e intraperitoneally injected with Pentobarbital 
40 mg/kg. In experiment 1, three clusters of mice were employed. Within each cluster, the animals were randomized into 3 groups, one injected with bortezomib, one with vehicle and one left untreated. The first cluster of animals was used for the electrophysiological analysis in the dorsal horn of the spinal cord, the second for the behavioral tests and Neurometer analysis and the third for the neurophysiological, histopathological and immunohistochemical analyses. In experiment 2, one cluster of animals was randomized into three groups: one group of mice was AZ-3146 exposed to the X-Ray immunosuppressive irradiation, one group was exposed to X-Ray immunosuppressive irradiation and then injected with bortezomib and a group was left untreated. Flow cytometer, histopathological, behavioral and neurophysiological analyses were performed. TR = treated; = number of mice/group; VEH = vehicle; BTZ = bortezomib; S.C = spinal cord. doi: 10.1371/journal.pone.0072995.t001 electrophysiological assessments. The second cluster was used for the behavioral and Neurometer tests. The third cluster was used for the peripheral nerve neurophysiological measures, the morphological observations of DRG, spinal cord, sciatic and caudal nerve, dorsal and ventral roots and for the immunohistochemical localization of the neuronal stress marker Activating Transcription Factor -3. In each experimental paradigm 9057848 mice were randomly assigned to 3 experimental groups of 8 mice each: one group was treated with bortezomib 0.8 mg/kg/i.v. twice weekly for 4 weeks, one group was treated with a dose-equivalent volume of vehicle i.v. twice weekly for 4 weeks and a group of nave mice was left untreated. In experiment 2, mice were randomly assigned to 3 groups and all housed under the conditions previously described for immunodeficient mice. One group of mice underwent X-Ray irradiation to deplete the immune cell-mediated response and one group of mice was exposed to the X-Ray irradiation and then treated with bortezomib according to the same schedule of experiment 1. A third group of nave mice was left untreated. Flow cytometer analysis of the PB and femoral BM was performed on days 26, 40 and 46 to assess the number of CD45-positive cells, a surface marker of both B and T cellderived subpopulations in mice. In both experiments the development and severity of PN was assessed by neurophysiological analysis of the peripheral nerves, morphological analysis of DRG and peripheral nerves and pain perception threshold using a behavioral test for mechanical sensitivity. 6: X-Ray Irradiation X-Ray irradiation was performed under sterile conditions using a RADGIL X-Ray treatment unit. Mice of experiment 2 were placed in a Rad Disk box and exposed to a 350 RAD sub-lethal dose of radiation exposure on day 1 followed by 22634634 three 100 RAD maintenance exposures on days 7, 15 and 23. In the 2 weeks after irradiation, mice were treated with Ciprofloxacin dissolved in their drinking water to prevent the development of opportunistic bacterial infections as a consequence of the irradiation-induced immunosuppression. ~37C using a heating pad. 4: Drug Bortezomib was prepared immediately before each administration and dissolved in a vehicle solution composed of absolute ethanol/tween80/saline. A 10 ml/kg volume was administered intravenously in the caudal vein. Bortezomib at 0.8 mg/kg or a dose-equivalent volume of vehicle solution was injected twice/ weekly for 4 w