Paclitaxel-induced increase in mitochondrial volume mediates dysregulation of intracellular Ca2+ in putative nociceptive glabrous skin neurons from the rat.

We have recently demonstrated that in a rat model of chemotherapy-induced peripheral neuropathy (CIPN), there is a significant decrease in the duration of the depolarization-evoked Ca2+ transient in small diameter, IB4+, and capsaicin-responsive neurons innervating the glabrous skin of the hindpaw. This change was specific to the transient duration and significantly smaller if not undetectable in neurons innervating the dorsal skin of the hindpaw or the skin of the inner thigh. Given the importance of mitochondria in intracellular Ca2+ regulation and the findings of chemotherapy-associated increase in mitotoxicity along the sensory neuron axons, we hypothesized that CIPN is due to both increases and decreases in mitochondria function, with changes manifest in distinct subpopulations of afferents. To begin to test this hypothesis, we used confocal microscopy and Ca2+ imaging in combination with pharmacological manipulations to study paclitaxel-induced changes in retrograde tracer-labeled neurons from naïve, vehicle-treated, and paclitaxel-treated rats. Paclitaxel treatment was not associated with decreased mitochondrial membrane potential or increased superoxide levels in the somata of putative nociceptive glabrous skin neurons. However, it was associated with significant increases in the relative contribution of mitochondria to the control of the evoked Ca2+ transient duration in putative nociceptive glabrous skin neurons, as well as increases in mitotracker and Tom20 staining which reflected an increase in mitochondrial volume. Furthermore, the relative contribution of the sarco-endoplasmic reticulum Ca2+ ATPase to the regulation of the duration of the depolarization evoked Ca2+ transient was also increased in this subpopulation of neurons from paclitaxel treated rats. Our results indicate that the paclitaxel-induced decrease in the duration of the evoked Ca2+ transient is due to both direct and indirect influences of mitochondria. It remains to be determined if and how these changes contribute to the manifestation of CIPN.

Paclitaxel-induced increase in NCX activity in subpopulations of nociceptive afferents: A protective mechanism against chemotherapy-induced peripheral neuropathy?

We recently demonstrated, in a rat model of chemotherapy-induced peripheral neuropathy (CIPN), that there is a significant decrease in the duration of the depolarization-evoked Ca(2+) transient in isolated somata of putative nociceptive afferents innervating the glabrous skin of the hindpaw, but no change in transient magnitude or the resting concentration of intracellular Ca(2+) ([Ca(2+)]i). Because the Na(+)-Ca(2+) exchanger (NCX) only contributes to the regulation of the duration of the evoked Ca(2+) transient, in putative nociceptive dorsal root ganglion (DRG) neurons, we hypothesized that an increase in NCX activity underlies the CIPN-induced change in this subpopulation of neurons. Acutely dissociated retrogradely labeled sensory neurons from naïve, vehicle-, and paclitaxel-treated rats were studied with fura-2 based Ca(2+) imaging. There was no difference in the relative level of NCX activity between glabrous neurons from paclitaxel-treated or control rats. However, in contrast to the relatively large and long lasting Ca(2+) transients needed to evoke NCX activity in neurons from naïve rats, there was evidence of resting NCX activity in glabrous neurons from both vehicle- and paclitaxel-treated rats. More interestingly, there was a paclitaxel-induced increase in NCX activity in putative nociceptive neurons innervating the thigh, neurons in which there is no evidence of a change in the depolarization-induced Ca(2+) transient, or a body site in which there was a change in nociceptive threshold. Furthermore, while the majority of NCX activity in glabrous neurons is sensitive to the NCX3-preferring blocker KB-R7943, the increase in NCX activity in thigh neurons was resistant to KB-R7943 but sensitive to the NCX1-preferring blocker SEA0400. These results suggest that a mechanism(s) other than NCX underlies the paclitaxel-induced decrease in the duration of the evoked Ca(2+) transient in putative nociceptive glabrous skin neurons. However, the compensatory response to paclitaxel observed may also explain why only subpopulations of sensory neurons are impacted by paclitaxel, raising the intriguing possibility that CIPN is due to the failure of injured neurons to appropriately compensate for the deleterious consequences of this compound.

Mechanisms underlying midazolam-induced peripheral nerve block and neurotoxicity.

BACKGROUND AND OBJECTIVES: The benzodiazepine midazolam has been reported to facilitate the actions of spinally administrated local anesthetics. Interestingly, despite the lack of convincing evidence for the presence of γ-aminobutyric acid type A (GABAA) receptors along peripheral nerve axons, midazolam also has been shown to have analgesic efficacy when applied alone to peripheral nerves.These observations suggest midazolam-induced nerve block is due to another site of action. Furthermore, because of evidence indicating that midazolam has equal potency at the benzodiazepine site on the GABAA receptor and the 18-kd translocator protein (TSPO), it is possible that at least the nerve-blocking actions of midazolam are mediated by this alternative site of action. METHODS: We used the benzodiazepine receptor antagonist flumazenil, and the TSPO antagonist PK11195, with midazolam on rat sciatic nerves and isolated sensory neurons to determine if either receptor mediates midazolam-induced nerve block and/or neurotoxicity. RESULTS: Midazolam (300 µM)-induced block of nerve conduction was reversed by PK11195 (3 µM), but not flumazenil (30 µM). Midazolam-induced neurotoxicity was blocked by neither PK11195 nor flumazenil. Midazolam also causes the release of Ca from internal stores in sensory neurons, and there was a small but significant attenuation of midazolam-induced neurotoxicity by the Ca chelator, BAPTA. BAPTA (30 µM) significantly attenuated midazolam-induced nerve block. CONCLUSIONS: Our results indicate that processes underlying midazolam-induced nerve block and neurotoxicity are separable, and suggest that selective activation of TSPO may facilitate modality-selective nerve block while minimizing the potential for neurotoxicity.

Heme oxygenase-1 protects human hepatocytes in vitro against warm and cold hypoxia.

BACKGROUND/AIMS: Hepatic injury induced by ischemia/reperfusion following surgery, transplantation, or circulatory shock combined with resuscitation is a major clinical problem. METHODS: In this study, hypoxic and inflammatory conditions were mimicked by exposing human hepatocytes to N(2) (at 4 and 37 degrees C) or to cytokines/endotoxin to investigate the potential protective effects of heme oxygenase-1 (HO-1). Incubation of human hepatocytes with single cytokines (IFN-gamma, IL-1beta, TNF-alpha) or LPS, as well as a combination of all four stimuli (CM, cytomix) caused a time-dependent HO-1 mRNA expression over 12h and a decline by 24 h. In parallel, we observed a time-dependent membrane leakage for LDH and AST and a maximum HO-1 protein expression between 3-24 h. RESULTS: Warm and cold hypoxia showed similar results in HO-1 mRNA and protein expression and the release of LDH and AST. CoPP, a potent HO-1 inducer, and bilirubin, a co-product of the HO-pathway, protected human hepatocytes from warm and cold hypoxia. HO-1 enzyme activity was highest during warm hypoxia, followed by cold hypoxia and CM which was confirmed by intracellular Fe(2+) formation. CONCLUSIONS: Taken together, we demonstrated, that HO-1 induction protected human hepatocytes against warm and cold hypoxia. Our results also suggest that HO-1 induction may have therapeutic potential against inflammatory insults.