Title

 

Authors

M.D.Sanna¹, N.Galeotti¹,C.Ghelardini<sup>1 

1</sup>Dept. of Farmacology "M.Aiazzi Mancini", University of Florence, Italy

 

Abstract

A major dose-limiting side effect of human immunodeficiency virus ⁄acquired immunodeficiency syndrome (HIV⁄AIDS) chemotherapies, such as the nucleoside reverse transcriptase inhibitors (NRTIs), is a small-fiber painful peripheral neuropathy. The mechanisms responsible for the induction of this neuropathy are not completely elucidated, even if an NRTIs-induced mitochondrial toxicity appears to be mainly involved. To investigate these mechanisms, we developed and characterized a model of 2′,3′ dideoxycytidine (ddC)-induced painful peripheral neuropathy in mice obtained by a single ip injection of ddC at the dose of 25 mg kg^-1. Mice were tested for thermal hyperalgesia by applying a thermal stimulus using the hot plate test (46, 48, 50, 52.5 °C) and cold allodynia evaluated by the cold plate test (4°C). These experiments revealed that ddC-treated mice showed a marked cold allodynia, but no detectable hyperalgesia appeared after applying an acute thermal stimulus. In vivo studies have enabled us to trace the time course of cold hypersensitivity induced by ip administration of ddC. The pain threshold slightly decreased in the first day of treatment, peaked at 3 days, persisted until the fifth day, and disappeared after 7-10 days. We, then, investigate into the cellular mechanisms underlying the pain hypersensitivity in this neuropathy. To this purpose we determined the involvement of a second messenger widely involved in pain modulation that contributes to the enhanced nociception in other models of neuropathic pain, the protein kinase C (PKC).The effect of ddC on the PKC expression and phosphorylation was investigated in cerebral areas highly involved in pain modulation,such as periaqueductal grey (PAG), thalamus, spinal cord and frontal cortex, by means of the immunoblotting technique performed in total protein and in cytosolic and membrane fractions. These studies showed a robust phosphorylation of the PKCgamma and PKCepsilon isoforms in all brain areas investigated, phosphorylation that was reversed by pretreament with the PKC blocker Calphostin C. However, in vivo experiments showed that neither single nor repeated administration of calphostin C prevented the ddC-induced hypersensitivity. Surprisingly, the cold allodynia induced by ddC was prevented by the i.c.v. administration of the calcium release blocker TMB-8. These results suggest that, conversely to other models of painful peripheral neuropathies, the ddc-induced cold allodynia is not related to a PKC activation, but it underlies a calcium-dependent mechanism. After excluding the involvement of PKC in the genesis of the ddC-induced hypersensitivity, we hypothesized that the PKC hyper-phosphorylation observed may be involved in the neuronal regeneration processes subsequent the neuronal insult produced by ddC. The activation of PKC may contribute not only to cell death, but is also able to modulate cell viability, resulting in protection of neuronal and non-neuronal cells. To evaluate the contribution of PKC to regeneration processes, we investigated the expression of GAP-43, a cytosolic protein substrate of PKC, which, when phosphorylated, is highly involved in neuronal development and regeneration. In the cytosolic fraction of the PAG, thalamus and spinal cord, GAP-43 levels progressively increased starting from 1 day after ddC administration up to 7 days,. Pretreatment with the PKC blocker calphostin C prevented this GAP-43 increase. In conclusion, we have developed a model of AIDS therapy-induced peripheral neuropathy in mice by a single ip administration of ddC that is characterized by an intense cold allodynia. An increased phosphorylation of the PKCgamma and PKCepsilon isoforms was detected in the central nervous system that was not related to the observed hypersensitivity. The relevant PKC-dependent increase of GAP43 expression detected let hypothesize a possible role of these PKC isoforms in neuronal regeneration.