Eleven beagle dogs approximately 2 years of age were used to evaluate PDX-induced neuropathy. The body weight of the dogs ranged from 7 to 11 kg. Two dogs were included in the control group and nine in the experimental group. Three of the dogs in the experimental group were included in the one-day post-PDX treatment group, three were included in the 1-week group post-PDX treatment group, and three were in the 4 weeks post-PDX treatment group (Fig. 6).
All dogs (purchased from Daeil, KyeongGi, Korea) were clinically judged to be in good health and neurologically normal. All experimental dogs had their own admission number (SNU-200908-18) from the Institute of Laboratory Animal Resources, Seoul National University (Korea). During the experiment, all dogs were cared for in accordance with the Animal Care and Use Guidelines of the Institute of Laboratory Animal Resources, Seoul National University.
PDX (Sigma, St. Louis, MO, USA) was diluted in a 0.9% sterile aqueous solution of sodium chloride and was administered subcutaneously (SC) once a day during the morning for seven consecutive days. The PDX treatment solution was prepared immediately prior to each injection. Animals in the control group received vehicle (iso-osmotic sterile aqueous solution of sodium chloride), while animals in the experimental group were administered 150 mg/kg of PDX in a volume of 100 mg/mL SC .
Body weight measurements and postural reaction assessments
The body weights of the test dogs were measured every morning during the PDX injection period, as well as at 1 week and 4 weeks after the last PDX treatment.
Postural reaction assessments (Table 1) were conducted on all dogs every morning during the PDX injection period, as well as at 1 week and 4 weeks after the last PDX treatment.
All dogs were pre-anesthetized with atropine (0.1 mg/kg of body weight, IM). Anesthesia was induced with diazepam and maintained with isoflurane (Baxter Healthcare, Deerfield, IL) through a semi-closed system. Subcutaneous temperature was maintained at 37–38ºC. A Neuropack2 system (Nihon Koden, Tokyo, Japan) was used for all recordings. All measurements were conducted in the left hindlimb of each dog.
The DEMP, M waves were recorded for the tibial nerve using a 1 Hz, 0.5 ms, supramaximal stimulus. Stimulating electrodes were positioned in the distal tibial nerve and recording electrodes were positioned in the plantar interosseous muscle, with a ground electrode positioned between the stimulating electrode and recording electrode. The recording electrode was a bipolar needle electrode. The REMP, H-reflexes were recorded using 1 Hz, 0.5 ms, submaximal stimulus. The stimulating electrode was positioned in the tibial nerve adjacent to the hook and a recording electrode and ground electrode were positioned in the same site of the tibial nerve where the M wave was measured. As previously described, at least 8 measurements were obtained . Electrophysiological recordings were conducted before the PDX treatment period and at 1 day, 1 week, and 4 weeks after the last PDX treatment.
For euthanasia, cephalic veins of the selected dogs underwent intravenous (IV) catheterization. The dogs were anesthetized with a high dose of propofol (5mg/kg body weight, IV). and tiletamine/zolazepam (10mg/kg body weight, IV). After confirmation of deep anesthesia, they were perfused transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4) and followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The DRGs of cervical, thoracic, lumbar spinal cords were removed and post-fixed in the same fixative for 12 h, after which they were dehydrated with graded concentrations of alcohol for embedding in paraffin. Next, the paraffin-embedded tissues were sectioned via microtome (Leica, Wetzlar, Germany) into 3 μm coronal sections and the sections mounted onto silane-coated slides. To elucidate the PDX-induced DRG damage, the samples were deparaffinized in xylene, rehydrated in a descending ethanol series, and stained with HE. In addition, CV staining was conducted as previously described . Briefly, CV acetate (Sigma) was dissolved at 1.0% (w/v) in distilled water, and glacial acetic acid was then added to this solution. Before and after staining for 2 min at room temperature, the sections were washed twice in distilled water, dehydrated with graded concentrations of alcohol at room temperature, and finally mounted with Canada balsam (Kanto, Tokyo, Japan). Images of all CV-stained neurons from the DRG were obtained by using a BX51 light microscope (Olympus, Tokyo, Japan) equipped with a digital camera (DP71, Olympus) connected to a personal computer monitor. DRG neurons were separated into three categories according to their sizes: small- (area 1000 μm2), medium- (area 1000–2000 μm2), and large-sized (>2000 μm2). The number of neurons in the DRG in each group was counted using an image analyzing system equipped with a computer-based CCD camera (software: Optimas 6.5, CyberMetrics, Scottsdale, AZ, USA). Mean cell counts were obtained by averaging the counts from sections taken from each animal. Immunohistochemistry for GFAP and Iba-1 was conducted to elucidate temporal changes in reactive gliosis after PDX treatment. The sections were sequentially treated with 0.3% hydrogen peroxide in PBS for 30 min and 10% normal goat serum in 0.05 M PBS for 30 min, after which they were incubated with diluted rabbit anti-GFAP (1:1000, Chemicon, Temecula, CA, USA) or rabbit anti-Iba-1 antibody (1:500, Wako, Osaka, Japan) overnight at room temperature and subsequently exposed to FITC-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch, West Grove, PA, USA) and FITC-conjugated anti-rat IgG (1:600; Jackson ImmunoResearch). The immunoreactions were observed under a BX51 microscope attached to a fluorescent lamp. Analysis of regions of interest in each of the three DRG regions was performed using an image analysis system. Images were converted into an array of 512 × 512 pixels corresponding to a tissue area of 140 × 140 μm (40× primary magnification). Individual pixel resolution included 256 gray levels. The intensity of GFAP and Iba-1 immunoreactivities was evaluated by determining the relative optical density (ROD), which was obtained after the transformation of the mean gray level using the formula: ROD = log (256/mean gray level). The ROD of the image background was determined using NIH Image 1.59 software in unlabeled portions and that value subtracted for correction, thereby yielding high ROD values in areas of preserved structures and low ROD values in areas of structural loss. The ROD ratio is presented as a percentage.
All data are expressed as mean ± SE or mean ± SEM values. Differences in the data were evaluated by applying Student’s t-test. Statistical significance was considered present at P < 0.05. A paired t-test was conducted for analysis of differences in body weights and M wave and H-reflex amplitudes before and after the pharmacologic treatment. The level of significance was set at P < 0.05.