodies, the specimens were rinsed with PBS and incubated with secondary antibodies diluted in blocker solution. Antibody and fluorochrome concentrations used in this study were: Alexa 546-conjugated goat anti-mouse IgG (1:200, Molecular Probes), rhodamine isothiocyanate (RITC)-conjugated goat anti-mouse IgG (1:200,
Southern Biotechnology, Birmingham, AL), Cy5-conjugated goat anti-mouse IgG (1:400, Jackson ImmunoResearch),

600 S.D. Grozdanic et al. / Experimental Eye Research 82 (2006) 597–607 biotinylated-horse anti-mouse IgG (1:200, Vector Laboratories) and Streptavidin Cy3 (1:10,000, Jackson ImmunoResearch). All primary and secondary antibodies were diluted in
5% goat serum with 0.4% BSA and 0.2% Triton X-100 in PBS.
2.9. Analysis of tissue sections
Tissue sections were examined with a Nikon Microphot
FXA photomicroscope (Nikon Corp. New York, NY). Adult hippocampal progenitor cells were analysed for their location, morphology and co-localization of antibody markers. Images were captured using a Kodak Megaplus Camera (Model 1.4;
Kodak Corp., San Diego, CA) connected to a Perceptics
Megagrabber framegrabber in a Macintosh computer (Apple
Computer, Cupertino, CA) using NIH Image 1.58VDM
software (Wayne Rasband, NIH, Bethesda, MD). A quantitative analysis was performed to determine the percentage of
GFP-expressing transplanted AHPCs co-expressing a neuronal
(MAP-2) or glial cell (GFAP) marker. Eight sections from eyes displaying transplanted AHPCs were examined (four sections each with MAP-2 or GFAP antibody). Four microscope fields
(300 m m X 600 m m) were examined per section. The number of
MAP-2 or GFAP-immunoreactive (-IR) GFP-expressing
AHPCs was pooled for eyes under each condition (acute ischemic and healthy eyes) and the percentage of MAP-2 or
GFAP expressing AHPCs was calculated.
Some sections were visualized and images captured using a
Lecia TCS-NT confocal scanning laser microscope (Leica
Microsystems, Inc., Exton, PA). As a control, single label studies were performed parallel to the multi-labelling studies to rule out that similar patterns were due to bleed-through and the other fluorescence channels were also examined to ensure that no bleed-through occurred. In addition, negative controls were performed in parallel by omission of the primary or secondary antibodies. No antibody labelling was observed in the controls.
Figures were prepared using Adobe Photoshop version 7.0 and
Macromedia Freehand version 10.0 for the Macintosh.
2.10. Statistical analysis
Statistical analysis was performed by using Student’s t -test,
Paired t -test, Kruskal–Wallis test and One Way ANOVA (as indicated in the text) with the GraphPad (GraphPad, San Diego,
CA) software.
3. Results
3.1. Assessment of retina and optic nerve function in acute ischemic rat eyes after AHPC transplantation
To test whether neural progenitor cells can survive and differentiate after grafting, and to determine whether or not they would improve or diminish the electroretinogram and pupil light reflex, we transplanted AHPCs into adult rat eyes damaged by acute retinal ischemia. Transplanted AHPCs were found in 8 of 15 acute ischemic eyes 62 days after surgery.
Monitoring of PLR amplitudes revealed no significant
Fig. 1. Transplantation and presence of AHPCs in acute ischemic eyes did not significantly improve retina and optic nerve function observed by pupil light reflex (PLR) ratios, compared to eyes, which received non-viable cell transplant. AHPC group: rats which had detectable transplanted cells during histological analysis; AHPC not detected group: rats which received AHPC but did not have detectable cells on histological analysis (separate analysis was performed for all transplanted eyes and there was no significant difference between all transplanted eyes and eyes which received non-viable cells).
PLRratio
Z pupil light reflex ratio between amplitudes recorded from the control (non-operated) eyes after stimulation of the operated, than control eye.
difference between acute ischemic rat eyes in which AHPCs were detected by histological analysis ( n
Z
8) and the control group, which received non-viable cells (
Fig. 1 ). We previously
demonstrated spontaneous, but temporary recovery of some function between 30 and 35 days postoperatively (ERG and
PLRs) in our model of acute retinal ischemia ( Barnett and
Grozdanic, 2004; Grozdanic et al., 2003b ). As such, we wanted
to be certain that possible changes in function were most likely due to the effect of the transplanted cells and not to intrinsic host retinal mechanisms, and therefore, we selected time points identical to the data from our previous studies.
Values for the PLR ratio
(ratio
Z indirect/direct PLR) are illustrated in
Table 1 . There was no statistical significance
between different groups after cell injection ( p O 0.1,
Table 1
Analysis of pupil light reflex amplitudes revealed no difference between different groups that received AHPCs or non-viable, control cells
Time
Preop
9 d
35 d
42 d
60 d
Pupil light reflex ratio (%)
NV AHPC
65.9
G
3.9
4.5
G
2.9*
32.5
G
8.5
17.7
G
8.7
21.3
G
9.1
74.2
G
3.9
20.7
G
8.4
24.2
G
8
21.7
G
7.8
28.1
G
9.4
ND
74.4
G
3.4
26.8
G
3.9
29.6
G
4.9
26.5
G
4.6
36.4
G
6.5
The group of rats that received AHPCs, but did not have histological evidence of viable cells at the end of the experiment had significantly greater amplitudes at 9 days postoperatively (time point before transplantation). NV, group which received non-viable cells; AHPC, group which had cells detected on histology;
ND, group which received AHPC, but no cells were detected on histology.
(* p !
0.05; Kruskal–Wallis test with Dunn’s post-test).

S.D. Grozdanic et al. / Experimental Eye Research 82 (2006) 597–607
Table 2
Analysis of PLR velocity ( D velocity
Z velocity ctrl K velocity operated
) showed no significant difference among different experimental groups ( p O 0.1; Kruskal–
Wallis test with Dunn’s post-test)
TIME
Preop
9 d
35 d
42 d
60 d
D Velocity (mm/s)
NV
0.38
G
0.09
2.4
G
0.1
1.8
G
0.2
2
G
0.4
1.8
G
0.3
AHPC
0.30
G
0.07
1.8
G
0.3
1.9
G
0.3
1.8
G
0.2
1.8
G
0.3
ND
0.35
G
0.04
1.7
G
0.3
1.5
G
0.1
1.4
G
0.1
0.98
G
0.2
NV, group which received non-viable cells; AHPC, group which had cells detected on histology; ND, group which received AHPC, but no cells were detected on histology.
Kruskal–Wallis test with Dunn’s post-test), however there was significant difference between the group of rats which received non-viable cells and the group that received transplanted cells, but did not have detectable cells at the
9 days postoperative time point (time point prior to the transplantation procedure).
We analysed velocity of the PLR ( D velocity
Z velocity ctrl K velocity operated
) and detected no significant difference among the different experimental groups ( p O 0.1; Kruskal–Wallis test with Dunn’s post-test,
Table 2 ).
By subtracting latency time values ( D latency
Z latency time oper K latency time ctrl
) we determined significantly increased latency time deficits at 42 and 60 days post-
operatively (
deficit was significant in the group of rats which received nonviable cells, compared to animals which received transplants but did not have any AHPCs detected histologically at the termination of the experiment ( with Dunn’s post-test). At 60 days, the control transplant group which received non-viable cells ( received AHPCs and had morphologically detectable cells
( p !
Table 3 ). At 42 days postoperatively, the latency
p p
!
!
0.05 Kruskal–Wallis test
0.01) and the group which
0.05), had significantly prolonged latency times when compared to animals which received transplants, but did not
601 have any cells detected histologically (Kruskal–Wallis test with Dunn’s post-test).
Pupillometric analysis of the transplanted acute ischemic eyes revealed no significant beneficial effect on the constriction amplitude. Furthermore, eyes that had morphologically detectable AHPCs had more significant latency deficits at
60 days postoperatively compared to the eyes that received viable cells, but did not have morphological evidence of AHPC survival.
Analysis of scotopic ERG amplitudes (62 days postoperatively) revealed no statistically significant difference between groups that received AHPCs and those animals that received non-viable cell transplants for any of the tested parameters (
Figs. 2 and 3 ).
The latency time of a-waves was not significantly different in AHPC transplanted eyes (50.8
G
0.5 msec) compared to eyes, that received AHPCs but were not histologically detected
(51.3
G
0.6 msec) and which received non-viable cells (50.5
G
1.2 msec, p
Z
0.8, Student’s t -test). The latency time of b-waves was not significantly different in AHPC transplanted eyes (48
G
1.6 msec) compared to eyes, that received AHPCs but were not histologically detected (53.7
G
3.3 msec) and those eyes that received non-viable cells (48.9
G
5 msec, p
Z
0.9, Student’s t -test).
The photopic flicker ERG amplitude ratios were 1.8
G
1.2%
(AHPC eyes), 0% (AHPC transplanted, but not detected) and
22.1
G
13.9% in eyes that received non-viable cells ( p O 0.1,
One Way ANOVA with Bonferroni’s Multiple Comparison
Test). The latency time of the flicker ERG was not significantly different in AHPC transplanted eyes (62.9
G
0.1 msec) compared to eyes that received non-viable cells (61.4
G
1 msec, p
Z
0.09, Student’s t -test). Oscillatory potentials were not detected in any of the experimental groups postoperatively due to severe inner retinal damage that occurs in this model of acute retinal ischemia. While there was no statistical difference in the ERG amplitudes between experimental groups, we observed a trend of better b-wave amplitudes in animals that had morphologically detectable AHPCs.
3.2. Assessment of retina and optic nerve function of healthy rat eyes after AHPC transplantation
Table 3
Analysis of PLR latency ( D latency Z latency ctrl K latency operated
) showed significantly larger latency deficits at 42 and 60 d postoperative time points for the group of animals which received non-viable cells when compared to animals which received AHPCs, but showed no evidence of cell survival
TIME
Preop
9 d
35 d
42 d
60 d
D Latency (msec)
NV
13.8
G
5.1
144
G
16.4
147.1
G
18.4
133.2
G
10.5*
144.1
G
11.8**
AHPC
18.8
G
4.9
137.2
G
15.6
116.6
G
16.2
116.6
G
13.1
121.4
G
12.5*
ND
16.6
G
3.6
114.2
G
14.8
88.9
G
8.4
76.6
G
6
50
G
14.3
Interestingly, rats that received AHPCs, but had evidence of the morphological survival of the transplanted cells, had significantly smaller latency deficits compared to the rats which received AHPCs and had morphological evidence of cell integration. NV, group which received non-viable cells; AHPC, group which had cells detected on histology; ND, group which received AHPC, but no cells were detected on histology. (* p !
0.05; ** p !
0.01; Kruskal–Wallis test with Dunn’s post-test).
To test whether neural progenitor cells can survive and differentiate after grafting, and to determine whether or not they would alter the electroretinogram and pupil light reflex, we transplanted AHPCs into normal healthy adult rat eyes.
Transplanted AHPCs were detected in 5 of 10 transplanted healthy rat eyes 30 days after transplant. As such, our functional analysis focused on this group of animals.
Monitoring of PLR amplitudes revealed no significant difference between preoperative and postoperative values
(
Fig. 4 (A)). Preoperative values for the PLR
ratio
(ratio
Z indirect/direct PLR,
Fig. 4
(A)) were 78.2
G
4.1 (mean
G
S
.
E
.
M
; %).
Fifteen days after transplantation the PLR ratio was 73.3
G
8.6
and was not significantly different compared to preoperative values ( p O 0.1, repeated measures ANOVA with Bonferroni post-test, n
Z
5). Thirty days after transplantation the PLR ratio

602 S.D. Grozdanic et al. / Experimental Eye Research 82 (2006) 597–607
Fig. 2. Electroretinographic analysis of the acute ischemic eyes revealed no functional recovery after AHPC transplant at the end of the experiment. (A–C) ERG analysis of one of the rats which received AHPC transplant and had detected cells histologically (A1 channel—control, non-operated eye, A2 channel—operated and transplanted eye with GFP-positive AHPCs on histological examination); (D–F) ERG analysis of a rat which received non-viable cells in the operated eye (A2 channel). (A), (D) scotopic flash ERG; (B), (E) scotopic oscillatory potentials; (C), (F) photopic flicker ERG.
was 79.3
G
2.3 and again was not significantly different compared to preoperative values ( p O 0.1, repeated measures
ANOVA with Bonferroni post-test).
We analysed latency time and velocity of the PLR and detected no significant decrease of the PLR velocity ( p O 0.1, repeated measures ANOVA with Bonferroni post-test) or the latency time ( p O 0.1, repeated measures ANOVA with
Bonferroni post-test) 15 and 30 days after transplantation.
Calculation of the velocity parameters (velocity ctrl K velocity transpl
) revealed the following values: preoperative
0.26
G
0.04 mm/s (mean
G
S
.
E
.
M
; n
Z
5), 15 days after transplantation the velocity deficit increased 0.46
G
0.03 mm/s, but the difference was not statistically significant when compared to the pre-transplant values ( p O 0.1, repeated measures
ANOVA with Bonferroni post-test). Thirty days post-transplant the velocity was 0.26
G
0.12 ( p O 0.1, repeated measures
ANOVA with Bonferroni post-test).
By subtracting latency time values (latency time oper K latency time ctrl
) we determined the interocular difference in latency values at the following time points: preoperative
Z
20
G
6.2 msec (mean
G
S
.
E
.
M
; n
Z
5), 15 days postoperatively
26.7
G
4.1 msec ( p O 0.1, repeated measures ANOVA with
Bonferroni post-test) and at 30 days 20
G
9.7 msec ( p O 0.1, repeated measures ANOVA with Bonferroni post-test).
Analysis of ERG amplitudes revealed no significant difference between transplanted and non-transplanted (control) eyes 30 days after transplantation (
Figs. 4(b) and 5 ). The
a-wave amplitudes were 191
G
7.3
m V (control eyes) and
163
G
16.9
m V in transplanted eyes ( p
Z
0.24, n
Z
5, Paired t -test). However, latency time of a-waves was significantly prolonged in transplanted eyes (23.9
G
0.74 msec) compared to control eyes (22.4
G
1.81 msec, p
Z
0.0479, Paired t -test). The b-wave amplitudes were 498
G
23.8
m V (control eyes) and
491
G
38.4
m V in transplanted eyes ( p
Z
0.86, n
Z
5, Paired t -test). Latency time of b-waves was not significantly prolonged in transplanted eyes (14 control eyes (14.1
time of 50.8
G
(transplanted eyes,
G
Isolated cone response (flicker ERG; deficits in transplanted eyes 30 days after surgery with average amplitude of 12
G
(transplanted eyes,
1.6
n
0.6 msec,
5.6
m V (control eyes) and 9.6
G n m
Z
V (control eyes) and 50.5
Z
5,
5, p p O
O p
Z
G
0.8 msec) compared to
0.71, Paired
0.1, Paired
0.1, Paired
Fig. 5
t t
(B)) revealed no
-test) and latency
-test).
t -test).
G
2.2
m V
1.8
Functional analysis of the majority of ERG and PLR parameters revealed no dramatic negative effects of the AHPC transplantation in healthy rat eyes. The only exception was minimal, but significantly increased latency time of the a-wave amplitudes, which may be attributed to the reactive host glial response to the presence of transplanted cells (
Fig. 6
(E)).
m V
Fig. 3. Statistical analysis of scotopic flash ERG amplitudes revealed no significant difference ( p O 0.1, One Way ANOVA with Bonferroni’s Multiple
Comparison Test) between operated rat eyes which received AHPC, eyes which received AHPC but cells were not detected histologically and non-viable cell transplant 62 days after acute ocular ischemia was induced.

S.D. Grozdanic et al. / Experimental Eye Research 82 (2006) 597–607 603
Fig. 4. (A) The dynamics of the PLR response after AHPC transplantation into healthy eyes. Monitoring of PLR amplitudes revealed no significant difference between preoperative and postoperative values 15 and 30 days after transplantation. (B) Bar histograms representing combined data for transplanted and control
(non-transplanted) eyes 30 days after transplantation. Difference in amplitudes was not statistically significant.
3.3. Morphological analysis of adult hippocampal progenitor cells following transplantation into the acute ischemic and healthy rat eyes
After the final pupillometry and ERG recordings, the eye tissue was prepared for immunohistochemical analysis (62d postop—acute ischemic eyes, 32d post-transplant—healthy rat eyes). The AHPCs were reliably identified following grafting based on their GFP fluorescence. Green fluorescent proteinexpressing AHPCs were observed throughout the posterior segment of the eye. Limited intra-retinal integration (predominantly inner retinal layers) was observed in acute ischemic rat eyes (
Fig. 6
(A)–(C)). Transplanted AHPCs were primarily found along the inner limiting membrane (ILM), in the vitreous and
around the lens ( Fig. 6 (D)–(F)) in the healthy rat eyes.
Specific antibodies were used to investigate the ability of grafted AHPCs to morphologically differentiate within the environment of transplanted eyes. Co-expression of GFP and one of the phenotypic antibody markers was used to evaluate neural differentiation of the AHPCs.
Subpopulations of GFP expressing AHPCs transplanted into the acute ischemic and healthy environment of the eye expressed the markers for MAP2ab and GFAP. Within the retina, MAP2ab immunoreactivity (-IR) is normally found within neurons of the
inner retina ( Caceres et al., 1986 ). MAP2ab-IR was observed in
AHPCs integrated within the inner retina, within the ILM and
GCL ( Fig. 6 (A) and (D)). In addition, some MAP2ab-IR AHPCs
appeared to have processes extending into the inner plexiform layer (IPL) (
Fig. 6
).
An antibody against GFAP was used to determine if grafted cells expressed this glial marker. GFAP is normally expressed in the astrocytes along the inner retina and in addition is expressed in reactive Mu¨ller glial cells. Extensive GFAP-IR was observed within GFP-expressing AHPCs located within the vitreal chamber, along and in the ILM and in the GCL
(
Fig. 6
(B), (C), (E) and (F)). Many of the GFP-expressing
AHPCs possessed processes that were strongly immunoreactive for the GFAP antibody marker (
Fig. 6 (F)).
As illustrated in
Fig. 6
, extensive GFAP-IR in the radially oriented Mu¨ller glial cells, as well as in the astrocytes along the inner retina was observed in transplanted eyes. The transplanted AHPCs tended to be restricted vitreal to the
ILM in healthy rat eyes and extensive co-expression of GFAP by many AHPCs integrated in the inner retinal layers was
Fig. 5. Original tracings from one of the healthy rats that received AHPC transplant—30 days postoperatively. (A) Scotopic flash ERG (L1-onset of the flash stimulus, L2-peak of the a-wave, L3-peak of the b-wave); (B) photopic flicker ERG (L1-onset of the flash stimulus, L2-L3- amplitude of the photopic flicker wave).

604 S.D. Grozdanic et al. / Experimental Eye Research 82 (2006) 597–607
Fig. 6. Confocal analysis of survival, integration and differentiation of AHPCs transplanted into acute ocular ischemic (A–C) and healthy (D–F) rat eyes. Merged images created by merging confocal images of GFP fluorescence (green) with antibody labeling (red). Expression of Map-2 (A, D) or GFAP (B, C, E, F) by transplanted AHPCs (arrows). Eyes subjected to acute ocular ischemia and subsequent transplantation displayed more AHPCs integrated into the retina (B, C) compared to transplants into healthy eyes (D–F). Transplanted AHPCs were found in and along the inner retina as well as integrated into other retinal layers. Dashed white lines delineate the inner retinal border (inner limiting membrane). White arrows indicate examples of transplanted cells expressing the marker of interest.
Arrowheads in (B) and (E) indicate examples of GFAP-immunoreactive Mu¨ller glia. Abbreviations: OFL, optic (nerve) fibre layer; GCL, ganglion cell layer; IPL, inner plexiform layer; Ret, neural retina; Vit, vitreous; Map-2, microtubule-associated protein-2; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein. Scale bar for (A–E): 50 m m, (F): 30 m m (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
clearly evident in acute ischemic rat eyes (
Fig. 6 (B) and (C)).
Although several other cell-specific antibodies were used in our analysis, only occasional transplanted AHPCs were observed labelled with the neuronal markers anti-neurofilament or anti-SV2 antibodies and no APHCs were observed labelled with antibodies directed against calretinin (neuronal), beta III class tubulin (neuronal), recoverin (photoreceptor) or
RIP (oligodendrocytes).
Morphological analysis revealed a similar percent of survival of transplanted AHPCs in the healthy (50%) and acute ischemic eyes (53%). Immunohistochemical analysis for the neuronal marker (MAP-2ab) revealed that 17% (acute ischemic eyes) and 13% (healthy eyes) of the transplanted
AHPCs were labelled with this neuronal marker. Analysis for the glial marker (GFAP) revealed that 49% (acute ischemic eyes) and 31% (healthy eyes) of the detected AHPC were positively immunoreactive. This immunohistochemical analysis of transplanted AHPCs suggests that the majority of cells were differentiating toward a glial rather then a neuronal lineage.
4. Discussion
A number of studies have examined the survival and morphological integration of transplanted neural progenitor cells in injured or healthy rat retinas (
Chacko et al., 2000; Guo et al., 2003; Kurimoto et al., 2001; Nishida et al., 2000;
Takahashi et al., 1998; Wojciechowski et al., 2002; Young et al., 2000 ). However, to date no studies have performed
detailed functional analysis of transplanted eyes to demonstrate whether transplanted cells integrated in the retinal circuitry or recovered function of eyes damaged by acute elevation of the intraocular pressure.
A previous study reported that transplanted neural progenitors can establish synaptic-like contacts (based on morphological criteria) with host neurons in mechanically injured retinas (
Nishida et al., 2000 ). We were interested to examine
whether transplanted neural progenitors can morphologically integrate, differentiate and recover function in damaged eyes.
Since acute retinal ischemia damaged retinal ganglion cells, we wanted to investigate whether intravitreally transplanted

S.D. Grozdanic et al. / Experimental Eye Research 82 (2006) 597–607 progenitors will predominantly integrate in the RGC layer. In a previous study (
Guo et al., 2003 ), which used a similar model
of acute retinal ischemia and GFP modified AHPCs, the authors demonstrated robust integration in different retinal layers after subretinal injection. However, since retinal ischemic diseases usually affect RGCs and the inner nuclear layer, it was our intention to use an approach which would deliver transplanted cells in close proximity to these layers and avoid possible complications and additional injury (retinal detachment) related to the subretinal delivery of cells. Indeed, the majority of transplanted cells were located in close proximity to the retinal ganglion cells, which is consistent with the possible presence of cues produced in the damaged inner retina which favoured predominant integration in the most damaged layer (RGC layer). Functional analysis using various pupil light reflex parameters (amplitude, velocity and latency time) revealed only significantly smaller latency deficits at 42 and 60d postoperatively in animals which received AHPC transplantation in eyes damaged by acute ischemia, however, amplitude and velocity were not significantly different among different tested groups during the course of the study. The absence of PLR deficits in healthy transplanted rat eyes is highly supportive of the rather passive role of the transplanted neural progenitors in healthy rat eyes, since we observed no disturbances in the retina and optic nerve physiological transmission which would be detected by a potential decrease in any of the tested PLR parameters
(amplitude, latency or velocity) as it is usually the case in conditions where retina and/or optic nerve might be damaged
(
Grozdanic et al., 2003a,b, 2004
).
The electroretinogram is an extracellular response, which arises during retinal activity because cell membranes become hyperpolarized or depolarised in response to photic stimulation. Any pathological event, which may affect electrophysiological properties of retinal cells, will affect the amplitude or the latency time (time between the onset of the light stimulus and the occurrence of electrical signals). We previously demonstrated the presence of significant ERG
deficits in the rat model of acute ocular ischemia ( Grozdanic et al., 2003b
). In this study, we did not detect any significant positive effect of neural progenitor cell transplantation on ERG function in acute ischemic rat eyes. However, transplantation of the neural progenitors in healthy rat eyes caused only mild, but significant a-wave latency time deficits in transplanted eyes
( p
Z
0.0479). Since, we did not detect significant integration of the neural progenitors in the outer nuclear layer it is difficult to speculate that direct contact of transplanted cells interfered with photoreceptor function. Although in some transplant recipient retinas we observed a radial pattern of GFAP-IR, indicative of reactive Mu¨ller glial cell labelling. It has been previously demonstrated that reactive Mu¨ller cells up-regulate production of bFGF (
Harada et al., 2002 ), which can attenuate
the transmission of the photoreceptor response to inner retinal cells (
Gargini et al., 1999 ). There is also a possibility that
transplanted cells induced immune reaction in recipient eyes as previously described in some retina transplantation experiments (
Anosova et al., 2001
). However, recent data revealed
605 that these neural stem/progenitor cells do not express the class
II MHC molecules, essential for the CD4
C mediated rejection of the transplanted tissue (
Hori et al., 2003; Klassen et al.,
2003
), a strong argument against the possibility that immunemediated changes caused the a-wave latency deficits observed in our study.
There are a number of possible explanations for the lack of functional recovery in our study. First, morphological and immunohistological data are highly suggestive that many of the progenitor cells differentiate into cells more characteristic of glia than neurons and this is further supported by lack of expression of the majority of the retinal neuronal markers and synaptic machinery proteins examined in our study. It is possible that differentiation into more neuronal elements may be accomplished in the future using specific growth factors and other signalling molecules that influence the path of cell differentiation. Second, non-modified neural progenitors release very little, if any, neurotrophic factors as previously reported for some other neural progenitors—(
Ourednik et al.,
2002
), which could not only facilitate neuronal differentiation, but also survival and functional recovery of damaged host neurons. Third, the use of neural progenitors of non-retinal origin may contribute to the decreased integration and differentiation capacity in the retinal environment, however, remarkable examples of morphological integration and at least partial differentiation have been described with the use of
neural progenitors of non-retinal origin ( Guo et al., 2003; Van
Hoffelen et al., 2003 ). Multiple studies which used retinal
progenitor transplantation in different models of retinal diseases demonstrated only modest integration and differentiation of the transplanted cells within the host retina (
Chacko et al., 2000, 2003; Coles et al., 2004; Klassen et al., 2004; Yang et al., 2002 ). While some studies have demonstrated the
presence of some molecular markers specific for neurons of retinal lineage, the general morphological appearance of these cells was not consistent with a true retinal neuron morphology.
Furthermore, the paucity of evidence of actual functional properties which could demonstrate that transplanted cells indeed fully differentiated into functional retinal neurons does not support an immediate advantage to restricting one’s use exclusively to retinal progenitors as the only source for retinal transplantation procedures at this time. Indeed, a recent study comparing mouse brain progenitors versus retinal progenitors found that the brain derived progenitors displayed a greater ability to morphologically integrate and respect the host architectural organization of the developing retina compared to the retinal progenitors (
Sakaguchi et al., 2004
). At this time it would seem vital to investigate the abilities of multiple stem and progenitor cell types for the purposes of cell based therapeutic approaches for the retina and CNS.
Until there is evidence to support the hypothesis that transplanted cells may integrate into the retinal circuitry to restore visual function, an alternative goal would be to use transplanted cells as a source for continued delivery of therapeutic substances, as previously demonstrated (
Wang et al., 2002 ). A number of studies have demonstrated the ability
of exogenously applied neurotrophic growth factors

606
5. Conclusion
Although neural progenitor cells were transplanted into rat retina with relative success, their differentiation into neuronal elements was limited. Most integrated into the inner retina and expressed glial cell markers. Monitoring of the retina and optic nerve function of transplant recipient eyes over time by electroretinography and pupillometry did not reveal any significant functional deficit in normal retinas, nor did it reveal any improvement in damaged retinas over the course of this study. Since, the ultimate goal of cell transplant based therapies is the restoration of function, it seems that a functional analysis, and not just the morphological appearance of the tissue, is likely to provide a better representation of the actual condition of the retina and optic nerve after neural stem/progenitor cell transplantation and should be incorporated into future studies of progenitor transplantation.
Acknowledgements
The authors wish to thank Dr F. H. Gage (Laboratory of
Genetics, The Salk Institute) who generously provided the
AHPCs. This work has been supported by an InterInstitutional
Grant from the College of Veterinary Medicine-Iowa State
University and the College of Medicine-University of Iowa, the
Iowa State University Biotechnology Foundation, the Iowa
Agriculture and Home Economics Experiment Station, Ames,
Iowa, project number 3205, was supported by Hatch Act and
State of Iowa Funds, The Glaucoma Foundation, NY, the
National Institutes of Health (NINDS NS 44007), an unrestricted grant from Research to Prevent Blindness (Dept. of
Ophthalmology, University of Iowa), NY and a Merit Review
Grant from the Veterans Administration (R.H.K.). R.H.K. is also a Lew Wasserman Scholar (Research to Prevent
Blindness). A.M.A. was funded by a summer internship from the Iowa State University Program for Women in Science and
Engineering.
S.D. Grozdanic et al. / Experimental Eye Research 82 (2006) 597–607
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