Twelve adult New Zealand White rabbits weighing 4,000-4,500 grams each were used. All rabbits were male and treated in accordance with the guidelines of the Korea Food and Drug Administration Guide for the Care and Use of Laboratory Animals, and the animal use protocol was approved by the Institutional Animal Care and Use Committee of Gyeongsang National University. All animals received general anesthesia using a combination of midazolam 1 mg/kg intramuscular (Roche, Basel, Switzerland) and ketamine 70 mg/kg intramuscular (Fada, Buenos Aires, Argentina). Under strict aseptic conditions, an 8.7 mL blood sample was drawn from the auricular artery of each rabbit with a 21 G needle. A 10 mL syringe preloaded with 1.3 mL of anticoagulant citrate dextrose solution was used to avoid coagulation. One millimeter of each sample was set apart for cell counting. All blood draws were performed by one investigator (SHW).

Each blood sample was centrifuged for 15 minutes at 72 g at 4℃, resulting in the three following layers: the inferior layer composed of red cells, the intermediate layer composed of white cells, and the superior layer made up of plasma. The 6 mL plasma layer was centrifuged for another 5 minutes at 1,006 g in order to obtain a two-part plasma sample: the upper part consisting of 5.5 mL of platelet-poor plasma (PPP) and the lower part consisting of 0.5 mL of PRP. The PPP was first aspirated to avoid its mixing with the PRP. The PRP was then gently aspirated with another pipette and placed in a sterile tube. The platelet count was carried out in the whole blood and in the PRP sample with a Neubauer camera using the Brecher and Cronkite direct manual method [6,7] (Fig. 1).

To analyze the morphologic and histological change, vocal folds were obtained from animals. The animals were sacrificed by administration of a high dose of pentobarbital sodium.

Morphologic examination of rabbit vocal folds was performed using a nasal speculum (7 cm, KASCO, Sialkot, Pakistan) and a direct endoscope (0°, 2 mm, 4 mm, Karl Storz, Tuttlingen, Germany). Once visualized, a CO₂ laser (15 W, 1 second; NIIC R204, Tokyo, Japan) was used to perform uniform bilateral vocal fold stripping at the posterior part of the vocal fold.

Immediately after laser scarring, the prepared PRP was injected into the right vocal fold (PRP-injected side) and normal saline was injected into the left vocal fold (normal saline-injected side). The method of injection was a 1 mL syringe with a 25 G spinal needle (length, 9 cm) and the injected volume was 0.5 mL on either side.

Endoscopic examination was performed using a nasal speculum and direct endoscope to assess the morphologic changes of the vocal folds at 2, 4, and 12 weeks after injection (Fig. 2). Morphological changes observed, including surface irregularities, granulation tissue formation, and atrophic change were investigated and graded by a two otolaryngologists, and were recorded on a subjective scale of 0-3, ranging from absent to severe (0, absent; 1, mild; 2, moderate; 3, intense). The analyses were performed on both normal saline-injected sites and PRP-injected sites. Images were captured with an endoscope and graded later.

Histopathological analyses of the specimens were done 12 weeks later the animals were euthanized and the tissues were stained using H&E for assessing inflammatory reaction and inflammatory cell infiltration, and masson trichrome (MT) for assessing collagen deposition. The slides were viewed with a Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan). Collagen was examined at original magnification ×40 in the lamina propria. Histological analysis was blindly checked and graded from 0 to 3 by two pathologists (0, absent; 1, mild; 2, moderate; 3, intense).

The analyses were performed on both normal saline-injected sites and PRP-injected sites, and statistical comparisons were performed between both sides using a McNemar test. A P-value<0.05 was considered statistically significant. For reliability of measurements, 10% of these slides were selected at random and measurements were repeated.

To analyze the relative expression of growth factors, vocal cords were obtained from two animals 14 days after surgery. The right vocal fold (PRP-injected side, treatment group) was dissected and the previous wound was located. The vocal fold fragments (5 mm square in size) were removed for western blot assay. After that the left vocal fold (normal saline-injected side, control group) was dissected using the same method.

The vocal folds were lysed in a lysis buffer (50 mM Tris [pH 7.5], 100 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol, leupeptin [50 lg/mL], aprotinin [50 lg/mL], 0.2 M PMSF, 1 mM Na3VO4, and 1 mM NaF) and incubated for 10 minutes on ice. Tissue lysates were collected and centrifuged for 10 minutes at 4℃ at 13,200 rpm. Protein concentration in tissue lysates was determined by a Quant-iT assay kit (Molecular Probes, Eugene, OR, USA). An equal amount (10 mg) of protein for each sample was resolved with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membranes were blocked with 1% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST), and sequentially incubated with specific antibodies for PDGF (1:1,000), EGF (1:1,000), VEGF (1:1,000), TGF-β (1:1,000), and glyceraldehyde 3-phosphate dehydrogenase (1:2,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The membranes were washed with TBST and processed using horseradish peroxidase-conjugated anti-rabbit antibodies. The antibodies were purchased from Abcam (Austin, TX, USA). Densitometry was performed using the ImageJ 1.38 (Windows version of NIH Image, http://rsb.info.nih.gov/nih-image/) software.

PRP was prepared in approximately 40 minutes. The concentration of platelets was found to be increased in the PRP compared to serum. In the PRP, the mean platelet concentration was 1,315,000 platelets/mm³ (range, 923,000 to 1,860,000 platelets/mm³; SD, 221,730 platelets/mm³) from an original blood concentration of 310,133 platelets/mm³ (range, 240,000 to 428,000 platelets/mm³; SD, 45,233 platelets/mm³).

As compared with the normal saline-injected vocal fold, the PRP-injected vocal fold showed a decrease in granulation tissue throughout the entirety of the injured vocal folds by visual inspection at the time of endoscopy (Table 1), but the difference in irregular surface and atrophic change was not significant (Fig. 3).

Collagen was found chiefly in the lamina propria of the vocal folds. As seen with MT for assessing collagen deposition, the normal saline-injected vocal fold showed a significant increase in collagen deposition throughout the entirety of the injured lamina propria (P<0.05). On the PRP-injected side, however, collagen was well organized and was distributed chiefly in the PRP-injected lamina propria without excessive deposition (Table 2). In contrast to the findings with collagen, there were no significant differences in the densities of fibrosis and inflammatory reaction between the normal saline-injected vocal fold and PRP-injected vocal fold (Fig. 4).

A western blot assay performed 14 days after surgery found that the PRP-treated group showed marked expression of several growth factors, including PDGF, TGF-β, VEGF, and EGF. These factors have been reported to prevent foreign body reactions, tissue necrosis, or extensive fibrosis (Fig. 5). However, a western blot assay after the 12 week later which did not marked expression of growth factors.