Experiments were conducted on 10 adult male rats (Rattus norvegicus albinus, Samtako®, Korea) that were approximately 8 weeks old and weighed 320 g at the time of surgery. All the rats were numbered using ear tags. The rats were housed in accordance with the National Institutes of Health guidelines; they were kept in a vivarium, maintained at 20-25 °C and 60% humidity with 12 hours alternating light–dark cycles (7 am-7 pm), and were provided food and water ad libitum.
The remnant-preserving ACL allograft
Arthrotomy was performed after intraperitoneal (IP) administration of general anaesthesia [a mixture of Zoletil (50 mg/kg, Virbac Laboratories, France) and Rompun (10 mg/kg, Bayer, Korea)]. In pairs, the Achilles tendon was obtained from a donor rat and used as an allograft for ACL reconstruction in the right knee of the recipient rat. Briefly, an approximately 1.5 cm longitudinal skin incision was made on the Achilles tendon insertion site in the left ankle. After longitudinally splitting 1.5 cm of the Achilles tendons of each rat, the tissue was fresh frozen at -80 °C for 5 minutes in a deep freezer. Then, the Achilles tendon was defrosted with 47 °C warm saline for 5 minutes (Fig. 1) and used as an allograft.
For the ACL reconstruction, an approximately 1.5 cm incision was made on the right knee. Then, the joint was exposed by transposing the patella laterally after a parapatellar incision was made with the knee in flexion. The ACL was detached at the femoral insertion, but the tibial insertion was maintained. Subsequently, the Achilles tendon was allografted onto the right ACL, which was partially detached at the femoral attachment site. Suture fixation was used on the femoral and tibial ACL anatomical attached sites (Fig. 2). Irrigation was performed on the joints, and the capsule and skin were closed with interrupted sutures.
The sham operation
A sham operation was conducted on the left knee as a control. The knee joints were exposed with the same incision. Additionally, irrigation was performed on the joints, and the capsule and skin were closed with interrupted sutures.
NGF injection and sample collection
A total of 10 µl of recombinant rat NGF (50 µg dissolved in 500 µl ddH2O, Invitrogen #50385MNAC50, CA, USA) was injected into both knee joints every week for 6 weeks after the operation using a 0.3 cc insulin syringe with a microfine needle. The presence of neural cells in the control group (sham operation), allografted Achilles tendon, and ACL remnants was examined 6 weeks post-surgery using haematoxylin and eosin (H and E) staining and immunofluorescent staining with anti-nestin antibody. The presence of neural cells was then compared among the Achilles allografts, ACL remnants, and normal ACL (sham operation) tissues.
H and E staining
Tissue samples were collected from the femoral insertion site of normal ACL tissues, the detached site of ACL remnants and the femoral insertion site of Achilles allografts. After dehydration with alcohol and washing with a tissue processor (Leica TP 1020, Leica, Germany), tissue pieces were fixed with masked formalin solution (mask form 2A, DANA Korea) for 24 hours and embedded in paraffin. The tissues were sectioned sequentially into 4 μm thick slices and stained with H and E. Mechanoreceptors were classified into four types according to a prior study (type I, a spherical or ovoid Ruffini corpuscle; type II, a columnar concentric circular Pacini corpuscle; type III, a spindle-shaped Golgi corpuscle; and type IV, a non-myelinated free nerve ending) . Sections with H and E staining were assessed for the presence of mechanoreceptors following the stated classification.
Sliced tissues on coverslips were washed three times with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 10 minutes, and permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature. After washing three times with PBS, sections were blocked with 1% bovine serum albumin (BSA) for 1 hour at room temperature. Sections were incubated with anti-nestin antibody (N5413, Sigma-Aldrich, St. Louis, MO) (diluted in blocking solution (1% BSA in PBS)) for 1 hour at room temperature in a shading box. Subsequently, the tissue sections were washed three times with PBS and incubated with secondary antibody, Alexa Fluor 555-conjugated rabbit anti-goat antibody (Invitrogen, Grand Island, NY) for 1 hour at room temperature. Alexa Fluor 488-conjugated phalloidin (Invitrogen, Grand Island, NY) was used for F-actin staining. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, Santa Cruz Biotechnologies, Santa Cruz, CA). Images were captured using a confocal microscope (Olympus, FV-1 mm).
Quantification with particle analysis
After immunofluorescent staining, quantitative analysis was performed on each image using ImageJ (National Institutes of Health, Bethesda, MD). The number and area of nuclei that were stained with DAPI and the neural cells that were stained with anti-nestin antibody were calculated. Several attached cells in each image were optically separated using a watershed separation tool provided by the ImageJ software. Following this, the area and number of cells were quantified using ImageJ particle analysis.
Statistical analysis was conducted using SPSS for Windows, version 12.0. The Kruskal-Wallis test was used to analyse the immunofluorescence data (with the 95% confidence level). P-values < 0.05 were considered significant. Where indicated, Mann-Whitney post hoc analysis was performed after the Kruskal-Wallis test, with a significance level set at p < 0.16. The power of group comparison was analysed using G*Power 188.8.131.52, where this study achieved a power of 0.68 for detecting differences with α = 0.05.