Abstract
purpose. A recent clinical report demonstrated that topical nerve growth factor
(NGF) treatment in patients affected by corneal neurotrophic ulcers
induced epithelial and stromal healing restoring corneal integrity.
Mechanisms(s) undergoing these clinical NGF actions are still unclear.
The aim of this study was to investigate the role of NGF in human and
rat cornea physiopathology.
methods. Expression of high-affinity NGF receptors, NGF-mRNA, and NGF protein
was evaluated in human and rat normal corneas, in human and rat corneal
epithelial cell cultures, in human corneal organ culture, and in the
rat cornea after an experimental model of epithelial injury, by means
of immunohistochemistry, in situ hybridization reverse
transcription–polymerase chain reaction, and enzyme-linked
immunosorbent assay.
results. The resultant data demonstrated that NGF is a constitutive
molecule present and produced in normal human and rat corneas. In vitro
human and rat corneal epithelial cells produce, store, and release NGF
and also express high-affinity NGF receptors (TrkA). In human organ
culture, epithelium, keratocytes, and endothelium have been shown to
bind exogenous radiolabeled NGF, and the epithelial cells’ binding was
increased after epithelium injury. In vivo, after rat corneal
epithelial injury, a transient increase of corneal NGF levels was
observed. Inhibition of endogenous NGF activity by neutralizing
anti-NGF antibodies delayed the corneal epithelial healing rate,
whereas exogenous administration of NGF accelerated healing.
conclusions. Taken together, the above findings show that NGF plays an important
role in corneal physiopathology and suggest that this neurotrophin may
exert therapeutic action in wide-spectrum corneal
diseases.
Corneal transparency is essential for the maintenance of visual
function and is contingent on the flawless integrity of all its
components: the epithelium, stroma, and endothelium.
1 Disruption of the epithelial anatomic barrier activates healing and
remodeling processes, which can predispose the tissue to stromal
ulceration and/or cause stromal opacification, ultimately leading to
irreversible visual deficit.
2 Epithelial/stromal integrity
is compromised by any insult to the ocular surface: infection, trauma,
chemical burns, contact lens wear, topical drug abuse, and
postoperative damage.
3 Despite the numerous studies
published in recent years that have indicated that cytokines, growth
factors, and neuropeptides can influence the epithelial proliferations
and differentiations
1 in vitro, a precise therapeutic
approach to modulate the healing process has not yet been
defined.
4 5
We recently reported that the topical administration of nerve growth
factor (NGF) in patients affected by neurotrophic corneal ulcer induces
complete corneal recovery.
6 This type of ocular disease is
characterized by an impairment of corneal sensitivity innervation
associated with a deficit of epithelial metabolism and vitality,
leading to inadequate healing even after minor injury.
7 8 9 In our study,
6 we provided consistent evidence that
topical NGF treatment restored stromal and epithelial integrity, but
the types of cells receptive to NGF, as well as the mechanism(s)
responsible for corneal healing, were not identified. Evidence that
this clinical effect was mediated by the action of NGF on the
epithelium was supported by the observations that human corneal
epithelium express high-affinity NGF receptors (TrkA)
10 and that in vitro NGF induce rabbit corneal epithelium to proliferate
and differentiate.
11 In the present study, the role of
NGF, particularly at the cellular and molecular levels, in normal and
epithelial injured human and rat corneas was investigated.
Eighteen human corneas (from 10 men and 8 women; age range, 49–72
years) were obtained from the Eye Bank of Veneto, Italy. The mean
cadaver time was 6 ± 4 hours. No subject had any history of
ocular surface disease. After removal of the corneas, they were frozen
at −70°C until processed for immunohistochemistry,
enzyme-linked immunosorbent assay (ELISA), reverse
transcription–polymerase chain reaction (RT–PCR), and in situ
hybridization (ISH; n = 6). For in vitro experiments,
corneal epithelium was debrided (human corneas = 4; rat
corneas = 8) or cultured for autoradiographic evaluation (human
corneas = 8). All procedures were conducted according to the
principles expressed in the Declaration of Helsinki.
For animal experiments, 76 adult Sprague–Dawley male rats, weighing
approximately 250 g, were obtained from Charles River Laboratories
(Como, Italy). Rats were deeply anesthetized by intraperitoneal
injection of ketamine (50 mg/kg) and xylazine (15 mg/kg). Animal care
and procedures were conducted in conformity with the ARVO Statement for
the Use of Animals in Ophthalmic and Vision Research.
To investigate the presence and production of NGF protein in human
(n = 6) and rat (n = 8) corneas, ELISA
was used to measure corneal NGF levels, RT–PCR to detect NGF mRNA, and
ISH and immunohistochemistry to identify which cells expressed NGF mRNA
and NGF protein, respectively.
The production, storage, and release of NGF from human and rat
epithelial cells were investigated. Epithelial cells were obtained by
mechanical removal from Eye Bank corneas (
n = 4) and
rat corneas (
n = 8). Cells were treated with trypsin
0.05% and EDTA 0.091% at 37°C for 3 hours, plated on lethally
irradiated 3T3-J2 cells (2.4 ×
10
4/mm
2), and then cultured
in 5% carbon dioxide in modified Dulbecco–Vogt Eagle’s and Ham’s
F-12 media (3:1 mixture).
12 After 24 hours in culture,
epithelial cells were fixed in 4% paraformaldehyde.
Immunohistochemical analysis for NGF and TrkA and ISH for NGF mRNA were
then performed.
13 14 Samples of culture medium were also
collected to measure NGF concentrations by ELISA.
15
To identify the human corneal cells that were receptive to exogenous
NGF, normal and injured human corneas were cultured in the presence of
1 μg/ml of radiolabeled NGF prepared as previously
described.
16 Eight human corneas were used; on four of
them 3-mm-diameter epithelial lesions were created using a trephine
incision and mechanical removal. Corneal discs were cultured in 60 ml
of medium (minimum essential medium supplemented with 2% fetal calf
serum, glutamine, HEPES buffer, antibiotics, and amphotericin B, all
purchased from GIBCO SRL, Life Technologies, Milano, Italy) in
conventional tissue culture bottles (Falcon; No. 3013E) and incubated
at 37°C in a humidified atmosphere containing 5% carbon
dioxide.
17 After 24 hours in vitro, all corneas were fixed
and evaluated by autoradiography.
A total epithelial corneal debridement was performed in 30 animals
using n-heptanol and mechanical removal.
18 NGF levels were
then measured at various time points post-injury: 2, 12, 24, 48, and
168 hours (
n = 6 animals/time point).
All rats were maintained on a strict 12-hour light/dark cycle (6 AM on;
6 PM off) for at least 1 week before each experiment, and throughout
the duration of the experiment, to synchronize their corneal epithelial
circadian rhythms.
19 Total corneal epithelial debridement
was performed unilaterally. Briefly, a cotton swab saturated with
n-heptanol was applied with moderate pressure to the cornea for 30
seconds. The corneal epithelium was then removed by means of a scraper,
and the cornea was washed with 10 ml of physiological solution (0.9%
NaCl). All wounds were made between 7 and 9 AM.
The corneal lesion was photographed immediately after the procedure and
every 2 hours thereafter until the healing process was
complete.
20 The circumference of the wound margin, as
projected onto the photograph, was traced on a digitizer, and the
lesion’s area was calculated using a computer image analysis system
(Videoplan, Kontron Elektronik, Germany). All measurements were
counted in a masked fashion, and the size of de-epithelialized area was
expressed as a percentage of the total corneal area.
In two groups of animals (
n = 12/group), the
corneal epithelium was removed as previously described. In the first
group, six animals were treated with topical administration of a 50μ
l drop of NGF (stock solution 100 μg/ml of physiological solution)
every 2 hours, and the other six rabbits (controls) were treated with
physiological solution every 2 hours.
6 Highly purified
active NGF form (coefficient of sedimentation = 2.5S) was
prepared from submaxillary glands of adult male mice, according to the
Bocchini and Angeletti method and further purified to remove all renin
activity as described.
21 22
In the second group of animals, six rabbits were treated with topical
administration of a 50 μl solution containing purified anti-NGF
antibody (1 mg/ml of physiological solution) every 2
hours,
23 and the other six animals (control) were treated
with a 50 μl solution of nonspecific rabbit immunoglobulins at the
same concentration every 2 hours.
The healing process was monitored by corneal photography every 2 hours
from the time the lesion was induced. All animals were killed after the
completion of corneal healing, and the corneas were histologically
evaluated.
Human and rat corneas were fixed, cut into 10-μm-thick sections
with a cryostat, and processed for immunohistochemistry using an
affinity-purified NGF monoclonal antibody, which recognizes human and
rodent NGFs,
24 or a specific rabbit polyclonal antibody
for TrkA (trk [763]; Santa Cruz, CA),
10 which recognizes
high-affinity NGF receptors. According to the manufacturer, antibody
trk(763) specifically recognizes TrkA without cross-reacting with other
Trk receptors.
25 26 To further assess specific binding of
NGF and TrkA, corneal sections were also exposed to nonspecific
purified immunoglobulins (IgGs).
ISH for NGF mRNA was performed on human and rat corneas as previously
described.
14 For hybridization, a 3′-end biotin-labeled
oligonucleotide complementary to bases 886 to 930 of the rat NGF mRNA
sequence,
27 or to bases 703 to 742 of human NGF
mRNA,
28 was used at a final concentration of 30 ng/ml.
NGF levels were measured in the human and rat corneas and in the
culture medium of human epithelial cells by a highly sensitive,
two-site ELISA with a sensitivity of 5 pg/ml, using anti-NGF antibodies
(Clone 27/21, Boehringer Mannheim, Mannheim, Germany), according to the
protocol previously described.
15
To further verify whether the NGF was locally produced, the presence of
its mRNA was also evaluated using a RT–PCR procedure on human corneas.
Total RNA was extracted by using the method of Chomczynski and
Sacchi
29 as modified in the TRIZOL kit (GIBCO SRL, Life
Technologies, Milano, Italy), and RT–PCR was performed as described
previously.
30 The PCR product, obtained using the sense
5′-CAGGACTCACAGGAGCAAGC-3′ and anti-sense 5′-GCCTTCCTGCTGAGCACACA-3′
primers, was a 349 base–long fragment corresponding to 511 to 889 of
the human NGF gene.
31
Human corneas were cultured in a medium containing 0.1 μg/ml of
125I-NGF. NGF was radioiodinated with
125I-Na (IMS30, 1 mCi; Amersham, Milan,
Italy) by the chloramine-T procedure and purified by Sephadex G-25
column chromatography.
16 The specific activity was 1.0 to
1.5 Ci/mmol. To assess specific NGF binding to noninjured
(
n = 2) and injured (
n = 2)
corneas they were preincubated with a 100-fold excess of
non-radiolabeled (cold) NGF. After 24 hours in vitro, all corneas were
fixed overnight with 4% paraformaldehyde in phosphate buffer 100 mM,
pH 7.4. The tissues were subsequently placed in 30% buffered sucrose
solution for 24 hours. Sections of cornea were cut at a thickness of 15μ
m for autoradiography. Slides were coated with the nuclear tracking
emulsion, Ilford K2 (Ilford Scientific Product, Knutsford, UK),
and, after 1 month of exposure, developed using a Kodak D19
developer.
16 Sections were counterstained with
toluidine blue and observed under light microscope (Axiophot; Zeiss,
Oberkochen, Germany) at magnifications ×40 and ×100.
ANOVA was calculated using the StatView package for Macintosh (SAS
Institute, Cary, NC). The effects of NGF or anti-NGF antibody (Ab-NGF)
on epithelial healing were analyzed by ANOVA considering the wound area
over time in the various treatment groups (saline versus NGF or
nonspecific IgGs versus Ab-NGF). Post-hoc comparisons within logical
sets of means were performed by the Tukey HSD test. Differences in NGF
concentration during the healing process were evaluated with the
nonparametric Mann–Whitney U test. P <
0.05 was considered statistically significant.
Under normal physiological conditions, human and rat corneas
produced and stored NGF. ELISA showed that NGF was present in
normal human corneas at a concentration of 1154 ± 376 pg/mg and
in the rat at a concentration of 388 ± 121 pg/mg. RT–PCR and in
situ hybridization for NGF mRNA and NGF immunohistochemistry indicated
that NGF was produced by epithelium, keratocytes, and endothelium in
humans and rats (
Figs. 1A 1C ). Indeed, as shown in
Figure 1D , rat corneal epithelium,
keratocytes, and endothelium all expressed immunopositivity for TrkA.
Results of ISH, immunohistochemistry, and ELISA showed that NGF
concentration in the growth media of in vitro human and rat corneal
epithelial cells were more than two times greater than control media
(27 ± 6 and 12 ± 4 pg/ml, respectively, versus <5 pg/ml),
evidence that these cells synthesized, stored, and released NGF (
Figs. 2A 2B 2C 2D ). Human and rat epithelial cells were also shown to
express TrkA
(Figs. 2E 2F) .
In corneal organ culture, cells of the epithelium, keratocytes, and
endothelium of human normal corneas were shown to bind radiolabeled
NGF. Monitoring the rate of healing after creation of a 3-mm-diameter
epithelial lesion revealed the healing process of the wounded area (7.1
mm
2 and a marked NGF positivity within 24 hours.
As illustrated in
Figures 3A and 3B , lesioned NGF-positive epithelial cells showed a stronger
binding of radiolabeled NGF than that seen in control non-lesioned
tissue, suggesting that these cells were highly responsive to NGF
activity. NGF-positive epithelial cells were not observed when corneal
tissue was preexposed to an excess of “cold” NGF, further
suggesting that these cells specifically bound NGF.
To learn more about NGF’s role in the corneal healing process, an
animal model of epithelial debridement was used in which healing was
initiated after 4 hours and completed within 24 hours. Time-course
studies showed that the corneal lesion induced a progressive increase
of NGF levels, reaching the highest value at 48 hours (3010 ± 284
pg/mg;
P < 0.01). Thereafter, NGF concentrations
progressively decreased, arriving at baseline values (706 ± 123
pg/mg;
P > 0.05;
Fig. 4 ) 7 days after induction of the lesion.
Figure 5A illustrates that exogenous NGF treatment accelerated healing of the
epithelium when compared with saline-treated eyes. This statistical
difference was confirmed by ANOVA (repeated measures × treatment:
F
5,50 = 20.615;
P = 0.0001) and
post-hoc comparisons (
P < 0.05). Four hours after
creation of the lesion, the NGF-treated cornea showed a reduction of
wound area (59% ± 11% of the cornea was de-epithelialized), whereas
the control eyes showed no sign of healing. The time occurring to
complete healing was 10 ± 1 hours in NGF-treated eyes versus
16 ± 2 hours in the control eyes. None of the NGF-treated corneas
showed changes in anterior stromal transparency, whereas 2 of the 6
saline-treated eyes presented a mild opacification.
Topical treatment with anti-NGF antibody (Ab-NGF) immediately after
epithelial debridement caused a significant delay in healing. ANOVA
(repeated measures × treatment: F
5,50 =
6.397;
P = 0.0001;
Fig. 5B ) and post-hoc comparisons
further indicated that this treatment delayed corneal healing
(
P < 0.05), reaching a maximum difference compared
with the control eyes, treated with nonspecific IgGs, 6 hours after
creation of the lesion (79% ± 9% versus 56% ± 9%;
P < 0.05;
Fig. 5B ). Moreover, 4 of 6 Ab-NGF–treated
eyes and 2 of 6 nonspecific IgG-treated eyes displayed a weak
opacification of the anterior stroma.
It was recently demonstrated that topical NGF treatment in
patients with corneal neurotrophic ulcers induces healing and
restoration of corneal sensitivity without any local or systemic side
effects.
6 To characterize this effect, the role of NGF in
normal corneas and in the healing process was investigated using in
vitro and in vivo models of corneal physiology and injury. These
experiments demonstrated that NGF was stored and produced in human and
rat normal corneas and that after corneal epithelial injury,
acceleration in healing was associated with an increase of NGF levels.
ISH and immunohistochemical analysis identified that cells of the
epithelium, keratocytes, and endothelium synthesized and stored NGF.
Furthermore, human and rat epithelial cells in vitro not only produced,
stored, and released NGF but also expressed TrkA, supporting the
hypothesis that its effect on corneal healing was mediated by NGF–NGF
receptor interaction.
These findings suggest that in normal corneas endogenous NGF is an
essential factor for the trophism and integrity of the corneal
epithelium. Moreover, the coexpression of NGF and its receptor on the
same corneal cell suggests the presence of an autocrine and/or
paracrine circuit that supports the survival and/or function of
epithelial cells. A similar mechanism has been reported in neuronal and
immune cells.
32 33 34 35 36 A clinical study has demonstrated that
NGF treatment accelerates the healing of corneal ulcers that had
developed subsequent to dysfunction of corneal sensitivity
innervation.
6 It is well known that impairment of corneal
sensitivity leads to decreased vitality and metabolism of the corneal
epithelium.,
7 8 9 37 with frequently associated epithelium
rupture and delay or absence of spontaneously healing.
Corneal NGF levels were also shown to increase after corneal epithelial
injury in the rat. It is possible that endogenous NGF plays an
important role in epithelial healing by modulating the proliferation
and differentiation of epithelial cells, a hypothesis also supported by
results of in vitro studies in rabbit corneal
epithelium.
11 Evidence from various studies further
strengthen this argument: in the present study, in vivo inhibition of
endogenous NGF by Ab-NGF significantly delayed the epithelial healing
process; animals with a targeted mutation for the NGF receptor develop
ulcers and mutilation of the feet and corneal
opacification
38 39 ; and concentrations of NGF are
decreased in ulcer tissue from patients affected by diabetes mellitus,
leprosy, and nerve trauma.
40 41
Both the previously reported clinical study and the findings of the
present study have indicated that NGF was essential for the epithelial
healing process and that its binding to corneal NGF receptor cells was
a crucial event triggering its pharmacological activity. This
hypothesis is consistent with the observation that in vitro human
corneal cultures exposed to radiolabeled NGF demonstrated epithelial
binding to exogenous NGF and that this binding was greatly enhanced in
injured tissue. In addition, exogenous NGF treatment in vivo in rats
with lesioned corneas significantly accelerated epithelial healing.
Although these data clearly indicated the existence of a direct action
of NGF on epithelial cells, the possibility of an indirect effect of
NGF through the production and release of specific neuropeptides, which
then acted as cell mediators capable of stimulating the healing
process, cannot be excluded.
4 5 42 43
A relevant observation in our in vivo studies was the presence of a
mild anterior stromal opacification in 4 of 6 Ab-NGF–treated corneas,
compared with 2 of 6 control eyes (both nonspecific IgG– and
saline-treated eyes) and 0 of 6 NGF-treated corneas. This finding
suggests a role for NGF in epithelium–stroma communication leading to
the induction of stromal healing and remodeling mechanisms, and/or the
promotion of a rapid epithelial healing to avoid the onset of a stromal
opacity. This attractive possibility needs to be explored in both in
vitro and in vivo studies.
Cumulatively, our structural, biochemical, and molecular evidence
support the previous clinical findings that NGF plays a crucial role in
the corneal healing process.
6 They also provide new
insight into the pathophysiological mechanisms of NGF effects and into
a potential therapeutic use of this neurotrophin in a wide spectrum of
corneal injuries. Our working hypothesis is that NGF treatment promotes
the healing process, avoiding the onset of the stromal remodeling
mechanisms that lead to superficial stroma opacification in corneal
disease.
The authors thank Rita Levi-Montalcini for encouragement
and advice during the course of the studies.