\set{final}

\def\Author{Jiang}
\def\author{jiang}
\def\vol{13}
\def\year{2007}
\def\anum{199}
\def\pages{1783-1792}
\def\txt_title{Intravitreal injections of GDNF-loaded biodegradable microspheres are neuroprotective in a rat model of glaucoma}
\def\txt_authors{Caihui Jiang, Michael J. Moore, Xinmei Zhang, Henry Klassen, Robert Langer, Michael Young}

\def\rcvd{13 August 2007}
\def\accept{21 September 2007}
\def\publ{24 September 2007}
\def\pdfsize{}
\def\PMID{}


\include{mvstyle.hsm}

\| External links
\def\imagej{http://rsb.info.nih.gov/ij}

\| Internal defs
\def\Moller{M\ouml ller}


\article{

\title{Intravitreal injections of GDNF-loaded biodegradable microspheres
are neuroprotective in a rat model of glaucoma}

\authors{\mailto{caihui.jiang@schepens.harvard.edu}{Caihui
Jiang},\sup{1} \mailto{mooremj@tulane.edu}{Michael J. Moore},\sup{1,2,4}
\mailto{xinmei.zhang@schepens.harvard.edu}{Xinmei Zhang},\sup{1}
\mailto{hklassen@uci.edu}{Henry Klassen},\sup{3}
\mailto{rlanger@mit.edu}{Robert Langer},\sup{2}
\mailto{michael.young@schepens.harvard.edu}{Michael Young}\sup{1}}

\institutions{\sup{1}Schepens Eye Research Institute, Department of
Ophthalmology, Harvard Medical School, Boston, MA; \sup{2}Department of
Chemical Engineering, Massachusetts Institute of Technology, Cambridge,
MA; \sup{3}Department of Ophthalmology, School of Medicine, University
of California, Irvine, Orange, CA; \sup{4}Department of Biomedical
Engineering, Tulane University, New Orleans,LA}

\correspondence{Michael Young, Schepens Eye Research Institute,
Department of Ophthalmology, Harvard Medical School, 20 Staniford
Street, Boston, MA 02114; Phone: (617) 912-7419; FAX: (617) 912-0101;
email: michael.young@schepens.harvard.edu}

\abstract

\abs_purpose{To evaluate the efficacy of intravitreal injection of
GDNF-loaded biodegradable microspheres in promoting the survival of
retinal ganglion cells (RGCs) and their axons in a rat model of
chronically elevated intraocular pressure (IOP).}

\abs_methods{Chronic elevation of IOP was induced in Brown Norway rats
through injection of hypertonic saline (1.9 M) into the episcleral
veins. After injection, IOP was measured twice a week in rats using
topical anesthesia. Poly DL-lactide-co-glycolide (PLGA) microspheres
were fabricated using a modified version of the spontaneous
emulsification technique. Two and ten percent of volume solutions of
microspheres loaded with glial cell line-derived neurotrophic factor
(GDNF) were injected into the vitreous cavity of rats with elevated IOP,
with injections of blank microspheres and PBS serving as controls.
Histological analysis was used to quantify surviving RGCs and axons and
provide comparison among different groups. In addition, the thickness of
the retinal inner plexiform layer (IPL) and the level of glial
fibrillary acidic protein (GFAP) expression within the retina and optic
nerve were quantitatively compared.}

\abs_results{IOP was significantly increased in eyes with episcleral
vein injection over untreated eyes (p\lt 0.001) but did not show a
significant difference among groups that received intravitreal
injections of GDNF microspheres, blank microspheres, or PBS (p=0.1852).
The duration of IOP elevation in this experiment was eight weeks.
Expression of GDNF and its receptors localizes to the adult rat RGCs.
Ten percent of the GDNF microsphere treatment significantly increased
RGC survival and axon survival (p\lt 0.001), reduced the loss of retinal
IPL thickness (p\lt 0.001), and decreased glial cell activation in the
retina and optic nerve (p\lt 0.001) compared with blank microspheres and
PBS. In addition, GDNF microsphere treatment moderately reduced cupping
of the optic nerve head.}

\abs_conclusions{Delivery of GDNF via biodegradable microspheres
significantly increased the survival of RGCs and their axons, preserved
IPL thickness, and decreased retina and optic nerve glial cell
activation in an experimental glaucoma model. This study suggests that
GDNF delivered by PLGA microspheres may be useful as a neuroprotective
tool in the treatment of glaucomatous optic neuropathy.}

\introduction

\p{Glaucoma is the second leading cause of blindness worldwide and the
prevalence of this disease is expected to grow as the population ages
[1,2]. In this disease, progressive death of the retinal ganglion cells
(RGCs) leads to optic nerve degeneration and vision loss. Therefore, a
major therapeutic aim is to facilitate the survival of RGCs [3].
Currently, glaucoma treatment relies on pharmacological or surgical
reduction of intraocular pressure (IOP). However, IOP can be difficult
to control and many patients suffer progressive visual field loss
despite what appears to be adequate control of IOP. For these reasons,
it is critical to develop treatment that actively prevents the death of
RGCs, which are at risk in this condition [4-11].}

\p{One approach has been delivery of neurotrophic factors to the inner
retina. Neurotrophic factors have the ability to promote the survival of
neurons and to influence the growth of neurons. Furthermore, retrograde
axonal transport of neurotrophic factors synthesized in target
structures has been specifically associated with RGC survival. For
instance, blocked transport of brain-derived neurotrophic factor (BDNF)
has been observed in glaucoma models and intravitreal injection of BDNF
has been known to increase RGC survival. Therefore, neurotrophic factor
deprivation has been proposed as one mechanism leading to RGC death in
glaucoma [12-17]. In addition, the growing recognition that glaucoma is
a form of optic neuropathy suggests that neuroprotection, therapy
directed at preventing neuronal loss, may represent an efficacious
adjunctive therapy in this setting [5].}

\p{Glial cell line-derived neurotrophic factor (GDNF) is a 20 kDa
glycosylated homodimer belonging to the TGF-\beta super family that was
first recognized for its ability to increase the survival of
dopaminergic neurons in animal models of Parkinson disease [18]. Recent
work has established that GDNF signals directly through the cell surface
receptor, GFR-\alpha, and indirectly through the transmembrane Ret
receptor, tyrosine kinase [19]. Both receptors have been identified on
embryonic chick RGCs as well as on amacrine and horizontal cells [20].
Exogenous GDNF also increased RGC survival in axotomized rats and in
mice following liquid injection and adenoviral transmission [21-24].
Studies in our laboratory showed that intravitreal microsphere-delivered
GDNF significantly increased long-term RGC survival in the DBA/2J mouse
glaucoma model [25].}

\p{Effective neuroprotection in glaucoma likely requires the consistent
availability of the active agent, such as GDNF, for prolonged periods of
time. Biodegradable microspheres are especially attractive as drug
delivery vehicles for several reasons. First, they are relatively inert
in the vitreous cavity, inciting only a minimal host immune response.
Furthermore, they can be formulated in ways so as to alter the duration
and magnitude of drug release. In addition, they can be reproduced with
a high consistency and at a low cost [25-27]. In this study, we induced
chronic elevation of IOP in rats through episcleral vein injection of
hypertonic saline [28,29] and used this model to investigate the
neuroprotective activity of Poly DL-lactide-co-glycolide (PLGA)
biodegradable microsphere-delivered GDNF on the survival of RGCs and
their axons.}

\methods

\subsection{Animals}

\p{All experiments were performed in compliance with the ARVO Statement
for the Use of Animals in Ophthalmic and Vision Research, and all
experimental protocols were approved by the Animal Care and Use
Committee of the Schepens Eye Research Institute. Adult male Brown
Norway rats (350-450 g, Charles River Laboratories, Wilmington, MA) were
housed with a 12 h light/dark cycle and with water and food provided
\i{ad libitum}. All rats were acclimatized to the environment and to IOP
measurements for at least two weeks before experiments.}

\subsection{Intraocular pressure elevation}

\p{Unilateral elevation of IOP was produced in adult male Brown Norway
rats as previously described by Morrison [28,29]. In brief, anesthesia
was induced by intraperitoneal injection consisting of ketamine (75
mg/kg), xylazine (10 mg/kg), and acepromazine (2 mg/kg; all from Phoenix
Pharmaceutical Inc., St. Joseph, MO). Hypertonic saline (1.9 M) was
injected into the episcleral vein of the left eye through a glass
micro-needle (generated in a pipette puller) while the right eye served
as a normal control. A syringe pump (Harvard apparatus, Holliston, MA)
was used to standardize the injection pressure, volume, and duration.
Two weeks later, the injection procedure was repeated on a second
episcleral vein on the opposite side of the same eye in all treated
animals.}

\subsection{Intraocular pressure measurement}

\p{IOP was measured using a TonoPen XL tonometer (Medtronic Ophthalmics,
Jacksonville, FL). Baseline IOP was obtained before the first hypertonic
saline injection and twice per week thereafter. All IOP measurements
were performed in conscious rats using only topical corneal anesthesia
(0.5% Proparacaine Hydrochloride, Akorn Inc. Buffalo Grove, IL). Fifteen
consecutive readings were taken, and the average served as the measured
IOP. In addition, all IOP measurements were performed around 2:00 PM to
minimize fluctuations due to diurnal variability [30,31].}

\subsection{Fabrication of microspheres}

\p{Microspheres containing GDNF were fabricated using a modification of
a spontaneous emulsion technique described previously [32]. To
summarize, 200 mg of 50:50 PLGA (DURECT Corp., Birmingham, AL) was
dissolved in 5 ml of a solution containing trifluoroethanol and
dichloromethane at a volume ratio of 4:1. Additionally, 8 mg magnesium
hydroxide was added to minimize protein aggregation during
encapsulation. As obtained from the manufacturer, 10 \mu g GDNF (R\and D
Systems, Minneapolis, MN) was reconstituted in 300 \mu l of a solution
containing 7 mg bovine serum albumin (BSA) and 100 mg docusate sodium
(Sigma-Aldrich, St. Louis, MO) dissolved in 3 ml PBS. The two solutions
were vortexed briefly, forming a fine emulsion that was poured into 200
ml of gently stirring 1% (w/v) polyvinyl alcohol (PVA, 88% hydrolyzed).
After stirring for three hours to allow microspheres to harden, they
were collected via centrifugation and washed three times to remove
residual PVA. The microspheres were then rapidly frozen in liquid
nitrogen, lyophilized for 72 h, and stored in a dessicator at -20 \deg
C. Blank microspheres were made in the same way except that additional
BSA was added in lieu of GDNF. Resulting GDNF and blank microspheres
exhibited average diameters of approximately 8 \mu m as determined by a
particle size.}

\subsection{Intravitreal injection}

\p{One week after the first hypertonic saline injection, eyes were
randomly chosen to receive intravitreal injections of either blank
microspheres, microspheres loaded with GDNF, or PBS alone. Intravitreal
injection was performed under general anesthesia using an ophthalmic
operating microscope (\Moller-Wedel GmbH, Wedel, Germany) and beveled
glass micro-needles with an outer diameter of approximately 100 \mu m.
Two and ten percent of (w/v) suspensions of microspheres were prepared
in PBS and briefly vortexed immediately before each injection to ensure
a uniform dispersion of microspheres in the injected fluid. A 30-gauge
hypodermic needle was used to perforate the sclera 1.5 mm behind the
limbus. Five microliters of test sample was then injected by way of this
passage into the vitreous using a 50 \mu l Hamilton Syringe (Hamilton
Co, Reno, NV). Care was taken not to damage the lens. Following
intraocular injections, the needle was held in place for one min and
withdrawn slowly. In addition, paracentesis was simultaneously performed
to relieve pressure and thereby prevent reflux. Animals with retinal
bleeding or lens injury following the injection procedure were excluded
from the study.}

\p{Microspheres labeled with the fluorescent marker rhodamine were
injected into the vitreous to verify the injection technique. Fundus
images were examined in vivo using epifluorescence microscopy.}

\subsection{Tissue preparation and histopathology}

\p{Rats were killed by CO\sub{2} inhalation eight weeks after the second
hypertonic saline injection. Enucleated eyes were immediately fixed in
4% paraformaldehyde (PFA). Optic nerve specimens were taken 1 mm
posterior to the globe and placed into a fixative consisting of 2.5%
glutaraldehyde and 2% formaldehyde in PBS.}

\p{The eyes, together with an additional subset of optic nerve (ON)
segments were fixed in 4% PFA overnight, cryoprotected in serial sucrose
solution, frozen in optimal cutting temperature compound (Tissue-Tek,
Miles Diagnostic Division, Elkhart, IN), sectioned in their entirety at
10 \mu m, mounted on Superfrost Plus slides (VWR Scientific, West
Chester, PA), and stored at -80 \deg C for further study.}

\p{ON segments were washed in 0.1 M cacodylate buffer and post-fixed in
2% aqueous osmium oxide. The segments were then dehydrated in graded
alcohols and embedded in epon. One micrometer section was cut and
stained with 1% toluidine blue in 1% borate buffer. Optic axons were
counted in nine fields per ON cross section viewed at 1000X
magnification. Axon survival percentage was calculated based on the
number of ON axons in elevated IOP eyes divided by the number of axons
in untreated contralateral eyes.}

\p{For immunohistochemical analysis, rabbit anti-GDNF antibody (1:100),
goat anti-GFR\alpha-1 antibody (1:50), goat anti-GFR\alpha-2 antibody
(1:50), goat anti-Ret antibody (1:50), goat anti-CD45 (1:100; all from
Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse anti-Neuronal
Nuclei (1:100, Chemicon, Temecula, CA), mouse anti-Neurofilament 200KD
(1:100, Chemicon, Temecula, CA), and rabbit anti-GFAP (1:200,
Invitrogen, Carlsbad, CA) were used on retina and ON frozen sections.
FITC-conjugated AffiniPure Goat anti-Mouse IgG (1:200), FITC-conjugated
AffiniPure Goat anti-Rabbit IgG (1:200), FITC-conjugated AffiniPure
Mouse anti-Goat IgG (1:200), FITC-conjugated AffiniPure Goat anti-Mouse
IgG (1:200), Cy3-conjugated AffiniPure Donkey anti-Goat IgG (1:800),
Cy3-conjugated AffiniPure Goat anti-Mouse IgG (1:800), and
Cy3-conjugated AffiniPure Goat anti-Rabbit IgG (1:800; All from Jackson
ImmunoResearch Laboratories, Inc., West Grove, PA) were used as
secondary antibodies. Negative controls for immunostaining consisted of
substituting normal serum in place of the primary antibodies.}

\p{Anti-NeuN positive cells in the RGC layer were counted from the
superior aspect of an average of five vertical sections through the
optic disc for each eye. A grid was placed over the retinal section and
the mean number of NeuN+ cells in the equivalent area of RGC layer from
each eye was calculated.}

\p{The fluorescence intensity of glial fibrillary acidic protein (GFAP)
expression in the retina and optic nerve cross section was measured by
\hot{\imagej}{ImageJ}.}

\subsection{Statistical Analysis}

\p{The data were expressed as means\pom SD. Data between groups was
compared using Student's t-test. The significance of RGC and axon rescue
was assessed by one-way ANOVA. Statistical significance was declared at
p\lt 0.05.}

\results

\subsection{Hypertonic saline injection elevates intraocular pressure}

\p{Normal control eyes in Brown Norway rats had an average IOP of
21.2\pom 3.6 mmHg. The saline injection method resulted in significant
IOP elevation at all time points (p\lt 0.001) with pressure levels
consistently approaching double that of control eyes by the third week
(\figref{1}). Overall IOP exposure (in mmHg-days, defined as the
integral of the IOP difference between the experimental and control eyes
during the follow-up) was not significantly different between treatment
groups which are those that received intravitreal injection of GDNF
(\figref{1}{B,C}), blank microspheres (\figref{1}{D}), or PBS
(\figref{1}{E}) on the first week (p=0.1852). The IOP elevation seen in
this study was sustained up until the end of the experiment at the 10
week time point, eight weeks beyond the second hypertonic saline
injection.}

\subsection{Injection technique delivers microspheres to the vitreal
cavity}

\p{The technique used for intravitreal injection of microspheres was
verified by in vivo fundus imaging. Injected microspheres were
visualized in the vitreous cavity, posterior to the lens, immediately
following injection. Diffusion of microspheres throughout the vitreous
was evident from using rhodamine-labeling four days and 10 days after
injection. These microspheres could still be observed 30 days later
after injection (data not shown).}

\subsection{GDNF, GFR\alpha-1, GFR\alpha-2, and Ret are expressed in the
rat retina}

\p{Immunoreactivity for GDNF was mainly detected in the ganglion cell
layer, inner nuclear layer, and inner segment of photoreceptors
(\figref{2}{A,D,G}). Immunoreactivities for GFR\alpha-1 and Ret were
mainly localized to the ganglion cell layer (\figref{2}{B,H}) while
immunoreactivities for GFR\alpha-2 mainly localized to the ganglion cell
layer and inner nuclear layer (\figref{2}{E}).}

\subsection{GDNF microspheres reduce retinal damage due to chronic
intraocular pressure elevation}

\p{Chronic IOP elevation resulted in substantial cupping of the optic
nerve head (ONH; \figref{3}{D,E}) compared with normal ONH architecture
(\figref{3}{A}). Ten percent of GDNF microspheres (\figref{3}{B})
reduced ONH excavation and 2% GDNF microspheres (\figref{3}{C})
moderately reduced the ONH excavation compared with either blank
microspheres (\figref{3}{D}) or PBS alone (\figref{3}{E}).}

\p{Anti-neurofilament 200 labeling was used to evaluate nerve fiber
layer (NFL) thickness in the vicinity of the optic disc. Compared to
normal rat eyes (\figref{3}{F}), eyes with chronic IOP elevation and
treated with blank microspheres (\figref{3}{I}) or PBS (\figref{3}{J})
showed substantially diminished NFL thickness. Ten percent of GDNF
microspheres (\figref{3}{G}) decreased the degree of NFL loss whereas 2%
GDNF microspheres (\figref{3}{H}) did not preserve the NFL to the same
extent.}

\p{Chronic IOP elevation also resulted in substantially diminished
retinal thickness (\figref{3}{L,M,N,O}) compared with normal retinal
architecture (\figref{3}{K}). The average thickness of the retinal inner
plexiform layer (IPL) in normal animals was 108.4\pom 4.9 \mu m, and
chronic IOP elevation resulted in a significant loss of IPL thickness
(\figref{3}{P}; p\lt 0.001). GDNF microspheres were associated with
significant preservation of the IPL with a thickness of 78.5\pom 3.8 \mu
m seen following 10% GDNF microspheres. In comparison, 2% GDNF
microspheres resulted in an IPL of 57.9\pom 3.3 \mu m thickness, which
was statistically greater than the 52.7\pom 2.7 \mu m seen with PBS
treatment alone (p\lt 0.01) but not significantly greater than blank
microspheres (p\gt 0.05).}

\subsection{GDNF microspheres increase retinal ganglion cell survival}

\p{The effects of chronic IOP elevation and GDNF microspheres on RGC
survival are shown in \figref{4}{A-E} and \figref{4}{P}. Eight weeks of
IOP elevation resulted in substantial loss of RGCs as labeled with the
anti-neuronal nuclear antibody NeuN (\figref{4}{B-E}) when compared to
eyes without IOP elevation (\figref{4}{A}).}

\p{The effects of GDNF microspheres on RGC survival were quantified by
counting anti-NeuN positive cells in the ganglion cell layer. In normal
eyes without IOP elevation, the average number of anti-NeuN positive
cells in the GCL was 64.2\pom 3.6/mm. Eight weeks of IOP elevation
resulted in a significant loss of the RGCs: 23.1\pom 2.1/mm with blank
microsphere treatment and 22.2\pom 2.4/mm with PBS treatment. GDNF
microsphere treatment (started one week after the first hypertonic
saline injection) resulted in dose-dependent preservation of RGCs:
41.1\pom 2.2/mm with 10% GDNF microspheres (25 ng/eye) compared with
25.9\pom 2.6/mm with 2% GDNF microspheres (5 ng/eye).}

\p{Chronic IOP elevation resulted in significant loss of RGCs compared
to uninjected control eyes without IOP elevation (p\lt 0.001). Ten
percent of GDNF microspheres significantly increased RGC survival
compared with either 2% GDNF microspheres, blank microspheres, or PBS
treatment (p\lt 0.001). Two percent of GDNF microspheres resulted in
significant preservation of RGCs compared with PBS treatment (p\lt
0.05). There were no significant differences between groups treated with
2% GDNF microspheres versus blank microspheres or with blank
microspheres versus PBS (p\gt 0.05).}

\subsection{GDNF microspheres increase optic nerve axon survival}

\p{The effects of chronic IOP elevation and GDNF microspheres on optic
axon survival are shown in \figref{4}{F-O} and \figref{4}{Q}.
\figref{4}{F} shows a semi-thin cross section of a normal ON with axons
stained using 1% toluidine blue. After eight weeks of IOP elevation,
there was evidence of substantial axon degeneration (\figref{4}{G-J})
compared with untreated normotensive controls (\figref{4}{F}). Axonal
degeneration can be identified by the appearance of swollen axons that
lack apparent axoplasm as well as axons that appear dark due to the
collapse of the myelin sheath. Degenerating axonal profiles such as
these occupied nearly the entire mass of the ON in eyes treated with
blank microspheres or PBS alone (\figref{4}{I,J}).}

\p{The effects of GDNF microspheres on axon survival were quantified by
counting intact axonal profiles (\figref{4}{Q}). The percentage of axon
survival was calculated from the number of axons in elevated IOP eyes
compared to the number of axons in the contralateral eye without IOP
elevation. The survival percentage was 61.58% with 10% GDNF microspheres
compared to 38.56% with 2% GDNF microspheres, 35.25% with blank
microspheres, and 33.12% with PBS alone (p\lt 0.001). Two percent of
GDNF microspheres were associated with significantly increased axonal
survival compared with PBS treatment (p\lt 0.05). However, there were no
significant differences comparing 2% GDNF to blank microspheres or
comparing blank microspheres to PBS alone (p\gt 0.05).}

\subsection{GDNF microspheres decrease GFAP expression of retina and
optic nerve}

\p{GFAP expression was mainly localized to the inner limiting membrane
in normal retina (\figref{5}{A}). Chronic IOP elevation resulted in
significantly increased GFAP expression in the retina (\figref{5}{B-E})
and optic nerve (\figref{5}{G-J}) compared with untreated control eyes
(\figref{5}{A,F}; p\lt 0.001). Ten percent of GDNF microspheres
significantly decreased the IOP-induced GFAP overexpression in both the
inner retina (\figref{5}{P}; p\lt 0.001) and optic nerve (\figref{5}{Q};
p\lt 0.001) while 2% GDNF microspheres were more difficult to
distinguish from treatment with blank microspheres and PBS alone (p\gt
0.05).}

\discussion

\p{This study demonstrates that intravitreal GDNF, delivered by way of
biodegradable PLGA microspheres, results in significant sparing of
retinal ganglion cells in a rat model of glaucoma. This phenomenon
includes preservation of IPL thickness as well as the sparing of optic
axons both within the nerve fiber layer of the retina and within the
orbital portion of the optic nerve. This sparing effect is dependent
upon the dose of GDNF microspheres used and is not replicated by blank
microspheres or injections of PBS alone. The glial activation that
accompanies IOP-associated injury in the inner retina and ON is also
mitigated by treatment with GDNF-laden microspheres, as evidenced by
dose-dependent suppression of GFAP expression in these regions, further
underscoring the beneficial effects of the treatment strategy used
here.}

\p{The inner retinal sparing seen in the present study can be attributed
to the neuroprotective effects of GDNF together with the sustained drug
delivery properties conferred by the incorporation of peptides in PLGA
microspheres. While the mechanism underlying the effects of GDNF on RGCs
has yet to be entirely delineated, it is known that both endogenous and
exogenous neurotrophic factors have neuroprotective effects on cells of
the rodent retina. With respect to retinal ganglion cells, there is
evidence to suggest that retrograde transport of neurotrophins from
retinorecipient regions may be essential for survival [12,13].
Furthermore, obstructed axonal transport of BDNF has been observed in
glaucoma models, and intravitreal injection of BDNF increases RGC
survival [14-17]. Adult rat RGCs are capable of taking up exogenous GDNF
placed in the superior colliculus and of retrogradely transporting this
molecule to their cell bodies in the inner retina [21]. Both the
GDNF-binding GFR\alpha\ surface receptor and transmembrane Ret receptor,
tyrosine kinase, are expressed by RGCs as well as other retinal neurons
[19,20]. GDNF and the GDNF receptor complex are present in the human
optic nerve head [33]. Studies have shown that GDNF interacts with a
GPI-linked cell surface receptor, GFR\alpha. In turn, GFR\alpha\
together with bound GDNF interacts with the tyrosine kinase receptor,
Ret. The binding of GDNF to the cell surface receptor activates the Ret
tyrosine kinase [34,35]. Available data suggest that GDNF must interact
with GDNF\alpha\ initially, after which it may interact with Ret
directly. In the present study, we have provided evidence that the
expression of GDNF and its receptors localizes to the RGCs of the adult
rat.}

\p{Effective neuroprotection in glaucoma likely requires the consistent
availability of the active agent for prolonged periods of time.
Neurotrophic factors present in the vitreous humor are rapidly degraded
by free extracellular proteases including any released as a consequence
of RGC degeneration. Additionally, neurotrophic factors may be taken up
and degraded in the retina by resident microglia. Repeated injections of
unprotected neurotrophic factors over the life of the patient might not
be sufficient to consistently confer a significant visual advantage and
could be expected to result in an unacceptable rate of serious
complications such as retinal detachment and endophthalmitis.}

\p{One strategy for improving the sustainability of peptide compounds in
vivo is to protect them from endogenous proteases and endocytotic
activity via incorporation into biodegradable polymers. Much progress
has recently been made in the field of polymeric drug delivery with
biodegradable microspheres emerging as one promising platform for use in
chronic neurodegenerative diseases [36-38]. Previous analysis of the
microspheres used in the current study indicated a cumulative GDNF
release of 35.4 ng/mg over 71 days in vitro [25]. This delivery is not
ideal, yet it represents a significant improvement over previous
examples. In particular, the burst release of GDNF in the first two days
was only 59% for these microspheres compared with the previous reported
burst release of 98% over the same interval [27]. More importantly, this
delivery profile provides sufficiently sustained delivery of GDNF to
protect mammalian RGCs in vivo as demonstrated here and in previous
studies [25].}

\p{GDNF selectively enhances the survival and development of
dopaminergic neurons with an ED\sub{50} of 40 pg/ml in vitro [18],
however, it can be difficult to determine the optimal in vivo dosages
for an experimental drug therapy such as the microsphere delivery
system. Previous studies have demonstrated significant rescue of
axotomized RGCs following treatment with 1 \mu g of GDNF in adult rats
[21,22]. However, phase I clinical trials have shown that high doses of
neurotrophins can be associated with significant side effects, including
chronic pain [39]. An advantage of a slow release system is the ability
to deliver low levels of the drug that achieves therapeutic relevance
via continuous presence in the target microenvironment. Furthermore, the
considerable expense of neurotrophic factors means that the slow-release
of small doses is more economically feasible. A previous study in our
laboratory has demonstrated that 1 \mu l of a 2% suspension of GDNF
microspheres (total theoretical release of GDNF=0.707 ng) significantly
increased long-term RGC survival in the DBA/2J mouse glaucoma model
[25]. In the present study, using a rat glaucoma model, we have shown
that the rescue effect of intravitreally administered biodegradable
microsphere-loaded GDNF is dose-dependent. Further dosage analysis of
microsphere-delivered neurotrophins in the vitreous would be a helpful
step toward achieving maximum pharmacological effect.}

\p{Increased expression of GFAP by glial cells is a nonspecific marker
of neuronal injury [40], and glial cell activation has been proposed as
an important factor contributing to RGC death in glaucoma [41,42]. In
the normal retina, GFAP expression is mainly localized to the vicinity
of the inner limiting membrane and nerve fiber layer. As with a variety
of insults, we found that chronic IOP elevation resulted in
significantly increased GFAP expression in the rat retina and the
retro-orbital ON, and the treatment with GDNF microspheres significantly
diminished this response in a dose-dependent fashion. While the
mechanism underlying this phenomenon remains to be revealed, it is
reasonable to note the potential association between the decreased
expression of GFAP seen following treatment with GDNF-containing
microspheres and the preservation of RGCs and their axons.}

\p{In summary, this study shows that PLGA microsphere-delivered GDNF
represents an important neuroprotective strategy in the experimental
treatment of glaucomatous optic neuropathy. Further studies will be
important to demonstrate the functional consequences of this treatment
and to extend this work to large animal models.}

\acknowledgements

\p{The authors thank Lenny Li, Scott Schmitt, and Julia Senior for help
with counting retinal ganglion cells, Dr. Morrison at Casey Eye
Institute, Oregon Health and Sciences University, for sending us very
useful instrumentation and instructions on how to produce this glaucoma
model, Dr. Grosskreutz at Mass Eye and Ear for allowing us to visit her
laboratory to gain first-hand knowledge of skills related to this rat
model, Dr. Calkins at Vanderbilt Eye Institute for advice on
histopathological processing of optic nerve axons, Gail and Richard
Siegal for their generous support of this study through the Richard and
Gail Siegal Foundation, the Minda de Gunzburg Center for Retinal
Transplantation, and the Lincy Foundation.}

\references

\p{1. Quigley HA. Number of people with glaucoma worldwide. Br J
Ophthalmol 1996; 80:389-93. \pubmed{8695555}}

\p{2. Quigley HA, Broman AT. The number of people with glaucoma
worldwide in 2010 and 2020. Br J Ophthalmol 2006; 90:262-7.
\pubmed{16488940}}

\p{3. Kuehn MH, Fingert JH, Kwon YH. Retinal ganglion cell death in
glaucoma: mechanisms and neuroprotective strategies. Ophthalmol Clin
North Am 2005; 18:383-95,vi. \pubmed{16054996}}

\p{4. Levin LA. Retinal ganglion cells and neuroprotection for glaucoma.
Surv Ophthalmol 2003; 48:S21-4. \pubmed{12852431}}

\p{5. Levin LA. Neuroprotection and regeneration in glaucoma. Ophthalmol
Clin North Am 2005; 18:585-96,vii. \pubmed{16314221}}

\p{6. Ritch R. Complementary therapy for the treatment of glaucoma: a
perspective. Ophthalmol Clin North Am 2005; 18:597-609.
\pubmed{16314222}}

\p{7. Ritch R. Neuroprotection: is it already applicable to glaucoma
therapy? Curr Opin Ophthalmol 2000; 11:78-84. \pubmed{10848224}}

\p{8. Osborne NN, Chidlow G, Layton CJ, Wood JP, Casson RJ, Melena J.
Optic nerve and neuroprotection strategies. Eye 2004; 18:1075-84.
\pubmed{15534592}}

\p{9. Thanos C, Emerich D. Delivery of neurotrophic factors and
therapeutic proteins for retinal diseases. Expert Opin Biol Ther 2005;
5:1443-52. \pubmed{16255648}}

\p{10. Quigley HA. New paradigms in the mechanisms and management of
glaucoma. Eye 2005; 19:1241-8. \pubmed{15543179}}

\p{11. Weinreb RN, Levin LA. Is neuroprotection a viable therapy for
glaucoma? Arch Ophthalmol 1999; 117:1540-4. \pubmed{10565524}}

\p{12. Pearson HE, Stoffler DJ. Retinal ganglion cell degeneration
following loss of postsynaptic target neurons in the dorsal lateral
geniculate nucleus of the adult cat. Exp Neurol 1992; 116:163-71.
\pubmed{1577124}}

\p{13. Schulz M, Raju T, Ralston G, Bennett MR. A retinal ganglion cell
neurotrophic factor purified from the superior colliculus. J Neurochem
1990; 55:832-41. \pubmed{2200848}}

\p{14. Quigley HA, McKinnon SJ, Zack DJ, Pease ME, Kerrigan-Baumrind LA,
Kerrigan DF, Mitchell RS. Retrograde axonal transport of BDNF in retinal
ganglion cells is blocked by acute IOP elevation in rats. Invest
Ophthalmol Vis Sci 2000; 41:3460-6. \pubmed{11006239}}

\p{15. Pease ME, McKinnon SJ, Quigley HA, Kerrigan-Baumrind LA, Zack DJ.
Obstructed axonal transport of BDNF and its receptor TrkB in
experimental glaucoma. Invest Ophthalmol Vis Sci 2000; 41:764-74.
\pubmed{10711692}}

\p{16. Ko ML, Hu DN, Ritch R, Sharma SC, Chen CF. Patterns of retinal
ganglion cell survival after brain-derived neurotrophic factor
administration in hypertensive eyes of rats. Neurosci Lett 2001;
305:139-42. \pubmed{11376903}}

\p{17. Martin KR, Quigley HA, Zack DJ, Levkovitch-Verbin H, Kielczewski
J, Valenta D, Baumrind L, Pease ME, Klein RL, Hauswirth WW. Gene therapy
with brain-derived neurotrophic factor as a protection: retinal ganglion
cells in a rat glaucoma model. Invest Ophthalmol Vis Sci 2003;
44:4357-65. \pubmed{14507880}}

\p{18. Lin LF, Doherty DH, Lile JD, Bektesh S, Collins F. GDNF: a glial
cell line-derived neurotrophic factor for midbrain dopaminergic neurons.
Science 1993; 260:1130-2. \pubmed{8493557}}

\p{19. Sariola H, Saarma M. Novel functions and signalling pathways for
GDNF. J Cell Sci 2003; 116:3855-62. \pubmed{12953054}}

\p{20. Karlsson M, Lindqvist N, Mayordomo R, Hallbook F. Overlapping and
specific patterns of GDNF, c-ret and GFR alpha mRNA expression in the
developing chicken retina. Mech Dev 2002; 114:161-5. \pubmed{12175504}}

\p{21. Yan Q, Wang J, Matheson CR, Urich JL. Glial cell line-derived
neurotrophic factor (GDNF) promotes the survival of axotomized retinal
ganglion cells in adult rats: comparison to and combination with
brain-derived neurotrophic factor (BDNF). J Neurobiol 1999; 38:382-90.
\pubmed{10022580}}

\p{22. Koeberle PD, Ball AK. Effects of GDNF on retinal ganglion cell
survival following axotomy. Vision Res 1998; 38:1505-15.
\pubmed{9667015}}

\p{23. Straten G, Schmeer C, Kretz A, Gerhardt E, Kugler S, Schulz JB,
Gravel C, Bahr M, Isenmann S. Potential synergistic protection of
retinal ganglion cells from axotomy-induced apoptosis by adenoviral
administration of glial cell line-derived neurotrophic factor and
X-chromosome-linked inhibitor of apoptosis. Neurobiol Dis 2002;
11:123-33. \pubmed{12460552}}

\p{24. Schmeer C, Straten G, Kugler S, Gravel C, Bahr M, Isenmann S.
Dose-dependent rescue of axotomized rat retinal ganglion cells by
adenovirus-mediated expression of glial cell-line derived neurotrophic
factor in vivo. Eur J Neurosci 2002; 15:637-43. \pubmed{11886444}}

\p{25. Ward MS, Khoobehi A, Lavik EB, Langer R, Young MJ.
Neuroprotection of retinal ganglion cells in DBA/2J mice with
GDNF-loaded biodegradable microspheres. J Pharm Sci 2007; 96:558-68.
\pubmed{17177208}}

\p{26. Giordano GG, Chevez-Barrios P, Refojo MF, Garcia CA.
Biodegradation and tissue reaction to intravitreous biodegradable
poly(D,L-lactic-co-glycolic)acid microspheres. Curr Eye Res 1995;
14:761-8. \pubmed{8529414}}

\p{27. Moritera T, Ogura Y, Honda Y, Wada R, Hyon SH, Ikada Y.
Microspheres of biodegradable polymers as a drug-delivery system in the
vitreous. Invest Ophthalmol Vis Sci 1991; 32:1785-90. \pubmed{2032801}}

\p{28. Morrison JC, Moore CG, Deppmeier LM, Gold BG, Meshul CK, Johnson
EC. A rat model of chronic pressure-induced optic nerve damage. Exp Eye
Res 1997; 64:85-96. \pubmed{9093024}}

\p{29. Morrison JC, Johnson EC, Cepurna W, Jia L. Understanding
mechanisms of pressure-induced optic nerve damage. Prog Retin Eye Res
2005; 24:217-40. \pubmed{15610974}}

\p{30. Moore CG, Milne ST, Morrison JC. Noninvasive measurement of rat
intraocular pressure with the Tono-Pen. Invest Ophthalmol Vis Sci 1993;
34:363-9. \pubmed{8440590}}

\p{31. Jia L, Cepurna WO, Johnson EC, Morrison JC. Effect of general
anesthetics on IOP in rats with experimental aqueous outflow
obstruction. Invest Ophthalmol Vis Sci 2000; 41:3415-9.
\pubmed{11006233}}

\p{32. Fu K, Harrell R, Zinski K, Um C, Jaklenec A, Frazier J, Lotan N,
Burke P, Klibanov AM, Langer R. A potential approach for decreasing the
burst effect of protein from PLGA microspheres. J Pharm Sci 2003;
92:1582-91. \pubmed{12884245}}

\p{33. Wordinger RJ, Lambert W, Agarwal R, Liu X, Clark AF. Cells of the
human optic nerve head express glial cell line-derived neurotrophic
factor (GDNF) and the GDNF receptor complex. Mol Vis 2003; 9:249-56
\mvref{9}{36}. \pubmed{12813408}}

\p{34. Jing S, Wen D, Yu Y, Holst PL, Luo Y, Fang M, Tamir R, Antonio L,
Hu Z, Cupples R, Louis JC, Hu S, Altrock BW, Fox GM. GDNF-induced
activation of the ret protein tyrosine kinase is mediated by
GDNFR-alpha, a novel receptor for GDNF. Cell 1996; 85:1113-24.
\pubmed{8674117}}

\p{35. Treanor JJ, Goodman L, de Sauvage F, Stone DM, Poulsen KT, Beck
CD, Gray C, Armanini MP, Pollock RA, Hefti F, Phillips HS, Goddard A,
Moore MW, Buj-Bello A, Davies AM, Asai N, Takahashi M, Vandlen R,
Henderson CE, Rosenthal A. Characterization of a multicomponent receptor
for GDNF. Nature 1996; 382:80-3. \pubmed{8657309}}

\p{36. Yang Q, Williams D, Owusu-Ababio G, Ebube NK, Habib MJ.
Controlled release tacrine delivery system for the treatment of
Alzheimer's disease. Drug Deliv 2001; 8:93-8. \pubmed{11400868}}

\p{37. Young MJ, Klassen HJ. Polymeric delivery of neuroprotective
compounds to the vitreous cavity: a potential treatment for glaucoma.
Arch Soc Esp Oftalmol 2006; 81:429-32. \pubmed{16933165}}

\p{38. Young MJ, Borras T, Walter M, Ritch R. Tissue bioengineering:
potential applications to glaucoma. Arch Ophthalmol 2005; 123:1725-31.
\pubmed{16344446}}

\p{39. Penn RD, Kroin JS, York MM, Cedarbaum JM. Intrathecal ciliary
neurotrophic factor delivery for treatment of amyotrophic lateral
sclerosis (phase I trial). Neurosurgery 1997; 40:94-9; discussion99-100.
\pubmed{8971830}}

\p{40. O'Callaghan JP. Assessment of neurotoxicity: use of glial
fibrillary acidic protein as a biomarker. Biomed Environ Sci 1991;
4:197-206. \pubmed{1910596}}

\p{41. Wang X, Tay SS, Ng YK. An immunohistochemical study of neuronal
and glial cell reactions in retinae of rats with experimental glaucoma.
Exp Brain Res 2000; 132:476-84. \pubmed{10912828}}

\p{42. Woldemussie E, Wijono M, Ruiz G. Muller cell response to
laser-induced increase in intraocular pressure in rats. Glia 2004;
47:109-19. \pubmed{15185390}}

\endreferences

}

\beginfigures

\figfile{1}{
\figtitle{1}{Time course of intraocular pressure in Brown Norway rats}

\p{IOP significantly increased in eyes with episcleral vein injection of
hypertonic saline on weeks 0 and 2 (\panel{B}, \panel{C}, \panel{D},
\panel{E}) compared with eyes without injection (\panel{A}; p\lt 0.001).
Overall IOP exposure was not statistically, significantly different
among groups that received intravitreal injection on week 1 of GDNF
microspheres (\panel{B}, \panel{C}), blank microspheres (\panel{D}), or
PBS alone (\panel{E}; p=0.1852). Data are expressed as the mean\pom SD
(n=9).}

\ctr{\gifimage{1}{700}{457}{24}}

}

\figfile{2}{
\figtitle{2}{Expression of GDNF and its receptors}

\p{Immunoreactivities of GDNF are shown on \panel{A}, \panel{D}, and
\panel{G}, respectively. (\panel{B}), (\panel{E}), and
(\panel{H})illustrate the immunoreactivities of GFR\alpha-1,
GFR\alpha-2, and Ret, respectively. Merged photos of GDNF and
GFR\alpha-1 (\panel{C}), GDNF and GFR\alpha-2 (\panel{F}), and GDNF and
Ret (\panel{I}) are also shown. Scale bars represent 100 \mu m.}

\ctr{\jpgimage{2}{800}{602}{410}}

}

\figfile{3}{
\figtitle{3}{GDNF microspheres reduce retinal damage due to chronic intraocular elevation}

\p{\panel{A}-\panel{E} Chronic IOP elevation resulted in the cupping of
the ONH (upper and middle row, scale bar: 200 \mu m): \panel{A} shows
the normal ONH architecture. Ten percent of GDNF microsphere (MS)
treatment (\panel{B}) reduced the ONH excavation, and 2% GDNF MS
treatment (\panel{C}) moderately reduced the ONH excavation compared
with blank MS treatment (\panel{D}) and PBS (\panel{E}).
\panel{F}-\panel{J} (Neurofilament 200 kDa, middle row): \panel{F} also
illustrates the normal ONH architecture. Ten percent of GDNF MS
treatment (\panel{G}) decreased the loss of NFL, and 2% GDNF MS
treatment (\panel{H}) moderately decreased the loss of NFL compared with
blank MS treatment (\panel{I}) and PBS treatment (\panel{J}).
\panel{K}-\panel{O} Effects of GDNF MS treatment on preservation of the
thickness of IPL after chronic IOP elevation (lower row, scale bar: 100
\mu m): \panel{K} shows the normal retinal architecture. Ten percent of
GDNF MS treatment (\panel{L}) resulted in the preservation of the
thickness of IPL, and 2% GDNF MS treatment (\panel{M}) moderately
resulted in a protection of IPL compared with blank MS (\panel{N}) and
PBS treatment (\panel{O}). (\panel{P}) shows the quantitative analysis
of GDNF MS treatment on the thickness of IPL. Chronic IOP elevation
resulted in a significant loss of the thickness of IPL compared with
that of the normal retina (p\lt 0.001). Ten percent of GDNF MS treatment
resulted in a significant reservation of the thickness of IPL compared
with 2% GDNF MS, blank MS, and PBS treatment (p\lt 0.001). Two perecent
of GDNF MS treatment resulted in significant reservation of the
thickness of IPL compared with PBS treatment (p\lt 0.01). There were no
significant differences between groups treated with 2% GDNF MS versus
blank MS nor between groups treated with blank MS versus PBS (p\gt
0.05). Three asterisks indicate p\lt 0.001 and a double asterisk denotes
p\lt 0.01. In the figure, MS represents microsphere.}

\ctr{\jpgimage{3}{800}{724}{469}}

}

\figfile{4}{
\figtitle{4}{GDNF microspheres increased retinal ganglion cells and
their axons survival}

\p{\panel{A}-\panel{E} Effects of GDNF microsphere (MS) treatment on the
survival of RGCs (anti-NeuN positive cells) due to chronic IOP elevation
(scale bars represent 100 \mu m): \panel{A} shows a normal retina
without IOP elevation. Ten percent of GDNF MS treatment (\panel{B})
resulted in the preservation of RGCs, and 2% GDNF MS treatment
(\panel{C}) resulted in moderate preservation of RGCs compared with
blank MS (\panel{D}) and PBS treatment (\panel{E}). \panel{F}-\panel{O}
Effects of GDNF MS treatment on axon survival due to chronic IOP
elevation: \panel{F} illustrates normal ON axons without IOP elevation.
Ten percent of GDNF MS treatment (\panel{G}) resulted in a preservation
of axons, and 2% GDNF MS treatment (\panel{H}) resulted in a moderate
preservation of axons compared with blank MS (\panel{I}) and PBS
treatment (\panel{J}). Degenerating axons occupied nearly the entire
mass of the ON of rats treated with blank MS (\panel{I}) and PBS
(\panel{J}). \panel{K}-\panel{O} are corresponding representative EM
photos. \panel{F}-\panel{J}: magnification 1000X; \panel{K}-\panel{O}:
magnification 7100X. \panel{P} shows the quantitative analysis of GDNF
MS treatment on the survival of RGCs. Chronic IOP elevation resulted in
a significant loss of the RGCs (p\lt 0.001). Ten percent of GDNF MS
treatment significantly increased the RGC survival compared with 2% GDNF
MS treatment, blank MS treatment, and PBS treatment (p\lt 0.001). Two
percent of GDNF MS treatment resulted in significant reservation of RGCs
compared with PBS treatment (p\lt 0.05). There were no significant
differences between groups treated with 2% GDNF MS versus blank MS and
between groups treated with blank MS versus PBS (p\gt 0.05). \panel{Q}
illustrates the quantitative analysis of GDNF MS treatment on the ON
axon survival. The survival percentage was 61.58% with 10% GDNF MS
treatment compared with 38.56% with 2% GDNF MS treatment, 35.25% with
blank MS, and 33.12% with PBS treatment (p\lt 0.001). Two percent of
GDNF MS treatment increased the survival percentage compared with PBS
treatment (p\lt 0.05). There were no significant differences between
groups treated with 2% GDNF MS versus blank MS and between groups
treated with blank MS versus PBS (p\gt 0.05). Three asterisks indicate
p\lt 0.001 and one asterisk denotes p\lt 0.05. In the figure, MS
represents microsphere.}

\ctr{\jpgimage{4}{800}{629}{398}}

}

\figfile{5}{
\figtitle{5}{GDNF microspheres decreased GFAP expression of retina and optic nerve}

\p{GFAP expression was mainly localized to the inner limiting membrane
in normal retina (\panel{A}). Chronic IOP elevation resulted in
increased GFAP expression (\panel{B}-\panel{E}). Scale bars represent
100 \mu m. Chronic IOP elevation increased the GFAP expression in an ON
cross section (\panel{G}-\panel{J}) compared with that of normal ON
without IOP elevation (\panel{F}). Ten percent of GDNF microsphere (MS)
treatment (\panel{G}) decreased the GFAP expression, and 2% GDNF MS
treatment (\panel{H}) moderately decreased the GFAP expression compared
with blank MS (\panel{I}) and PBS treatment (\panel{J}).
\panel{K}-\panel{O} illustrate the corresponding optic nerve section
stained with DAPI. Scale bars represent 200 \mu m. Quantitative analysis
of GDNF MS treatment on the GFAP expression of retina (\panel{P}) and ON
(\panel{Q}). Chronic IOP elevation resulted in significantly increased
GFAP expression in the retina (p\lt 0.001) and optic nerve (p\lt 0.001)
compared with that of normal tension eyes. Ten percent of GDNF
microspheres significantly decreased the IOP-induced GFAP overexpression
in both the inner retina (p\lt 0.001) and ON (p\lt 0.001) while 2% GDNF
microspheres were more difficult to distinguish from treatment with
blank microspheres and PBS alone (p\gt 0.05). Three asterisks indicate
p\lt 0.001. In the figure, MS represents microsphere.}

\ctr{\jpgimage{5}{800}{634}{335}}

}