Decrease in activated AMPK in the neural retina during inflammation and its prevention by AICAR.

<p>(A, B) Immunoblot analyses. The ratio of p-AMPK/t-AMPK was significantly lower in the retina of vehicle-treated EIU mice than controls, 1.5 hours after LPS injection. AICAR administration significantly increased the ratio during EIU. Both p-AMPK and t-AMPK were measured after normalized to α-tubulin. (C) Immunohistochemistry of p-AMPK. Under control condition, p-AMPK was expressed throughout the retina and retinal pigment epithelium, but the staining was sparse in the retina of vehicle-treated EIU mice, 1.5 hours after LPS injection. The retina of AICAR-treated EIU mice showed clear expression of p-AMPK at the same time point. A negative control staining in the retina of a control animal was also shown. (A, B) *P<0.05. **P<0.01. Control, n = 8; EIU with vehicle treatment, n = 7; EIU with AICAR treatment, n = 8. p-AMPK, phosphorylated AMPK (activated form); t-AMPK, total AMPK; IPL, inner plexiform layer; OPL, outer plexiform layer; OS, outer segment.</p

AMPK-NF-κB Axis in the Photoreceptor Disorder during Retinal Inflammation

<div><p>Recent progress in molecular analysis has revealed the possible involvement of multiple inflammatory signaling pathways in pathogenesis of retinal degeneration. However, how aberrant signaling pathways cause tissue damage and dysfunction is still being elucidated. Here, we focus on 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK), originally recognized as a key regulator of energy homeostasis. AMPK is also modulated in response to inflammatory signals, although its functions in inflamed tissue are obscure. We investigated the role of activated AMPK in the retinal neural damage and visual function impairment caused by inflammation. For this purpose, we used a mouse model of lipopolysaccharide-induced inflammation in the retina, and examined the effects of an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). During inflammation, activated AMPK in the neural retina was decreased, but AICAR treatment prevented this change. Moreover, the electroretinogram (ERG) a-wave response, representing photoreceptor function, showed visual dysfunction in this model that was prevented by AICAR. Consistently, the model showed shortened photoreceptor outer segments (OSs) with reduced levels of rhodopsin, a visual pigment concentrated in the OSs, in a post-transcriptional manner, and these effects were also prevented by AICAR. In parallel, the level of activated NF-κB increased in the retina during inflammation, and this increase was suppressed by AICAR. Treatment with an NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) preserved the rhodopsin level during inflammation, suppressing NF-κB. These findings indicated that AMPK activation by AICAR and subsequent NF-κB inhibition had a protective effect on visual function, and that AMPK activation played a neuroprotective role during retinal inflammation.</p></div

Protective effect of AICAR on the rhodopsin level and OS length during inflammation.

<p>(A, B) Immunoblot analysis. Rhodopsin protein in the retina was decreased during EIU, and this decrease was attenuated by AICAR, 24 hours after LPS injection. (C) Real-time PCR. rhodopsin mRNA was constant 24 hours after LPS injection with or without AICAR treatment. (D) The OS length was shortened during EIU, and this influence was suppressed by AICAR. (E) Relative OS length was measured in the mid-peripheral retina. *P<0.05. **P<0.01. Scale bar, 50 µm. (A–C) Control, n = 4; EIU with vehicle treatment, n = 5; EIU with AICAR treatment, n = 5. (D, E) All groups, n = 6. ONL, outer nuclear layer; IS, inner segment; OS, outer segment.</p

Protective effect of AICAR on visual function during inflammation.

<p>(A–E) ERG data 24 hours after LPS injection. (A) Representative wave responses of scotopic ERG intensity series from an individual mouse. The amplitudes of the a-wave (B) and b-wave (C) were decreased during EIU, but AICAR treatment clearly prevented the decrease. The implicit times of the a-wave (D) and b-wave (E) were prolonged during EIU, but this effect was significantly avoided by AICAR. *P<0.05. **P<0.01. All groups, n = 8.</p

Preservation of rhodopsin level by an inhibitor of NF-κB activation, DHMEQ, during inflammation.

<p>(A, B) Immunoblot analysis. The rhodopsin level measured 24 hours after LPS injection was preserved by DHMEQ treatment. (C, D) The shortening of OS length during EIU was avoided by DHMEQ. Relative OS length was measured in the mid-peripheral retina. (E, F) Immunoblot analysis. The level of p-NF-κB p65 was decreased by DHMEQ in the retina 1.5 hours after LPS injection. *P<0.05. **P<0.01. Scale bar, 50 µm. (A, B) All groups, n = 8. (C, D) All groups, n = 5. (E, F) Control, n = 8; EIU with vehicle treatment, n = 8; EIU with AICAR treatment, n = 7. p-NF-κB p65, phosphorylated NF-κB p65.</p

Suppressive effect of AICAR on NF-κB activation during inflammation.

<p>(A, B) Immunoblot analysis. The level of p-NF-κB p65 was increased in the retina 1.5 hours after LPS injection. AICAR significantly blocked the increase of the p-NF-κB p65 level during EIU. *P<0.05. All groups, n = 7. p-NF-κB p65, phosphorylated NF-κB p65.</p