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Carageenan evoked P-Akt in deep dorsal horn neurons is prevented by loss of neurokinin1 positive neurons in superficial dorsal horn

Sorkin LS, Choi J-I, Koehrn FJ (2010) Carageenan evoked P-Akt in deep dorsal horn neurons is prevented by loss of neurokinin1 positive neurons in superficial dorsal horn. Neuroscience 2010 Abstracts 81.21/VV19. Society for Neuroscience, San Diego, CA.

Summary: P-Akt expression increases in dorsal horn after prolonged noxious stimulation and participates in synaptic strengthening. Recent work from our lab, showed separate p-Akt peaks in superficial (45 min) and deep dorsal horn (DH) neurons (2 hrs) after paw carrageenan. It has been suggested that NK1 receptor-expressing projection neurons (NK1+) in superficial DH are necessary for deep DH neuronal sensitization, possibly via a spino-bulbo-spinal loop. In this study, we examined whether the pattern of p-Akt expression was modulated by elimination of superficial DH NK1(+) neurons. Male Holtzman rats (250-275 g) were injected over the lumbar enlargement with substance P-saporin conjugate (SSP-SAP ([Sar9Met(O2)11] 100 ng/µl, n=8), SAP (n=8), or BSA vehicle (n=3). Catheters were removed 20 min post-injection. Two weeks later, carrageenan (2%, 100 µl) was injected into the hindpaw. Animals were perfused 45 min or 2 hrs after carrageenan (or sham) injection. Immunohistochemistry was performed on frozen sections (20mm). After correction for background density, NK-1 immunoreactivity was measured as number of bright pixels (intensity value >50 of 256)/total pixels within user-defined boxes in laminae I-III and IV-V. Neuronal p-Akt was measured using double labeling with rabbit anti-p-Akt ser 473 and mouse anti-NeuN; cells immunopositive for both were counted separately for lamina I-III and IV-V. For both measures, values from four random sections taken from segments L4 and 5 were averaged for each animal. The histologist was blinded as to spinal treatment. Density of NK-1 immunoreactivity was markedly reduced in laminae I-III in rats treated with SSP-SAP compared to SAP or BSA (24.7± 5.6% vs 78.3 ± 3.2%, 76.9 ± 2.0% respectively; p<0.01) with no differences in lamina IV-V (72.2 ± 3.4% vs 75.9 ± 1.7%, & 76.5 ± 3.4%. Counts of p-Akt neurons did not differ at any time point among animals with no pretreatment, BSA or SAP prior to carrageenan. However, the carrageenan-evoked increases in p-Akt neurons seen in laminae I-III at 45 min (9.3 ± 1.0 SAP and 2.4 ± 1.6 SSP-SAP at 45 min) and in laminae IV-V at 2 hour (11.3±1.0 SAP and 3.5± 0.9 SSP-SAP) were totally blocked by loss of NK1 neurons. Selective ablation of NK1+ neurons in superficial DH blocked peripheral inflammation-induced increase of p-Akt expression in both superficial and deep DH neurons. While some p-Akt reduction in laminae I-III was probably due to neuronal loss, i.e. NK1 receptor bearing neurons become p-AKT positive, we propose that reduction in deeper laminae was due to elimination of the first leg of a facilitatory spino-bulbo-spinal loop although loss of local NK+ interneurons could also have contributed.

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