The study protocols were cleared and approved by our institute’s animal care and use committee (approval number: BA1608-206/050-01) prior to the start of the study and reported according to guidelines ARRIVED. All animal experiments were performed in accordance with the Principles for the Care of Laboratory Animals (NIH Publication Number 85-23, revised 1996) and approved guidelines. This experimental study was conducted from July 2018 to August 2019.

Animals

Twenty-five female New Zealand White rabbits weighing 3.6 ± 0.4 kg (range, 3.0–4.0 kg) were obtained from DooYeol Biotech (Seoul, Korea) and Saeronbio (Uiwang-si, Korea ). The rabbits were acclimated to the animal experimental platform for 1 week before the experiments. Animals were housed in individual cages under standard laboratory conditions, including a controlled light cycle (12 h light/12 h dark) and controlled temperature (21 ± 2°C). Tap water and standard laboratory food were provided ad libitum.

We originally planned to use 20 rabbits. However, a total of 25 rabbits were used in the experiment, as 5 of them died for unknown reasons immediately after anesthesia or during transport (Fig. 1). Rabbits were randomly assigned to an experimental group (n=10, PLGA group) or a control group (n=10, triamcinolone acetonide (TA) group). To assess local steroid retention in the lumbar segments over time, we sacrificed rabbits from both groups at timed intervals from the day of epidural injection, i.e. day 1 (n=1 for each group), and at week 1, 2 and 4 (n = 3 for each group and each time point).

Preparation of PLGA microspheres

TA-loaded polymeric microspheres were prepared using a Buchi mini spray dryer (model B-290, Buchi Labortechnik AG, Flawil, Switzerland). TA powder (2000 mg), PLGA polymer (4000 mg, lactide/glycolide ratio 50:50, molecular weight 38,000–54,000 Da, 5050 DLG 4A, Lakeshore Biomaterials, Birmingham, AL, USA) and lecithin (200 mg , Avanti Polar Liqids, Alabaster, AL, USA) were completely dissolved in dichloromethane (260 mL, 99.9% HPLC grade, dichloromethane, Daejung, Seoul, South Korea) at 250 rpm for 2 h at l using a magnetic stirrer. The organic solution was then drained into the spray dryer at a feed rate of 5 ml/min. The organic solvent was evaporated with inlet and outlet temperatures of 40°C and 28°C, respectively. Vacuum capacity has been set to 100%. Collected TA-loaded PLGA microspheres were stored in a desiccator at room temperature. For epidural injection, the spray-dried particles were redispersed in an aqueous medium containing polysorbate 80 (0.04% v/v, TWEEN 80, Sigma-Aldrich, St. Louis, MO, USA), carboxymethylcellulose sodium (0.63% v/v%, Sigma-Aldrich, St. Louis, MO, USA) and sodium chloride (0.66% w/v, Sigma-Aldrich, St. Louis, MO, USA), as a dispersant, suspending agent and isotonic agent, respectively.

Particle size and homogeneity of TA-loaded PLGA microspheres were determined using a laser diffraction particle size analyzer (LA-950, Horiba Ltd., Kyoto, Japan). The TA-loaded PLGA microspheres fabricated by the spray-drying procedure were highly spherical, with a smooth and homogeneous surface (Fig. 2). The loading efficiency of the TA microcrystals on the PLGA microsphere was greater than 95%.

Morphological and physical characteristics of TA-loaded PLGA microspheres. (a) Particle size distribution of TA-loaded PLGA microspheres prepared using the spray-drying technique. (b) Electron microscopy image of PLGA microspheres.

Experimental design and epidural injection

All experiments were conducted on fully anesthetized animals. Anesthesia was achieved by intramuscular injection of alfaxalone (Alfaxan, 10 mg/ml; Jurox Pty, Ltd., Rutherford, Australia; 5 mg/kg body weight) and xylazine (Rompun, 23.32 mg/ml ; Bayer Korea, Ansan, Korea ; 5 mg/kg body weight) for the 20 rabbits before the ESI. The anesthetized rabbit was placed on a fluoroscopy table in a supine position. The target level, the interspinous space between the L6 and L7 vertebrae, was identified by both manual palpation and fluoroscopic guidance. Epidural puncture was performed using a 25-gauge Quincke spinal needle after sterilization. Correct positioning of the needle tip in the epidural space was confirmed by injecting 0.5 ml of contrast material (iohexol, Omnipaque 300, 300 mg iodine per ml; GE Healthcare Co., Ltd., Shanghai, China) under fluoroscopy (Fig. 3). In the experimental group, 1.5 ml of PLGA microspheres (containing 40 mg of TA suspended in 1.5 ml) were manually injected via the extension line. Rabbits in the control group received 1.5 ml of TA suspension (1 ml TA [Triam, 40 mg/ml; Shinpoong Pharmaceuticals, Seoul, Korea] and 0.5 ml of normal saline) in the same way. After injection of each formulation, 0.5 ml of contrast medium was infused through the extension tubing to inject the drug remaining in the tubing. All epidural injections were performed by a single radiologist (SJ, 3 years of experience) under the supervision of an experienced radiologist (JWL, 20 years of experience). After the first injection, the animals were housed in an animal experimentation platform and cared for by veterinarians.

Epidural injection of steroids by the interlaminar route. (a) Adequately anesthetized rabbit is placed on a fluoroscopy procedure table in the supine position. (b,vs) The epidural injection is administered at the level of the vertebral segment L6/L7 under fluoroscopic guidance. The target level is confirmed on both sides (D) and anteroposterior (e) views.

Euthanasia and tissue removal

Tissue was harvested from animals sacrificed at predetermined time intervals after epidural injection. In each group, rabbits were anesthetized with consecutive intramuscular and intravenous injections of alfaxalone (Alfaxan, 10 mg/ml; Jurox Pty, Ltd., Rutherford, Australia) and xylazine (Rompun, 23.32 mg/ml ; Bayer Korea, Ansan, Korea) as follows: intramuscular injection of alfaxalone (0.7 ml/kg) and xylazine (0.3 ml/kg) followed by intravenous injection of alfaxalone (0.35 ml/ kg) and xylazine (0.15 ml/kg). Fully anesthetized rabbits were euthanized by intravenous injection of potassium chloride (150 mg/ml; JW Pharmaceutical, Seoul, Korea; 150 mg/kg body weight). After confirmation of death by loss of heart rate, two consecutive lumbar segments including the injection segment (L6/L7) ​​and the adjacent segment (L4/L5) were harvested (Fig. 4). All tissue samples were kept separately on ice until further chemical analysis was performed. All instruments used for the dissection were thoroughly rinsed before processing the next sample.

Tissue collection. (a) The euthanized rabbit is placed in a supine position. Two consecutive vertebrae consisting of the ESI level (black arrow) and the level just above (white arrow) are harvested simultaneously. (b) A single vertebral segment was removed. (vs) The axial view of the harvested vertebral segment shows a potential epidural space (white arrowhead) just below the bony neural arch (small black arrow). The neural spine (black arrowhead) of the rabbit vertebra was located on the dorsal side and the vertebral body (large black arrowhead) is located on the ventral side. The spinal cord (asterisk) was visualized in the neural canal.

Tissue processing and quantification of triamcinolone acetonide

Residual steroid concentration (RSC) in the lumbar segments was analyzed using a high-performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS) test in a chemical laboratory (Biological Mass Spectrometry Group at the Dankook University, Cheonan, Korea). First, the tissue was processed by adding 50 mL of methanol to each bottle containing the tissue and shaking for 24 h in a shaking incubator. Ten microliters of the supernatant obtained by centrifugation of the diluted solution at 14,000 rpm for 10 min were analyzed by an HPLC–MS/MS system (Prominence LC-20 HPLC system; Shimadzu, Tokyo, Japan) and a mass spectrometer API 2000 triple quadrupole (AB/SCIEX, Poster City, CA). Chromatographic separation was performed using a Phenomenex Luna C18 column (2.0 × 150 mm, particle size 5 μm). The mobile phase had a flow rate of 0.25 ml/min and was composed of 0.1% formic acid, with the same volume ratio of water and acetonitrile. The electrospray ionization technique with positive ion mode was used to analyze the steroid compound eluted from the column, after being transferred into the MS/MS instrument, under the following conditions: gas temperature, 400℃; ion sputtering voltage, 5500 V; curtain gas pressure, 16 psi; and collision gas pressure, 6 psi. The AT was monitored and confirmed based on the transitions (transition m/z 435.1 to 415.0 for monitoring; 435.1 to 171.4 and 435.1 to 397.1 for confirmation). The test showed excellent linearity (R² value of 0.9973) over the TA concentration range. Each sample was analyzed three times in the same way.

statistical analyzes

The normality of the data distribution was assessed using the Spiro-Wilk normality test. Data with normal distribution were expressed as mean and standard deviation (SD), while data not normally distributed were presented as median and interquartile range (IQR). Experimental data was presented descriptively as percentages and graphs. Generalized estimating equations were applied to assess the difference in CSR between drugs over time. Analyzes were performed with R software (version 4.0.2; The R Project for Statistical Computing), and P-values ​​less than 0.05 were considered statistically significant.