To detect central neuron activation, expression of the transcription factor Fos and phosphorylation of the protein kinase ERK (pERK) can be visualized by immunocytochemistry. These approaches have been extensively used to quantify the activation of nociceptive neurons in the spinal dorsal horn (DH) following peripheral stimulation in vivo. Here we propose an alternative and simplified in vitro model to investigate Fos and pERK expression based on the stimulation of acutely dissected spinal cord slices to mimic acute inflammatory changes in DH. Transverse slices were obtained from postnatal (P8-P12) CD1 mice and were treated for 5 min with capsaicin (CAP, 2 μM). CAP induces a strong release of glutamate from primary afferent terminals which, in turn, excites spinal DH neurons. Since ERK phosphorylation and Fos expression occur following different time frames, two distinct protocols were used to detect their activation. Thus, for studying Fos immunoreactivity CAP-treated slices were left for 3 hours in Krebs solution after stimulation. Instead, for studying pERK immunoreactivity slices were maintained in Krebs solution for only 15 min after stimulation. Both Fos and pERK were significantly up-regulated following CAP challenge. To validate our model we tested the efficacy of octreotide (OCT, 1 μM) in preventing the CAP effect on Fos and pERK expression. OCT is a synthetic antinociceptive analogue of somatostatin, one of the neuropeptides involved in the negative modulation of pain signals in DH. After CAP, OCT reduced the response to both Fos and pERK. Our data validate the use of Fos and pERK immunoreactivity in vitro to investigate the activation of spinal nociceptive pathways and testing potentially antinociceptive molecules.

Fos and pERK immunoreactivity in spinal cord slices: comparative analysis of in vitro models for testing putative antinociceptive molecules.

FERRINI, Francesco Maria;SALIO, Chiara
2014-01-01

Abstract

To detect central neuron activation, expression of the transcription factor Fos and phosphorylation of the protein kinase ERK (pERK) can be visualized by immunocytochemistry. These approaches have been extensively used to quantify the activation of nociceptive neurons in the spinal dorsal horn (DH) following peripheral stimulation in vivo. Here we propose an alternative and simplified in vitro model to investigate Fos and pERK expression based on the stimulation of acutely dissected spinal cord slices to mimic acute inflammatory changes in DH. Transverse slices were obtained from postnatal (P8-P12) CD1 mice and were treated for 5 min with capsaicin (CAP, 2 μM). CAP induces a strong release of glutamate from primary afferent terminals which, in turn, excites spinal DH neurons. Since ERK phosphorylation and Fos expression occur following different time frames, two distinct protocols were used to detect their activation. Thus, for studying Fos immunoreactivity CAP-treated slices were left for 3 hours in Krebs solution after stimulation. Instead, for studying pERK immunoreactivity slices were maintained in Krebs solution for only 15 min after stimulation. Both Fos and pERK were significantly up-regulated following CAP challenge. To validate our model we tested the efficacy of octreotide (OCT, 1 μM) in preventing the CAP effect on Fos and pERK expression. OCT is a synthetic antinociceptive analogue of somatostatin, one of the neuropeptides involved in the negative modulation of pain signals in DH. After CAP, OCT reduced the response to both Fos and pERK. Our data validate the use of Fos and pERK immunoreactivity in vitro to investigate the activation of spinal nociceptive pathways and testing potentially antinociceptive molecules.
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Francesco, Ferrini; Arianna, Russo; Chiara, Salio
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/142578
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