For the induction of peripheral sensory damage, postnatal day 3 rat pups received subcutaneous injection (at the nape of the neck) of vehicle (10% ethanol, 10% polyoxyethylene sorbitan, and 80% PBS [0.1 M, pH 7.4]) or capsaicin (Sigma, St. Louis, MO) at a dose of 50 mg/kg of body weight (injection volume, 50 uL). All injections were performed between 9 and 12 hours, under hypothermic conditions.
The effects of capsaicin treatment were evaluated at 3 weeks of age by: the hot-plate test; corneal nerve count by gold chloride staining; tear secretion by modified Schirmer test; NGF pathway modifications by immunohistochemistry, Western blot, ELISA, and RT-PCR; and alterations in corneal stem cells by p63 expression, quantification, and localization.
For the evaluation of peripheral nerves' function, peripheral sensitivity was evaluated using the hot-plate test: rats treated with vehicle or capsaicin were placed in the center of a hot-plate apparatus (Basile model D837; Basile, Comerio, Italy) maintained at 52.0 ± 0.1°C. Nociceptive heat sensitivity was assessed by scoring latency time for forelimb licking. The number of episodes of this behavior was also recorded. Latency time was determined by a digital stopwatch to the nearest millisecond (cutoff time was 60 seconds).
23
For the visualization of corneal sensory nerves, we used the gold chloride staining technique.
24 Briefly, the cornea was excised in toto and rinsed twice in 0.9% saline solution. Four cuts were made radially into the cornea so that it would be flat on a slide when mounted. The cornea was then placed into 100% pure unfiltered lemon juice for 15 minutes and transferred into a 1% aqueous gold chloride solution (chloroauric acid; Sigma) for 45 minutes. Thereafter, the cornea was transferred to acidulated water for 5 minutes, dehydrated with graded alcohol, cleared in xylene, cover-slipped, and observed under the microscope. The number of subbasal corneal nerves was measured both in control and capsaicin-treated corneas in a masked fashion by counting the number of gold chloride–positive subbasal nerve fibers intersecting a 1-mm–line drawn perpendicular to the long axis of the nerves in a 1-mm
2 central area for each corneal quadrant, as previously described.
25 Results are expressed as mean ± SEM (standard error of the mean) unless otherwise specified.
For the evaluation of tear secretion, modified Schirmer test was performed on the rats at as previously described.
26 Briefly, following anesthesia, modified Schirmer strips were inserted in the inferior conjunctival fornix. Lacrimal function was measured based on the length of paper strip wetted after 5 minutes.
For the evaluation of NGF pathway, we investigated the expression of NGF and its receptors TrkA and p75.
27 Specifically, for NGF determination tissues were homogenized with ultrasonication in extraction buffer, centrifuged at 4°C for 20 minutes at 13,000 rpm, then the supernatant was recovered and used for NGF determination as suggested by the instructions provided by the manufacturers. NGF ELISA kits were purchased from Promega (Emax ImmunoAssay System; Madison, WI). Assays were performed in duplicate and the data are expressed as concentration of growth factors pg/mg of total proteins.
For TrkA and p75 protein quantification, we performed Western blotting as follows: tissue samples were homogenized in buffer at 4°C. After 12,000-rpm centrifugation for 20 minutes, the supernatants were submitted to Western blotting. Samples (30 μg of total protein) were dissolved in loading buffer, separated by 8% or 12% SDS-PAGE, and electrophoretically transferred to polyvinylidene difluoride membranes overnight. The membranes were incubated for 1 hour at room temperature with blocking buffer constituted by 5% BSA for TrkA or nonfat dry milk in TBS-T for p75. Membranes were washed three times for 10 minutes each at room temperature in TBS-T followed by incubation at 4°C with primary antibodies overnight (anti-TrkA, or p75) at the concentration indicated by the manufacturers (Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were washed three times for 10 minutes each at room temperature in TBS-T and incubated for 1 hour with horseradish peroxidase-conjugated anti-rabbit IgG or horseradish peroxidase-conjugated anti-mouse IgG as secondary antibody (Cell Signaling Technology, Beverly, MA). The blots were developed with an ECL chemiluminescent horseradish peroxidase (HRP) substrate as the chromophore (Millipore, Billerica, MA). The Java-based image processing software (ImageJ; National Institutes of Health [NIH], Bethesda, MD) was used to evaluate band density, which was expressed as arbitrary units of gray level. The Java-based image processing software (ImageJ; NIH) determines the optical density of the bands using a grayscale shareholding operation. The optical density of β-actin bands was used as a normalizing factor. For each gel per blot, the normalized values were then expressed as percentage of relative normalized controls and used for statistical evaluation. Statistical evaluations were performed using statistical software (StatView package for Windows; BrainPower Inc., Fremont, CA), with data expressed as mean ± SEM.
Localization of NGF, TrkA, and p75 was performed by immunohistochemistry as follows: whole eyes of each experimental group were removed and fixed in 4% paraformaldehyde dissolved in 0.1 M-phosphate buffer, pH 7.4, for 24 hours and then left overnight in the same buffer containing 20% sucrose. Coded sections of each eye were then sectioned with a cryostat, at 10-μm thickness. Sections were first exposed to 0.03% of hydrogen peroxide and 10% of methanol W/V for 20 minutes, followed by exposure to 0.1 M PBS containing 10% of horse or goat serum for 1 hour, then incubated overnight at 4°C with antibodies against NGF, TrkA (Upstate Chemicon, Temecula, CA), and p75 (Santa Cruz Biotechnology) at the concentrations suggested by the manufacturers. Sections were then exposed to biotinylated anti-mouse or anti-rabbit IgG (Vector Laboratories, Burlingame, CA), with 2% of goat or horse serum for 2 hours at room temperature, and then to avidin-conjugated horseradish peroxidase complex in PBS 0.1% Triton for another 2 hours at room temperature and for 15 minutes with a solution of 3,3′-diaminobenzidine. Stained sections were visualized using a microscope (Zeiss Axiophot; Carl Zeiss, Jena, Germany) equipped with a 40× objective. Positive cells were counted in a masked fashion in a randomly selected microscopic field and analyzed using a computerized image analysis system (IAS 2000; Delta Sistemi, Rome, Italy). Control sections were immunostained without primary antibody.
TrkA and p75 mRNA expression was evaluated using RT-PCR as follows: total RNA from the limbal area was extracted using an SV total isolation system (Promega Italia, Milan, Italy). RNA was quantified by spectrophotometer at 260 nm. RNA was converted into cDNA in a 25-μL reverse transcription reaction containing total RNA; 1× reverse transcriptase buffer; 0.5 mM dNTPs; 200 ng oligo (dT); 0.5 U ribonuclease inhibitor (RNasin; Promega); and 200 U of RT-PCR (M-MLV RT; Promega). Reactions were incubated at 42°C for 60 minutes, heated at 95°C for 5 minutes, then cooled at 4°C for a minimum of 5 minutes and a maximum of 30 minutes.
The polymerase chain reaction (PCR) was analyzed using a PCR system (7900HT Fast Real-Time PCR System; Applied Biosystems, Foster City, CA) and FAM-labeled probes (Applied Biosystems) specific for the NTrk1 (NM_021589.1) and Ngfr (NM_012610.1). Designed primers and a FAM-labeled probe for rat GAPDH were included in the reactions as control. The cDNA was amplified under the following conditions: 1 cycle at 50°C for 2 minutes and at 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and at 60°C for 1 minute. The amount of mRNA of each gene was calculated using the standard curve method and adjusted for the expression of GAPDH. The data are presented as change in relative expression as compared with control groups.
For the evaluation of corneal stem cells, we performed Western blot, RT-PCR, and immunohistochemistry evaluation of p63.
21 Briefly, Western blot and immunohistochemistry for p63 were performed as described above using antibodies against p63 (Neomarkers, Fremont, CA) at the concentration suggested by the manufacturer. RT-PCR was performed as described above using custom designed primers (Tp63, Rn01404783_m1) purchased from Promega.