Cover Article: Carrageenan evoked P-Akt in deep dorsal horn neurons is prevented by loss of neurokinin1 positive neurons in superficial dorsal horn

Contributed by L.S. Sorkin, J.-I. Choi, and F.J. Koehrn, Univ. California San Diego, La Jolla, CA

Paw inflammation with carrageenan elicits phosphorylation of Akt in spinal cord neurons with an unusual time course (Choi et al., 2010). Activation of Akt is seen first in neurons of the superficial dorsal horn and in α-motor neurons, followed by its appearance as much as 2 hrs after injection in large, deep (lamina V) dorsal horn neurons. P-Akt is thought to be an indicator of neuronal activity and a marker of sensitization. In the present study, we determined whether carrageenan-induced expression of P-Akt in the deeper lamina of the dorsal horn and in ventral horn α- motor neurons required a linkage through neurons in the superficial laminae (I-III).

Rats were given a spinal injection of 100 ng of either non-targeted Saporin or [Sar⁹Met(O₂)¹¹] substance P coupled to saporin (SSP-SAP, Cat. #IT-11). Injection was at the level of the thoraco-lumbar junction. It has been reported that this treatment results in loss of neurons with NK1 receptors (substance P receptors) in the superficial, but not the deep dorsal horn (Wiley et al., 2007). Two weeks post-injection, rats were tested for locomotor ability using a rotorod or for carrageenan-induced cutaneous sensitization to mechanical stimuli. Some animals with paw carrageenan were perfused at 45 min or 2 hrs post injection and their lumbar spinal cords processed and reacted for NK1 receptor, P-Akt and a variety of cell markers.

Immunohistological staining demonstrated that NK1 receptor was gone from lamina I-III (p≤ 0.01) of the dorsal horn with no loss in lamina V compared to SAP-pretreated animals. SSP-SAP animals had no carrageenan-associated induction of P-Akt in any spinal lamina at any time point. Behavioral testing indicated a significant loss in mechanical sensitization compared to the SAP animals (p≤ 0.001), with no loss of motor ability. Despite this, SSP-SAP animals still had substantial mechanical sensitization (p≤ 0.001), which peaked 2 hrs after paw injection. We interpret these data as meaning that loss of NK1 receptor bearing neurons in the superficial dorsal horn blocks a large component of spinal sensitization. It is likely that paw carrageenan- induced expression of P-Akt in motor neurons requires an excitatory interneuronal link, which is not required for normal locomotor activity. Inflammation-induced activation of P-Akt in lamina V also requires a superficial dorsal horn linkage (either via excitatory interneurons or the lamina I projection neurons). Allodynia seen in the SSP-SAP animals is probably due to peripheral sensitization of primary afferent fibers and a resultant heightened afferent drive, rather than to a significant spinal sensitization component.

Panels A, C, E and G are spinal cord sections taken from SAP-treated animals; B, D, F and H are from SSP- SAP-treated animals. Panel A vs. B shows loss of NK1 receptor (green) from the superficial, but not deep, dorsal horn, with SSP- SAP treatment. Panels C/D (dorsal horn) and G/H (ventral horn) were obtained 45 min after paw carrageenan. In SAP, but not SSP-SAP animals, carrageenan induces an increase in P-Akt (green- for C-H) compared to BSA. The same pattern of carrageenan- induced P-Akt prevented by SSP-SAP pretreatment was seen in deeper laminae of the dorsal horn. Bar in A applies to Panels A-F = 100 mm; Bar in G applies to G-H = 50 mm; Red staining = NeuN.