The study was carried out in accordance with the recommendations of the Guide for the Care and the Use of Laboratory Animals of the National Institutes of Health (National Research Council (U.S.) , the Committee for the Update of the Guide for the Care and Use of Laboratory Animals, and the Institute for Laboratory Animal Research (U.S.), 2011). The study was approved by the Ethical Committee for laboratory animals of the Republic of Slovenia (Permit Number: U34401-21/2013/6) following European directives on the use of laboratory animals in research and the ARRIVE guidelines.
Animal housing and induction/confirmation of diabetes
Six weeks old C57BL/6J-OlaHsd female mice (n = 36, weight 25-30 g) were obtained from Harlan Laboratories – Envigo (Italy) and reared at the Centre for Laboratory Animals of the Biotechnical Faculty of the University of Ljubljana. All mice were housed individually in ventilated cages (IVC system) with temperature maintained at 23 ± 1° C, humidity maintained at 40-60%, and a 12-hour light/12-hour dark cycle.
At the age of 8 weeks, after 2 weeks of quarantine and acclimatization period with free access to clean water and standardized diet (Mucedola, Milan, Italy), diabetes type 1 was induced in mice by intraperitoneal injection of 200 mg kg-1 STZ in accordance with the protocols for achieving STZ-induced diabetes in mice [21, 29]. STZ is an alkylating agent that induces degeneration in pancreatic β islets [18, 30]. Diabetes was confirmed by measuring a fasting glucose level n blood samples collected from the tail vein using Bayer Contour glucose meter (Ascensia Diabetes Care Holdings AG, Switzerland) three weeks after STZ injection. Animals with a fasting glucose level of more than 25 mmol l-1 were considered diabetic, while those with less than 8 mmol l-1 were considered nondiabetic .
Verification of diabetic neuropathy
To confirm the presence of peripheral sensory neuropathy, tail flick and paw withdrawal tests were performed using Combination Plantar/Tail Flick Analgesia Meter (IITC Life Science, California, USA) with infrared intensity set at 40% and 50%, and cut-off times of 4.00 and 15.00 seconds, respectively . The paw withdrawal test was performed two days before the STZ application and two days prior to the sciatic nerve block, whereas the tail flick test was performed two days prior to the sciatic nerve block. Heat stimulation was repeated 3 times at 5 minute-interval; the mean value of the two measurements was used as the baseline . The plantar method is based on Hargreaves method of quantifying the heat thresholds in the hind paws of rodents upon application of radiant or infrared heat stimulus . The tail flick test involved the application of a heat stimulus to the tail after which the time for the tail to ‘‘flick’’ or twitch was recorded. We used a tail temperature option with an automatic temperature trigger at the start of the tests. Once the pre-set temperature was reached, the timer was automatically triggered and stopped after the tail flicks and the light had stopped. The automatic readouts of the start and end temperatures, and the test time improved a repeatability of the measurements. This option has solved the problem associated with “tail temperature prior to and at the end of testing” . While the recent reviews discussed advantages as well as disadvantages of both tests, the two methods are still considered as relevant stimulus-evoked nociception tests .
Eighteen STZ-induced diabetic and eighteen nondiabetic mice were randomized into the three treatment groups. According to the group assignment, both diabetic and nondiabetic groups included 6 mice treated with 35 mg kg-1 1.3% liposomal bupivacaine (EXPAREL), 6 mice treated with 7 mg kg-1 0.25% bupivacaine HCl (AstraZeneca UK Ltd, UK), and 6 mice treated with saline (NaCl Braun, 9 mg ml-1 injection solution, B Braun Melsungen AG, Germany).
Sciatic nerve block
The mice were anaesthetized with isoflurane up to 4% in a nitrous oxide/oxygen mixture (N2O/O2) via a face-mask. Sciatic nerve blocks were performed by injecting local anaesthetics or saline perineurally using a 29-gauge needle (Omnican®A, B. Braun Melsungen AG, Germany) while held in a lateral recumbent position with paws in a right angle with the trunk. The needle was introduced posteromedially towards the greater trochanter in an anteromedial direction. After encountering the ischial tuberosity, 85 µl of testing solution was injected by a single research staff member, blinded to the study group assignment [13, 36, 37]. The success of the sciatic nerve block was evaluated 20 min after using the paw withdrawal test.
Histopathological evaluation of the sciatic nerve
The animals were sacrificed by cervical dislocation one week after the nerve block in order to allow enough time for nerve pathohistological changes to manifest . At the site of local anaesthetic injection and contralaterally, five mm-long sections of the sciatic nerve were harvested and processed for Epon-embedding for histomorphometric evaluations. After initial fixation in Karnovsky’s KII Solution (2.5% glutaraldehyde, 4.0% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4), the nerve sections were post-fixed in an 1:1 solution of 2% aqueous osmium tetroxide and 3% potassium ferrocyanide. Dehydration was accomplished with graded ethanol solutions and propylene oxide following Epon embedding. A high-resolution light microscope (Eclipse E800; Nikon, Tokyo, Japan) was used to study the prepared 0.5 µm toluidine blue stained cross-sections with images captured by a digital camera (DXM1200F™, Nikon, Tokyo, Japan) connected to the microscope. Images were analysed by a single operator blinded to group assignment.
Morphometric analysis was performed using the Ellipse program (ViDiTo, version 220.127.116.11, 2004, Košice, Slovakia) . Randomly selected areas of the nerve were analysed. The outer border of the nerve fibres and the inner border of the myelin sheaths were assessed at high magnification followed by measurement of the nerve fibre density, proportion of large fibres (percent of fibres where the myelin sheet is visible and can be circumscribed), large fibre diameter, axon diameter and myelin width. Furthermore, G-ratio defined as the ratio of the inner to the outer diameter of the myelin sheath was also calculated . The images were analysed by a trained evaluator blinded to group assignment.
Histopathological evaluation was also employed to assess inflammatory cell infiltration in the histological specimens. Frozen samples of the sciatic nerve were sliced into 10 µm transverse sections processed for immunohistochemistry for leucocyte receptor-type tyrosine-protein phosphatase C (CD45) labelling with anti-CD45 antibody (MCA1388, Bio-Rad Laboratories Inc., San Francisco, CA, USA) and revealed by a secondary antibody P0260 (Dako, Glostrup, Denmark). Positive and negative tissue controls were included with each batch of slides as a check on correct tissue preparation and staining techniques. Sections of mouse thymus served as positive control for the presence of leukocytes. For negative controls, the sections in which the primary antibody was replaced with phosphate-buffered saline were used. The images were analysed by a trained evaluator blinded to group assignment.
The Shapiro-Wilk test was used to evaluate the groups for normality. If normality and equal variance assumptions were met, differences in histomorphometric parameters among treatment groups were tested by two-way analysis of variance (ANOVA) followed by Bonferoni post-hoc tests that corrected the p-values for the subgroup analyses. The dependent t-test for paired samples was used to test differences in histomorphometric parameters between treated and untreated sciatic nerves in the same animal. One-way ANOVA, followed by Tukey post-hoc tests was used for paw withdrawal test. Independent t-test was used to compare tail flick test results, body mass and fasting glucose. Statistical analysis was performed with the IBM SPSS Statistics for Windows, version 25 (IBM Corp, NY, USA). Differences were deemed statistically significant at P < 0.05. Data are presented as means (standard deviation).
The sample size calculation was based on the primary research hypothesis that the STZ-induced diabetic and nondiabetic nerves would differ in their fibre density as an indicator of local anaesthetic neurotoxicity . Using the difference in mean fibre density (14,000 fibres per mm2), pooled standard deviation (1600 fibres per mm2), Type I alpha (0.01), and a desired power (0.90), the sample size was estimated at 6 animals in each treatment group for this two-sided test of a completely crossed 2x3 ANOVA (diabetic/nondiabetic by liposomal bupivacaine/bupivacaine HCl/saline).