Peripheral inflammation induces tumor necrosis factor dependent AMPA receptor trafficking and Akt phosphorylation in spinal cord in addition to pain behavior

Abstract

In the present study, intraplantar carrageenan induced increased mechanical allodynia, phosphorylation of PKB/Akt and GluR1 ser 845 (PKA site) as well as GluR1, but not GluR2 movement into neuronal membranes. This change in membrane GluR1/GluR2 ratio is indicative of Ca(2+) permeable AMPA receptor insertion. Pain behavior was reduced and biochemical changes blocked by spinal pretreatment, but not post-treatment, with a tumor necrosis factor (TNF) antagonist, Etanercept (100microg). Pain behavior was also reduced by spinal inhibition of phosphatidylinositol 3-kinase (PI-3K) (wortmannin; 1 and 5microg) and LY294002; 50 and 100microg) and Akt (Akt inhibitor IV; 3microg). Phosphorylated Akt was found exclusively in neurons in grey matter and in oligodendrocytes in white matter. Interestingly, this increase was seen first in superficial dorsal horn and alpha-motor neurons (peak 45min) and later (peak 2h post-injection) in deep dorsal horn neurons. Akt and GluR1 phosphorylation, AMPA receptor trafficking and mechanical allodynia were all TNF dependent. Whether phosphorylation of Akt and of GluR1 are in series or in parallel or upstream of pain behavior remains to be determined. Certainly, TNF-mediated GluR1 trafficking appears to play a major role in inflammatory pain and TNF-mediated effects such as these could represent a path by which glia contribute to neuronal sensitization (spinal LTP) and pathological pain.

Figure 1

Intrathecal Etanercept (TNF antagonist) dose-dependently reduced mechanical allodynia. Rats were pretreated with intrathecal Etanercept or saline 1 hour prior to carrageenan injection into paw. A, Time course; B, Area under the curve (AUC). C. Rats post-treated (90 min) with 100 µg etanercept * p<0.05, ^# p<0.01, significantly different from vehicle. n=6–7 rats/group

Figure 1

Intrathecal Etanercept (TNF antagonist) dose-dependently reduced mechanical allodynia. Rats were pretreated with intrathecal Etanercept or saline 1 hour prior to carrageenan injection into paw. A, Time course; B, Area under the curve (AUC). C. Rats post-treated (90 min) with 100 µg etanercept * p<0.05, ^# p<0.01, significantly different from vehicle. n=6–7 rats/group

Figure 2

Inhibitors of PI3K/Akt pathway reduced mechanical allodynia evoked by carrageenan. Carrageenan was injected into the paw just after the intrathecal injection of wortmannin (PI3K inhibitor), LY294002 (PI3K inhibitor), Akt inhibitor IV (Akt inhibitor), or vehicle. Vehicle was 5% DMSO for wortmannin and Akt inhibitor IV and 2.5% EtOH and 5% DMSO for LY294002. A–C, Time course; D–F, Area under the curve (AUC). *p<0.05, ^#p<0.01, +p<0.001, significantly different from vehicle. n= 7 rats/group

Figure 3

Intraplantar carrageenan induced a TNF dependent increase in GluR1, but not GluR2 in membrane fractions of cellular homongenates. Histograms illustrate that intraplantar carrageenan induced an increase in GluR1 in membrane fractions 1 h after intraplantar injection. GluR2 in the same fractions showed no change in levels compared to controls. In cytoplasmic fractions obtained from the same tissue, GluR1 showed a marked tendency to decrease, but it did not reach significance. Spinal pretreatment with Etanercept (100 µg) blocked the carrageenan induced GluR1 trafficking indicating the TNF dependence of the process.

Figure 3

Intraplantar carrageenan induced a TNF dependent increase in GluR1, but not GluR2 in membrane fractions of cellular homongenates. Histograms illustrate that intraplantar carrageenan induced an increase in GluR1 in membrane fractions 1 h after intraplantar injection. GluR2 in the same fractions showed no change in levels compared to controls. In cytoplasmic fractions obtained from the same tissue, GluR1 showed a marked tendency to decrease, but it did not reach significance. Spinal pretreatment with Etanercept (100 µg) blocked the carrageenan induced GluR1 trafficking indicating the TNF dependence of the process.

Figure 4

Intraplantar carrageenan induced a time dependent increase in P-Akt in dorsal spinal cord. A. Level of P-Akt increased at 1 and 2 h after bilateral carrageenan injections. Representative P-Akt blots along with β-actin loading controls are illustrated. + is a P-Akt positive loading control. Animals did not have i.t. catheters. B. Histogram illustrates P-Akt response 2 h after a unilateral carrageenan injection. This increase in PAkt was blocked by spinal pretreatment with Etanercept (ETA, 100mg) 1 hour prior to carrageenan injection. Equivalent increases and ETA antagonism were observed 1 h after carrageenan. *p<0.05, significantly different from naïve or control. n=4–7 rats/group C. Graph illustrates co-variance between Akt phosphorylated at ser 473 and thr 308 in the same samples run on the same membrane. Samples were obtained from spinal saline-injected animals with and without paw carrageenan. Samples were collected 2 h after the paw injection time. Axes are in arbitrary units.

Figure 5

Representative sections double labeled for NeuN (red) and P-Akt (green). Panels A–E were taken from animals perfused at 0, 0.75, 1.3, 2 and 3 h respectively, after bilateral carrageenan injection into the paws. F. Histogram illustrating time course of P-Akt within various regions of the dorsal horn. Neurons positive for P-Akt and NeuN were counted by a blinded observer, all counted neurons had a visible nucleus. Counts are averages of 4 separate sections/animal. Counts are of 1 hemisection. N=4 animals/group for all bars. Panel G is an enlargement of the lamina I neuron indicated by the arrowhead in panel B (0.75 h). Neuron has the large, flat morphology typical of a lamina I projection neuron. Panel H is an enlargement of lamina V neurons indicated by the arrowhead in panel D (2.0 h). P-Akt appeared to be confined to the cytosol and in many cases to extend into the dendrites. Size bar = 100 µm panels A–E and 20 µm panels F and G

Figure 6

Animals were perfused 2 h after carrageenan injection and sections stained for A. APC, B. GFAP or C. OX-42 (red) to identify oligodendrocytes, astrocytes and microglia, respectively. Sections were all double stained for P-Akt (green). A. Oligodendrocytes in dorsal columns were frequently double labeled for P-Akt. This was also true for other white matter tract areas, including the lateral funiculus (not shown). However, there was no co-localization within the grey matter. Lack of co-localization was observed under naïve conditions as well as 0.75 and 2 h post-injection. B. Although there was plentiful P-Akt in the deeper lateral dorsal horn, it did not co-localize with GFAP. This was true for all areas of the spinal cord and 0.75 h post-injection. WM = white matter C. Although there was plentiful P-Akt in the deeper lateral dorsal horn, it did not co-localize with OX-42. This was true for all areas of the spinal cord and at 0.75 h post-injection. Size bar = 50 µm, magnification was the same in all 3 panels.

Figure 7

Representative sections double labeled for NeuN (red) and P-Akt (green). Micrographs were obtained from ventral horn using the same animals as in Figure 6. Panels A–C were taken from animals perfused at 0, 0.75 and 2 h respectively, after bilateral carrageenan injection into the paws. Panel D illustrates the time course of P-Akt within the -motor neurons. All counted neurons had a visible nucleus, α-motor neurons had a diameter greater than 25 µm across. Size bar = 100 µm,

Figure 8

Intraplantar carrageenan induced an ipsilateral increase in P-GluR1 ser 845 at 1 (illustrated) and 2 (not shown) h after a single intraplantar carrageenan injection. Representative P-GluR1 ser845 blots along with β-actin loading controls are also illustrated. This increase in P-GluR2 ser 845 was blocked by spinal pretreatment with Etanercept (ETA, 100µg) 1 hour prior to carrageenan injection. *p<0.05, significantly different from naïve or control. n=3–4 rats/group