LC3 II is a marker for autophagic vacuoles

e the weight-based bortezomib dose. 5: Experimental Design For each assay, the investigator was blinded as to the experimental condition of mice. The experimental plan was divided into two parts: the first part was carried out to fully characterize the bortezomib-induced painful PN in BALB/c mice; the second part aimed at investigating the role of the immune response in the development of painful PN. In experiment 1, all of the studies used the same schedule of bortezomib administration in 3 clusters of 24 mice each. The first cluster of mice was used for spinal cord 8: Bone marrow and PTK/ZK Peripheral blood collection The femoral BM and PB were collected under deep anesthesia from 3 mice/group on days 26, 40 and 46 of experiment 2. The same mice were not analyzed more than once to avoid repeated invasive procedures. The BM was 3 Experimental Bortezomib Peripheral Neuropathy doi: 10.1371/journal.pone.0072995.g001 collected by femoral aspiration using a 300l micro-fine insulinsyringe with a 30-gauge needle and analyzed to determine the number of CD45-positive cells using a flow cytometer. A PB sample was drawn from the submandibular plexus by a single puncture using an 18-gauge 10336542 needle and analyzed to determine the white blood cell count. After the BM and PB drawing procedures, the mice did not show any signs of distress. At the completion of all of the experiments, the mice were euthanized and the BM cells processed by crushing the femurs using a mortar and pestle. The cells were phenotypically analyzed as described below. 10: Neurophysiological analysis of the peripheral nerves 8832224 Two days after the last bortezomib administration, caudal and digital NCVs and action potential amplitudes were measured using an electromyography instrument as previously described. Briefly, caudal NCV was measured by placing two proximal recording needle electrodes on the tail and two stimulating needle electrodes 3.5 cm distal to the recording electrodes. The digital NCV was measured in the hind paw by placing the recording electrodes near the ankle and the stimulating electrodes in the fourth toe. Both the caudal and digital NCVs were calculated as a ratio of the distance between stimulating and recording electrodes and the time latency from the stimulus artifact to the onset of the elicited action potential. Serial stimulations with different amplitudes were performed to achieve the maximal action potential feedback. Ten responses per stimulation amplitude were averaged for each recording. 9: White blood cells count and flow cytometer analysis PB and BM samples were collected as described above in experiment 2. Cellularity was assessed using a Coulter AcT diff hematology analyzer instrument. Red blood cells were lysed with ammonium chloride solution to purify leukocytes. Cells were stained with the following mouse antibodies: CD45.2-PE or PerCPCy5.5, CD19-PE, CD3-APC, Gr1-APC, NK1.1-PE ; and subsequently analyzed by flow cytometry using a FACS Calibur and Cell Quest Pro software, Italy). 11: Behavioral tests 11.1: Mechanical stimulation: Dynamic Aesthesiometer test. The nocifensive behavior of paw withdrawal from a mechanical stimulus was used to assess the development of mechanical allodynia in experiment 1 and 2. The Dynamic Aesthesiometer test, which generates a linearly increasing mechanical force, was performed weekly from baseline through the end of bortezomib administration. Before performing the test at baseline, the mice were acclimated to the instrument