Experimental and clinical evidence of neuroprotection by nerve growth factor eye drops: Implications for glaucoma

Abstract

Elevated intraocular pressure (IOP) in glaucoma causes loss of retinal ganglion cells (RGCs) and damage to the optic nerve.
Although IOP is controlled pharmacologically, no treatment is available to restore retinal and optic nerve function. We evaluated
the effects of NGF eye drops in a rat model of glaucoma. We also treated 3 patients with progressive visual field defects
despite IOP control. Glaucoma was induced in rats through injection of hypertonic saline into the episcleral vein. Initially,
2 doses of NGF (100 and 200 μg/mL) were tested on 24 rats, and the higher dose was found to be more effective. Glaucoma was
then induced in an additional 36 rats: half untreated and half treated with 200 μg/mL NGF QID for 7 weeks. Apoptosis/survival
of RGCs was evaluated by histological, biochemical, and molecular analysis. Three patients with advanced glaucoma underwent
psychofunctional and electrofunctional tests at baseline, after 3 months of NGF eye drops, and after 3 months of follow-up.
Seven weeks of elevated IOP caused RGC degeneration resulting in 40% cell death. Significantly less RGC loss was observed
with NGF treatment (2,530 ± 121 vs. 1,850 ± 156 RGCs/mm2) associated with inhibition of cell death by apoptosis. Patients treated with NGF demonstrated long lasting improvements
in visual field, optic nerve function, contrast sensitivity, and visual acuity. NGF exerted neuroprotective effects, inhibiting
apoptosis of RGCs in animals with glaucoma. In 3 patients with advanced glaucoma, treatment with topical NGF improved all
parameters of visual function. These results may open therapeutic perspectives for glaucoma and other neurodegenerative diseases.

Full text

Experimental and clinical evidence of neuroprotection
by nerve growth factor eye drops: Implications
for glaucoma
Alessandro Lambiase
a
, Luigi Aloe
b
, Marco Centofanti
c,d
, Vincenzo Parisi
d
, Flavio Mantelli
a
, Valeria Colafrancesco
d
,
Gian Luca Manni
c,d
, Massimo Gilberto Bucci
c,d
, Stefano Bonini
a,1
, and Rita Levi-Montalcini
e,1
a
Centro Integrato di Ricerca, Department of Ophthalmology, University of Rome ‘‘Campus Bio-Medico’’ and Fondazione Alberto Sordi, 00128 Rome, Italy;
b
Institute of Neurobiology and Molecular Medicine, National Research Council, 00143 Rome, Italy;
c
Department of Ophthalmology, University of Rome ‘‘Tor
Vergata’’, 00133 Rome, Italy;
d
Gian Battista Bietti Eye Foundation, Rome, Italy; and
e
European Brain Research Institute Foundation, 00143 Rome, Italy
Contributed by Rita Levi-Montalcini, June 17, 2009 (sent for review May 15, 2009)
Elevated intraocular pressure (IOP) in glaucoma causes loss of retinal
ganglion cells (RGCs) and damage to the optic nerve. Although IOP is
controlled pharmacologically, no treatment is available to restore
retinal and optic nerve function. We evaluated the effects of NGF eye
drops in a rat model of glaucoma. We also treated 3 patients with
progressive visual field defects despite IOP control. Glaucoma was
induced in rats through injection of hypertonic saline into the epis-
cleral vein. Initially, 2 doses of NGF (100 and 200
␮
g/mL) were tested
on 24 rats, and the higher dose was found to be more effective.
Glaucoma was then induced in an additional 36 rats: half untreated
and half treated with 200
␮
g/mL NGF QID for 7 weeks. Apoptosis/
survival of RGCs was evaluated by histological, biochemical, and
molecular analysis. Three patients with advanced glaucoma under-
went psychofunctional and electrofunctional tests at baseline, after 3
months of NGF eye drops, and after 3 months of follow-up. Seven
weeks of elevated IOP caused RGC degeneration resulting in 40% cell
death. Significantly less RGC loss was observed with NGF treatment
(2,530 ⴞ 121 vs. 1,850 ⴞ 156 RGCs/mm
2
) associated with inhibition of
cell death by apoptosis. Patients treated with NGF demonstrated long
lasting improvements in visual field, optic nerve function, contrast
sensitivity, and visual acuity. NGF exerted neuroprotective effects,
inhibiting apoptosis of RGCs in animals with glaucoma. In 3 patients
with advanced glaucoma, treatment with topical NGF improved all
parameters of visual function. These results may open therapeutic
perspectives for glaucoma and other neurodegenerative diseases.
NGF 兩 optic nerve 兩 retina
G
laucoma is the leading cause of irreversible blindness in the
world (1). This chronic and progressive optic neuropathy is
characterized by loss of axons of the retinal ganglion cells (RGC)
that constitute the optic nerve (2). Elevated intraocular pressure
(IOP) is the primary risk factor for glaucoma, responsible for
long-term damage to the optic nerve (3). Patients with glaucoma
typically lose their visual field and become blind if untreated.
Reduction of IOP, the only modifiable causative factor, slows the
onset and progression of the disease, yet no actual treatment is
available to restore optic nerve damage (4).
Neuroprotection has gained substantial interest in recent years as
a therapeutic approach to preventing neuronal degeneration and
loss of function in glaucoma (4). Neuroprotective therapies cur-
rently under investigation to restore retinal/neural function include
memantine, neurotrophins, erythropoietin, reactive oxygen species
scavengers, and even vaccine therapies (4–6). Nevertheless, results
of the se randomized clinical trials have so far been inadequate.
Nerve growth factor (NGF) is an endogenous neurotrophin that
exerts trophic and differentiating activity on neurons of the central
and peripheral nervous systems with protective and/or regenerative
effects observed in degenerative diseases or following injury (7–9).
Intracerebral administration of NGF has been shown to be bene-
ficial in Parkinson’s and Alzheimer’s patients (10–12), and intraoc-
ular administration of NGF in animal models has been shown to
inhibit RGC degeneration after mechanical, ischemic or hyperten-
sive injury (13–15). NGF applied topically to the eye has also been
shown to restore sensory nerve function to the ocular surface of
patients with neurotrophic keratitis (16). Interestingly, absorption
studies have demonstrated that topical ocular NGF reaches the
retina, optic nerve, and brain in animals (17, 18).
In the present study, we demonstrate that topical application of
exogenous murine NGF to the eye prevents RGC degeneration in
an experimental rat model of glaucoma. Based on these findings, we
used the same dosage regimen to treat 3 patients with rapid and
progressive visual field loss despite successful treatment of ocular
hypertension.
Results
Effects of Episcleral Venous Injection of Hypertonic Saline. At baseline
time 0, the mean IOP in SD rats was 24.6 ⫾ 2.1 mm Hg and 24.7 ⫾
2 mm Hg in the control and experimental eyes, re spectively.
Significant unilateral elevation of IOP was successfully induced in
the glaucomatous eyes by saline injection into the episcleral veins
(Fig. 1), as shown by mean IOP values measured weekly for 7 weeks
starting 1 week after treatment (Fig. 2A). Mean IOP in the saline
injected eyes was 35.8 ⫾ 3.2 mm Hg, compared to 24.7 ⫾ 2.2 mm
Hg in the contralateral, sham operated eyes (P ⬍ 0.01).
Effects of Elevated IOP on RGC. Histological evaluation indicated that
compared to normal healthy retinas (Fig. 2B), 7 weeks of chroni-
cally elevated IOP induced approximately a 40% decrease in the
number of RGCs (Fig. 2C; 1,861 ⫾ 106 RGC/mm
2
vs. 3,155 ⫾ 98
RGC/mm
2
, respectively, P ⬍ 0.01, Fig. 2D). To assess whether the
reduced RGC number was due to cell death through apoptosis,
biomarkers involved in cell death and cell survival were studied. As
shown in Fig. 3 A–C, anti-TUNEL staining, a biomarker for
apoptotic cell death, was greater in RGCs of rats with elevated IOP
compared to healthy controls (6 ⫾ 0.9 vs. 0.4 ⫾ 0.3 per mm
2
, P ⬍
0.01). Moreover, molecular analysis of Bcl-2, a biomarker of cell
survival, and Bax, a marker of cell death, demonstrated a lower
mRNA Bcl-2/Bax ratio in the experimentally induced, untreated
glaucomatous eyes (Fig. 3D, Ct values inversely correlated with
mRNA expression values). The results of western blot analysis
reported in Fig. 3E confirmed the molecular data indicating a lower
Bcl-2/Bax protein ratio in glaucomatous eyes.
Effects of Topical NGF in the Animal Model of Glaucoma. Preliminary
experiments comparing 100 and 200
␮
g/mL NGF eye drops showed
Author contributions: A.L., L.A., M.C., V.P., M.G.B., S.B., and R.L.-M designed research; A.L.,
L.A., M.C., V.P., F.M., V.C., and G.L.M. performed research; L.A. and S.B. contributed new
reagents/analytic tools; A.L., L.A., M.C., V.P., F.M., V.C., G.L.M., M.G.B., and S.B. analyzed
data; and A.L., L.A., M.C., V.P., F.M., S.B., and R.L.-M. wrote the paper.
The authors declare no conflict of interest.
1
To whom correspondence may be addressed. E-mail: s.bonini@unicampus.it or
scientific.assist@ebri.it.
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a significantly higher biologic effect of 200
␮
g/mL NGF eye drops
in protecting RGC loss in retinal sections from glaucomatous rat
eyes, as demonstrated by E/E staining (2,145 ⫾ 102 vs. 2,623 ⫾ 138
RGCs/mm
2
, respectively, P ⬍ 0.05). Thus, in subsequent experi-
ments, the 200-
␮
g/mL concentration was used.
Histological analysis showed that topical ophthalmic administra-
tion 4 times daily with 200
␮
g/mL NGF for 7 consecutive weeks
protected RGCs of rats with glaucoma (Fig. 4 A–H). Specifically,
glaucomatous eyes treated with NGF had significantly more RCGs
than a parallel group of glaucomatous eyes not treated with NGF
(2,530 ⫾ 121 vs. 1,850 ⫾ 156 RGCs/mm
2
, P ⬍ 0.01). NGF-treated
glaucomatous eyes also had significantly less anti-TUNEL staining
of RGCs (1.2 ⫾ 0.6 vs. 6 ⫾ 0.9 per mm
2
, P ⬍ 0.01), and greater RGC
survival, as shown by the significantly higher Bcl-2/Bax ratio (Fig.
4G, Ct values inversely correlated with mRNA expression values).
The re sults of western blot protein analysis confirmed the signifi-
cantly higher Bcl-2/Bax ratio in NGF-treated glaucomatous eyes,
indicating greater RGC survival compared to the RGCs of un-
treated glaucomatous eyes.
No statistically significant differences were observed between
NGF-treated glaucomatous eyes and control eyes in TUNEL
staining (1.2 ⫾ 0.6 vs. 0.4 ⫾ 0.3 per mm
2
) and Bcl-2/Bax ratio.
Although RGC cell count showed a protective effect of NGF in
glaucomatous eyes, a significantly higher number of RGCs was still
observed in control eyes as compared to NGF-treated glaucoma-
tous eye s (3,155 ⫾ 98 vs. 2,530 ⫾ 121, P ⬍ 0.05).
Effects of Topical NGF in Patients with Glaucoma. A ll 3 patients
treated with 200
␮
g/mL NGF showed improvements in psy-
chofunctional and electrofunctional parameters after 3 months
of treatment. This effect was sust ained even after a subsequent
3 months without NGF therapy. The patients with glauc oma had
severe dysfunction of the inner most retinal layers and a delay of
neural conduction along the postretinal visual pathways, as
indicated by PERG P50 and VEP P100 values w ith longer
time-to-peaks and longer RCT and PERG P50-N95 with re-
duced amplitudes with respect to control data (19). A progres-
sive improvement of inner retinal layer function and postretinal
neural conduction was observed during NGF treatment. This
enhanced neuronal function was then maintained even 3 months
af ter discontinuation of NGF treatment (Table 1 and Fig. 5).
These electrophysiological changes were accompanied by
improvements in clinical parameters as well. Visual field mean
defects (MD) improved f rom 0% to 5% in all patients by the end
of NGF treatment (Table 1 and Fig. 6) and a further 1% to 15.8%
3 months after NGF discontinuation.
Contrast sensitivity at 12 cyc/deg in Patient 1 improved from 0.91
(baseline) to 1.080 (15.7%) (end of NGF treatment); in Patient 2,
from 0.91 to 1.080 (15.7%), and in Patient 3 from 1.080 to 1.250
(13.6%). These values remained unchanged 3 months after discon-
tinuation of NGF treatment (Table 1).
The best corrected visual acuity in Patient 1 improved from 0.4
to 0.7 (42.8%), in Patient 2, from 0.4 to 0.8 (50%), and in Patient
3 from 0.5 to 0.8 (37.5%). These visual acuity values remained
unchanged 3 months after discontinuation of NGF treatment
(Table 1).
No side effects were observed during NGF treatment and during
the follow-up period, with the exception of a transient (1 week)
burning sensation in 1 patient.
Discussion
This study demonstrated that murine NGF administered topically
to the eye re scued RGCs from apoptosis in rats. We used a
well-characterized experimental model of glaucoma, in which a
single injection of hypertonic saline into the episcleral veins of rat
eyes induced chronic elevation of IOP, optic-nerve degeneration,
and selective RGC loss by apoptosis, the sum effects of which
resemble human glaucoma (20–22). The beneficial effect of NGF
on RGC survival was demonstrated to be due to inhibition of
apoptosis, as shown by the reduction in TUNEL RGC immuno-
staining and the greater retinal Bcl-2/Bax ratio.
It is known that RGCs express NGF receptor (TrkA) and that
NGF binding to TrkA up-regulates Bcl-2 protein, which protects
cells from apoptosis by preventing caspase activation (21, 23).
Furthermore, intravitreal NGF delivery to the retina and optic
nerve is crucial to the survival of RGCs and NGF is known to be
responsible for functional recovery of the retina following ocular
ischemia and hypertension in animal models (13–15). Lastly, an
ophthalmic solution of NGF administered topically to the ocular
surface has been shown to reach the retina and optic nerve where
it is biologically active (17).
A
D
B
C
Fig. 2. Measurement of intraocular pressure (IOP) demonstrated significant
increases (P ⬍ 0.01) in rats that received episcleral injection with hypertonic saline
solution (A). Hematoxylin/eosin staining of normal (B) and glaucomatous (C)
retinas showed a significantly lower (P ⬍ 0.01) number of RGCs (arrows) in
glaucomatous eyes (D).
Fig. 1. Glaucoma was induced in adult SD rats by single injection of 50
␮
L
hypertonic saline solution (1.75M NaCl) into the superior episcleral vein. Once the
rats were anesthetized, episcleral veins were isolated (arrow) under a led micro-
scope and injections were performed using custom-made microneedle glass
syringes (asterisk).
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Three months of topical, ocular NGF treatment in 3 patients with
advanced glaucoma at risk of vision loss resulted in long-lasting
improvement in visual field, contrast sensitivity, and best corrected
visual acuity. Despite successful IOP control by medical therapy,
these patients had progressive visual field defects and severe
abnormalities in PERG and VEP response s that indicated dysfunc-
tion of the innermost retinal layers, delay in visual cortical re-
sponses, and delay in neural conduction along postretinal visual
pathways (24, 25). In glaucoma, up to 20% of patients show
progression of visual field defects with RGC and optic nerve
degeneration despite successful management of ocular hyperten-
sion (26). In fact, elevated IOP is thought to be only the primum
movens that triggers a cascade of events leading to optic nerve
damage (4). An approach that would vastly improve the treatment
of this challenging disease would involve neuroprotection with
exogenous neurotrophic factors (6, 27).
PERG and VEP amplitudes and times-to-peak were the first
electrofunctional parameters improved in our patients, sugge sting
a functional recovery of RGCs and an improvement of neural
c onduction along the postretinal visual pathways. The observed
increase in CSV at 12 cyc/deg further supports the efficacy of
NGF treatment on RGC (28). These effects are in line with the
cr ucial role of neurotrophins in modulating RGC function and
visual cortical neuronal activity reflected by receptive field size,
orient ation selectiv ity, v isual acuity, response latency, and ha-
bituation (29–31).
NGF treatment also improved mean visual field defects in 2
patients (patient 1 and 2), and stabilized the defect in the third
patient. Improvement of visual field persisted 90 days after discon-
tinuation of treatment, indicating that changes induced by NGF had
a prolonged duration. Two patients were actually followed up after
18 months, at which time improvements in visual field were still
stable. This ‘‘long-term’’ NGF effect may be related not only to a
protective activity against neural apoptosis, but also to the forma-
tion of new neural pathways, since it is known that NGF promote s
neural plasticity and axonal regeneration (8, 32–34). In fact, NGF
A
D
E
C
B
Fig. 3. Anti-TUNEL immunostaining of RGCs (arrows) in
normal (A) and glaucomatous (B) eyes showed a signifi-
cantly greater (P ⬍ 0.01) number of apoptotic RGCs in
glaucomatous eyes (C). Molecular analysis of glaucoma-
tous compared to normal, healthy retinas showed
greater mRNA expression of Bax (a biomarker of cell
apoptosis) and lower expression of Bcl2 (a biomarker for
cell survival), as illustrated by the Bcl-2/Bax ratio (D). The
results of western blot protein analysis confirmed the
significantly lower Bcl-2/Bax ratio in glaucomatous reti-
nas (E).
AB
DE
C
F
HG
Fig. 4. Hematoxylin/eosin staining of reti-
nas from untreated (A) and NGF-treated (B)
glaucomatous eyes showed significantly less
(P ⬍ 0.05) loss of RGCs (arrows) in animals
that received 200
␮
g/mL NGF eye drops (C).
Anti-TUNEL immunostaining of retinas from
untreated (D) and NGF-treated (E) glaucoma-
tous eyes showed significantly less (P ⬍ 0.05)
apoptotic RGCs (arrows) in animals that re-
ceived NGF (F). Molecular analysis showed
significantly lower mRNA expression of Bax
(a biomarker of cell apoptosis) associated
with greater expression of Bcl2 (a biomarker
for cell survival), as illustrated by the Bcl-2/Bax
ratio (G), in glaucomatous eyes treated with
NGF compared to untreated glaucomatous
eyes. Western blot analysis of Bcl and 2/Bax
(H) confirmed this protective effect of NGF.
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acts on numerous levels to promote neuronal recovery following
ischemic and chemical injuries: through a neosynaptogenetic mech-
anism, by directly affecting precursor cells and/or by induction of
other growth factors, including BDNF (8, 35–37). These multiple
activities may cause the progressive improvement in visual acuity
observed in our 3 patients during and after NGF treatment (Table 1).
The neuroprotective effects of NGF in glaucoma demonstrated
in this study, together with the recently gained knowledge of NGF’s
ability to reach the brain when topically administered to the eye,
allude to exciting possibilities for the treatment of neurodegenera-
tive diseases of the central nervous system (18, 38). A major
challenge in treating neurodegenerative disorders such as Alzhei-
mer’s disease has been the difficulty of delivering neurotrophic
factors across the blood-brain barrier (8). This obstacle might be
overcome by ophthalmic topical NGF treatment, and absorption
and diffusion studies following this premise should be undertaken.
Many similarities between glaucoma and Alzheimer’s disease go
far beyond the challenges encountered in their treatment: (i) RGCs
die by apoptosis in glaucoma through activation of specific caspases,
which are also activated in Alzheimer’s; (ii) caspase activation with
cleavage of APP has been shown to up-regulate amyloid-beta
production in Alzheimer’s and in animal models of glaucoma; (iii)
age-related mitochondrial dysfunction plays a key role in the
etiology of both neurodegenerative disorders; (iv) elevated gluta-
mate and nitric oxide synthase up-regulation with reactive oxygen
species formation have been implicated in both glaucoma and
Alzheimer’s neurotoxicity; and (v) glutamate toxicity is involved in
both glaucoma and Alzheimer’s synaptic dysfunction (39, 40). All
of these similarities have led glaucoma to be dubbed the ‘‘ocular
Alzheimer’s disease’’ (39). The obvious benefit to this likeness is the
combining of forces in identifying new strategies to treat either
disease.
In summary, this study indicates that topical NGF treatment may
be an effective adjunct therapy for glaucoma, reducing neuron
death and nerve loss. These encouraging results merit further
investigation of topical NGF in controlled clinical trials in glaucoma
and other forms of neurodegenerative disorders.
Fig. 5. Results of NGF eye drop treatment in 3 glauco-
matous patients. Presented are relative changes in the
electrophysiological parameters that reflected function
of the innermost retinal layer (Panels A and B: pattern
electroretinogram: PERG P50 time-to-peak and P50-N95
amplitude), the bioelectric visual cortical response (Panel
C: visual evoked potential: VEP P100 time-to-peak) and
neural conduction along the postretinal visual pathways
(Panel D: retinocortical time: RCT) observed after 30, 60,
and 90 days of NGF treatment and after another 90 days
of follow-up (time 1, 2, 3, and 6) with respect to the
baseline condition (time 0). The relative changes are ex-
pressed as percentage increase in amplitude or percent-
age decrease in time-to-peak from baseline. Percentage
increases in PERG P50-N95 amplitude and percentage
decreases in PERG P50 time-to-peak indicated a reduc-
tion in ganglion cell dysfunction after NFG treatment.
Percentage decreases in VEP P100 time-to-peak and per-
centage decreases in RCT indicated a reduction of the
neural conduction delay along visual pathways after
NFG treatment.
Table 1. Effects of NGF treatment in 3 patients affected by advanced glaucoma
Age,
Sex
IOP,
mmHg
Visual
field,
MD
PERG
P50
latency,
ms
PERG
P50-N95
amplitude,
␮
V
VEP
P100
latency, ms
VEP
N75–100
amplitude,
␮
V
Retinocortical
time, ms CSV 3° CSV 6° CSV 12° CSV 18° Visual
Baseline
Patient 1 74, M 15 ⫺32.90 63 1.0 167 1.3 104 1.085 1.210 1.080 0.470 0.40
Patient 2 59, M 15 ⫺33.90 69 0.8 145 0.7 76 1.085 1.290 0.910 0.640 0.40
Patient 3 82, F 18 ⫺34.27 60 0.8 179 1.4 119 1.160 1.210 0.910 0.640 0.50
At 1 month of NGF treatment
Patient 1 17 ⫺33.14 57 1.5 150 2.9 93 1.085 1.290 0.910 0.640 0.40
Patient 2 15 ⫺33.15 60 1.2 122 2.4 62 1.160 1.210 0.910 0.640 0.40
Patient 3 15 ⫺34.40 63 1.1 168 1.5 105 1.085 1.210 1.080 0.470 0.50
At 3 months of NGF treatment
Patient 1 15 ⫺31.50 52 1.6 145 5.2 93 1.010 1.290 0.910 0.640 0.40
Patient 2 12 ⫺32.10 67 1.5 109 2.8 42 1.850 1.540 0.910 0.710 0.70
Patient 3 17 ⫺34.30 59 1.4 174 2.6 115 1.010 1.210 0.910 0.470 0.60
At 3 months of NGF treatment discontinuation
Patient 1 14 ⫺27.70 52 1.6 145 5.2 93 1.630 1.850 1.080 0.640 0.70
Patient 2 12 ⫺29.20 67 1.5 109 2.8 42 2.000 1.530 1.080 0.800 0.80
Patient 3 16 ⫺33.90 59 1.4 174 2.6 115 1.010 1.210 1.250 0.470 0.80
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Materials and Methods
For this study, we used pathogen-free, adult male Sprague-Dawley (SD) rats (n ⫽
78) maintained on a 12-h light-dark cycle and provided with food and water ad
libitum. All procedures regarding housing, care and experimental procedures
were carried out following the guidelines of the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research, International law (EEC council direc-
tive 86/609, OJ L 358, 1, December 12, 1987), and the Italian National Research
Council’s Ethical Commission on Animal Experimentation (1992).
Preliminary histological studies aimed to identify the best NGF concentration
were performed on 24 rats: 6 untreated normal rats (control group), 6 untreated
rats with glaucoma, 6 100
␮
g/mL NGF-treated rats with glaucoma, and 6 200
␮
g/mL NGF-treated rats with glaucoma. NGF was administered 4 times daily for
7 consecutive weeks. Based on these results, a second set of experiments was
performed on an additional 54 rats: 18 normal controls, 18 NGF-treated (200
␮
g/mL) rats with glaucoma and 18 untreated rats with glaucoma. NGF was
administered with the same dosage regimen, 4 times daily for 7 weeks.
Animal Model of Glaucoma. Glaucoma was induced as described by Morrison et al.
(20) Briefly, was injected once into the superior episcleral vein of 1 eye (Fig. 1),
indicated as the ipsilateral glaucomatous eye, while the contralateral eye served
as a sham, non-glaucomatous control eye. Glaucoma was defined as a significant
loss of RGCs by apoptosis (20, 21). Rats were housed in single cages in a constant
low-light environment (40 –90 lux) to minimize IOP circadian oscillations and
treated as indicated below.
IOP was measured weekly with a TonoPen XL tonometer (Mentor) under
topical anesthesia and the values recorded were the mean of 10 valid measure-
ments, expressed as TonPen readings. Mean changes were then calculated for
each eye ⫾ the standard deviation of the mean (SD). NGF treatment was initiated
immediately at time 0, and results were compared among the 3 parallel groups:
healthy eyes, glaucomatous eyes, and glaucomatous eyes treated with NGF.
Histological Evaluation. Rats were euthanized with an overdose of Nembutal after
7 weeks. Eyes were removed and fixed with 4% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.4, for 24 h. For light histological analysis of the retina, eyes
were fixed in Bouin’s fluid for 1 week, and then immersed for 3 days in phosphate
buffer containing 20% sucrose, pH 7.4. Retinal sections 20
␮
m in width were cut
with a cryostat at 4 °C, and stained with haematoxylin-eosin. Using ImageJ image
processing and analysis software, RGCs were counted in a masked fashion in 4
quadrants of the retinal sections approximately 2 mm from the center of the optic
disc. Counts were taken from comparable areas under a Zeiss Microscope at 400⫻
magnification. The results were averaged and converted to cells per mm
2
.
Analysis of Cell Death. For the determination of RGC death and survival, sections
of the retina were immunostained with TUNEL (TdT-dUTP Terminal Nick-End), a
marker of apoptotic cell death, combined with western blot and molecular
analyses for Bcl-2 and Bax. Briefly, retinal sections were first incubated in a
blocking solution (3% H
2
O
2
in methanol) for 10 min at 15–25 °C, incubated in a
permeabilization solution containing 1⫻ PBS 0.1% Triton X-100 for 2 min at 4 °C,
and then labeled with an in situ cell death detection kit (Roche Diagnostic,
Boheringer) according to the manufacturer’s instructions. For TUNEL-positive
cells, DNA strand breaks were labeled and visualized with 0.4% DAB-H
2
0
2
.
TUNEL-positive cells with nuclear condensation or fragmentation were consid-
ered as apoptotic cells.
For Bcl-2 and Bax western blot experiments, protein concentrations were
determined using the Micro BCA protein assay kit. After determination of protein
concentrations, equivalent amounts of retinal lysates (50
␮
g) were denatured in
sample buffer (final concentration of 2% SDS, 10% glycerol, and 2% 2-mercap-
toethanol, pH 6.8) and electrophoresed through 10% denaturing polyacrylamide
gels. Following SDS polyacrylamide gel electrophoresis, samples were transferred
electrophoretically to nitrocellulose membranes in transfer buffer. Membranes
were blocked for1hin1⫻ TBS/0.1% Tween-20 with 5% defatted milk powder.
Anti-Bcl-2 and anti-Bax primary antibodies (Santa Cruz Biotechnology) were
incubated with the appropriate membranes at a dilution of 1:500 overnight. The
GAPDH primary antibody (Sigma) was used at a dilution of 1:5,000. Appropriate
HRP-conjugated secondary antibodies, all diluted to 1:2,500, were incubated
with the membranes for 1 h. After incubation with secondary antibody, mem-
branes were washed 3 times in 1⫻ TBS (pH 7.4) with 1% Tween-20, and then
developed using chemiluminescence. Images were digitalized in a Kodak Imager
Station and bands were subjected to densitometric analysis using 1D Kodak
software.
Molecular Analysis/Real-time PCR. Bcl-2 and Bax mRNA were measured in rat
retinas (average 0.010 mg wet weight for each sample). Tissues were pretreated
with proteinase K (20 mg/mL; Finnzyme) in HIRT buffer at 56 °C/3 h, and total RNA
was extracted from samples using the Puregene RNA purification kit (Gentra
Systems). The resulting total RNA was re-suspended in 25
␮
L diethyl pyrocarbon-
ate-treated water (ICN) and treated with RNase-Free DNase I to eliminate any
genomic DNA contamination according to the supplier’s protocol (2 U/
␮
L Turbo
DNA free kit AM-1907; Ambion Ltd.). Total RNA samples were checked for both
RNA quantity (Nanodrop; Celbio), purity (⬎1.6) and absence of any RNA degra-
dation (RIN ⬇8). Equivalent amounts of RNA (3
␮
g) per sample were used as a
template in normalized cDNA synthesis. Reverse transcription was performed
according to the standardized Mu-MLV protocol (final volume reaction of 20
␮
L
using 50 pM oligo dT-primer, 1 mM dNTP mix, and 200 U reverse transcriptase;
Mu-MLV, F-605L; Finnzyme) in a PTC-100 programmable thermocycler (MJ Re-
search). The resulting cDNA was amplified using the SYBR Green PCR core reagent
kit (Applied Biosystems) and an Opticon2 MJ Research system (MJ Research). The
reaction contained 10
␮
L SYBR reagent, 3
␮
L cDNA (for the target) or 1
␮
L cDNA
(for the referring gene), and 20 nM primers in a 20-
␮
L final volume. The temper-
ature profile included initial denaturation at 95 °C/15 min, followed by 35– 47
cycles of denaturation at 95 °C/30 s, annealing at 55– 60 °C/25 s (the annealing
time depended on the primer’s Tm), elongation at 72 °C/30 s, fluorescence
monitoring at 60–90 °C, and further incubation at 72 °C/5 m. Specific previously
published primers for Bcl2 were used for this study (21). Primer specificities were
further confirmed by the single melting curves obtained during each amplifica-
tion. Negative controls (without template) were produced for each run. Experi-
ments were performed in duplicate for each data point. Quantitative values were
obtained from the threshold cycle value (Ct), which is the point where a signifi-
cant increase of fluorescence is first detected. According to the REST© software,
results are expressed as N-fold difference (increase or decrease) in target gene
expression. Lastly, ratios between Bcl-2/Bax were calculated according to the
single Ct values.
NGF Eye Drop Preparation and Treatment. NGF was obtained from murine salivary
glands as previously described, following the Bocchini and Angeletti method (41).
Briefly, gel filtration at pH 7.5 was performed on the aqueous gland extract of
adult mice, followed by dialysis at pH 5.0 and fractioning by cellulose-
chromatography. In the present study, the biologically active form of highly
Fig. 6. A representative visual field illustrate changes
from baseline (A) to 1 month of NGF treatment (B), to
3 months of NGF treatment (C) and to 3 months after
discontinuation of NGF (D) in a patient affected by
advanced glaucoma.
Lambiase et al. PNAS
兩
August 11, 2009
兩
vol. 106
兩
no. 32
兩
13473
MEDICAL SCIENCES

purified murine NGF weighing 26 kDa was used, dissolved in a sterile 0.9% NaCl
solution at 2 different concentrations (100 and 200
␮
g/mL) (17).
Selection of Patients. Three patients (69 ⫾ 6 years old, 2 males and 1 female)
affected by advanced and progressive glaucoma (disease duration 21 ⫾ 9 years),
with impending risk of vision loss, despite good pharmacological control (timolol
0.5% and pilocarpine 2% in a fixed combination BID and latanoprost QID) of
intraocular pressure (measured by applanation tonometry) were included in the
study. Advanced glaucoma was defined by the following functional criteria: a
mean deviation less than ⫺24 dB, the presence of only a central or temporal island
remaining in the visual field gray scale (42); and optic disk rim deterioration as an
additional morphological criterion (43).
Treatment Regimen in Patients with Glaucoma. Based on the dosage regimen used
in the animal model, the 3 patients were treated topically with 1 drop (⬇50
␮
L)
of highly purified murine NGF, 200
␮
g/mL, instilled into the conjunctival fornix of
1 eye only 4 times daily for 3 months.
The tenets of the Declaration of Helsinki were followed in this study.
Informed consent was obtained from the subjects after explanation of the
nature and possible consequences of the study. All patients were at imminent
risk of irreversible and complete vision loss for uncontrolled progression of
glaucoma despite adequate IOP control and were, therefore, treated on a
compassionate basis.
Electrofunctional and Psychosensorial Evaluation of Patients. Patients were eval-
uated at baseline, every month during treatment and 3 months post-therapy by
complete ocular examination including visual acuity, tonometry, optic disk pho-
tography, contrast sensitivity (CSV-1000, Vector Vision), visual field (Humphrey,
program 10/2), and and electrofunctional tests (Pattern Electroretinigram, PERG,
Visual Evoked Potentials, VEPs). Static perimetry was also performed and re-
peated 3 times using a Humphrey field analyzer (model 740, central 10–2 ach-
romatic full threshold strategy, showing fixation losses, false positive rate, and
false negative rates each less than 20%, and numeric loss of sensitivity). The mean
defect (MD) defined the mean obtained in all tested points, considering the
increasing scatter of sensitivity values with respect to the data obtained in normal
subjects according to eccentricity, and therefore indicating the severity of global
damage (44). We used the central 10° with a finer grid pattern to improve
resolution of the remaining visual field and to reduce testing time.
Foveal contrast sensitivity was tested using a commercially available chart
(CSV1000; Vector Vision). At the testing distance of 8 feet, the translucent chart
presents 4 spatial frequencies, each on a separate row: 3, 6, 12, and 18 cyc/deg.
According to the Pomerance and Evans procedure (28), the sensitivity threshold
was measured twice, allowing only a few seconds between measurements. The
second measurements were considered for analysis. The test-retest variability was
consistent with that previously reported.
Simultaneous recordings of VEPs and PERGs were assessed using a previously
published method (19). Transient VEP was characterized by 3 peaks that ap-
peared after 75, 100, and 145 ms and had negative (N75), positive (P100), and
negative (N145) polarity, respectively. The transient PERG was characterized by 3
peaks that appeared after 35, 50, and 95 ms and had negative (N35), positive
(P50), and negative (N95) polarity, respectively. Amplitudes (in mV) and time-to-
peaks (in ms) were measured. Simultaneous recordings of VEPs and PERGs iden-
tified an index of neural conduction along the postretinal visual pathways,
defined as retinocortical time (RCT, the difference between the VEP P100 and the
PERG P50 time-to-peak).
Data Analysis. Statistical analysis was performed using the SuperANOVA package
for Macintosh (Abacus Concepts Inc.) and the Tukey-Kramer comparison; a P
value of less than 0.05 was considered statistically significant. Animal data of
parallel control groups were evaluated and compared at endpoint. While the
group of 3 patients was too small to elaborate statistically, the data presented are
individual changes over time from baseline.
ACKNOWLEDGMENTS.This work was supported by Italian Ministery of Health
Grant RF-FGB-2005-625679, Italian Ministery of University and Scientific Research
Grant 2007AF3XH4, and an unrestricted research grant from Fondazione Rome.
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www.pnas.org兾cgi兾doi兾10.1073兾pnas.0906678106 Lambiase et al.