sed to chronic bortezomib treatment has to be elucidated. Given that the bortezomib-treated mice developed neurophysiological, morphological and molecular changes from the peripheral nerves to the dorsal column of spinal cord, we then investigated the electrical activity of WDR neurons in the lumbar enlargement of the spinal dorsal horn, since a variety of neuropathic pain models exhibit increased evoked WDR neuron activity after nerve injury. Here we found that WDR neuron activity in response to innocuous, moderate and noxious hind paw stimulation was significantly increased, in complete agreement with the observed development of 14 Experimental Bortezomib Peripheral Neuropathy doi: 10.1371/journal.pone.0072995.g012 mechanical allodynia. The increased activity of the WDR neurons in our model might be due to increased neurotransmitter release in the spinal dorsal horn and/or to a decreased frequency of inhibitory synaptic events, leading to altered synaptic transmission and exaggerated excitatory activity. Therefore, our results, which are similar to those obtained in other chemotherapy-induced neuropathy models, may reflect physiological changes in 20832753 the spinal dorsal horn as well as in primary afferent fibers. In the second part of the study, we explored the effect of bortezomib administration in immune-deficient mice to test the hypothesis that the immune response might be relevant to the development and/or severity of bortezomib-induced PN. The main reason for this pathogenetic investigation is that nerve biopsies of neuropathic patients that underwent bortezomib therapy showed an increase in the reactivity for the CD3 surface antigen and for the antigen CD68, as well as the presence of perivascular activated T cells and endoneural macrophages. These cells can release pro-inflammatory cytokines that can sensitize primary sensory afferents and modify afferent input to the spinal dorsal horn to facilitate pain. On this basis, immune modulation has been proposed as a possible doi: 10.1371/journal.pone.0072995.g013 15 Experimental Bortezomib Peripheral Neuropathy doi: 10.1371/journal.pone.0072995.g014 16 Experimental Bortezomib Peripheral Neuropathy treatment for bortezomib-induced PN. Moreover, in a recent clinical study, a cohort of MM patients that received a bortezomib-based chemotherapy regimen was tested for their neurological, neurophysiological and inflammatory status. Some of the patients with neuropathic pain and neurophysiological abnormalities showed significant modifications in Th1 and Th2 cell subsets and a concomitant increase of IL-6 levels. To address this issue in BALB/c mice, we developed a new ad hoc immunodeficient mouse model based on X-Ray irradiation at sub-lethal doses. This type of X-Ray exposure results in pancytopenia and, eventually, in a complete inhibition of innate and acquired immunity, antibody production and GW 501516 site cell-mediated response. After irradiation, the immunodeficient animals were exposed to the same 4 week-treatment with bortezomib used in the first part of the study. Behavioral tests, neurophysiological analysis in peripheral nerves and morphological observation of sciatic and caudal nerves and DRG were performed and demonstrated that the immunodeficient animals 12176911 treated with bortezomib developed a painful PN with the same features observed in the immunocompetent mice, thus making it very unlikely that the immune response is a key factor in the pathogenesis of the severe damage induced by bortezomib i