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.