The present pilot study (EudraCT n. 2008-004561-26) followed the tenets of the Declaration of Helsinki and was approved by the ethics committee of the institution. All the enrolled patients were fully informed as to the nature and goals of the study. Written informed consent was obtained from all patients.
Patient recruitment and Inclusion criteria
Sixteen eyes of 8 patients (6 males, 2 females; average age 49.7 ± 14.3 years) affected by RP were included in the study (Table 1). All patients had progressive forms of RP based on history, clinical findings and ERG abnormalities. Furthermore, patients met the following inclusion criteria: (1) typical RP with a rod-cone pattern of retinal dysfunction, as determined by standard Ganzfeld electroretinography, dark-adapted Tuebinger perimetry, and classic fundus appearance. (2) Advances stage of the disease (at baseline: central portion of visual field with Goldman V/4e <15 deg; fERG <1 uV). (3) Known inheritance pattern and/or genotype under study. (4) At least 1 years of fERG and clinical examination follow-up, with a minimum of three visits. (5) No or minimal ocular media opacities. (6) No concomitant ocular (e.g. glaucoma, amblyopia) or systemic diseases. Patients with non- Usher syndromic sub-types of RP, Leber’s congenital amaurosis or early onset RP with atypical functional patterns were excluded.
Measures of ocular function and electroretinography
A full general and ophthalmologic examination (including detailed family history, anterior segment biomicroscopy, BCVA, direct and indirect ophthalmoscopy, intraocular pressure measurement) was performed on each patient at baseline.
Best-corrected visual acuities were obtained with a projected Snellen chart. Kinetic visual fields were measured to the V4e white test light of the Goldmann perimeter against the standard background of 31.5 apostilbs. Goldmann visual fields were digitized and total visual field areas were calculated.
Cone focal ERGs (fERG) were recorded from the central 18° region using a uniform red field superimposed on an equiluminant steady adapting background, used to minimize stray-light modulation [19, 20]. The stimulus was generated by a circular array of eight red LEDs (λ maximum, 660 nm; mean luminance, 93 cd/m2) presented on the rear of a Ganzfeld bowl (white-adapting background). A diffusing filter in front of the LED array made it appear as a circle of uniform red light. fERGs were recorded in response to the sinusoidal 95 % luminance modulation of the central red field. Flickering frequency was 41 Hz. Patients fixated monocularly at a 0.25° central fixation mark, under the constant monitoring of an external observer. Pupils were pharmacologically (1 % tropicamide and 2.5 % phenylephrine hydrochloride) dilated to a diameter ≥8 mm, and all subjects underwent a pre-adaptation period of 20 min to the stimulus mean luminance. fERGs were recorded by an Ag–AgCl electrode taped on the skin over the lower eyelid. A similar electrode, placed over the eyelid of the contralateral patched eye, was used as reference (inter-ocular recording). fERG signals were amplified (106-fold), bandpass filtered between 1 and 100 Hz (6 dB/oct), and averaged (12-bit resolution, 2-kHz sampling rate, 200–600 repetitions in 2–6 blocks). Off-line discrete Fourier analysis quantified the amplitude and phase lag of the response fundamental harmonic (1st harmonic) at 41 Hz.
Ocular and systemic complications potentially related to ngf administration
During the entire period of assessment (40 days; see below) particular attention was paid to detect ocular and/or systemic side effects. Potential ocular complications included inflammation (external or uveitis), pain, development of lens opacities, and increased intraocular pressure. Systemic complications previously reported in the literature include allergic reactions, systemic pain as well as weight loss [21].
A comprehensive medical evaluation was carried out by a general physician at day zero, and at the end of the NGF treatment. All patients received oral and written information about the experiment procedures before signing the informed consent.
Nerve growth factor isolation
NGF (2.5S) was purified from male mouse submandibular glands as already described [15, 22]. Briefly, the extract of submandibular glands of adult male mice was passed through subsequent cellulose columns, to separate NGF by adsorption. NGF-containing fractions were analyzed by spectrophotometry and Western blot analysis. NGF purity (>95 %) was estimated by high-performance liquid chromatography, while its biological activity was evaluated by neurite outgrowth stimulation in rat PC12 cells. Purified NGF was dialyzed, lyophilized under sterile conditions, and stored at −20 °C until used. At the time of use, purified NGF was dissolved in 0.9 % sterile saline solution in concentrations of 200 µg/mL. The concentration of NGF in this solution was stable over the 10 day treatment time.
NGF administration schedule
A total of 1 mg of NGF diluted in 5 mL of saline solution was administered in the form of eye drops onto the conjunctiva of both eyes for 10 consecutive days 3 times a day. This amount is considered sufficient to reach and stimulate NGF receptors in most cerebral cholinergic areas of the brain and optic pathways, as previously reported [13]. We preferred to use murine NGF, instead of human-recombinant NGF, because contrasting results have been reported on the efficacy of the latter, mainly due to a lack of in vivo studies [16].
Testing schedule
fERG examinations were performed at baseline, at the end of the 10 days period of NGF administration and 30 days later. BCVA measurement and Goldmann visual field examination were performed at baseline and 30 days after the end of NGF administration.
Data analysis
Changes in BCVA and fERG amplitude obtained after treatment were evaluated as individual changes, as well as in the contest of test–retest variability data obtained from a large cohort of RP patients followed clinically at the Visual Electrophysiology Service of the Institute of Ophthalmology at Universita’ Cattolica del S. Cuore, which have been subject of a long term follow-up study [23].
In each patient, the pre and post treatment total areas of Goldmann visual fields were compared. A percentage difference >20 % was considered clinically significant according to previous studies on test–retest variability in patients with RP [24].