A proposed model of rapamycin’s neuroprotective effect during retinal inflammation.

<p>The basal level of activated mTOR promoted NF-κB activation during inflammation, which induced the retinal expression of the inflammatory cytokines, interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). Activated NF-κB elevated the activated and phosphorylated mTOR (pmTOR) level during inflammation, which may have exacerbated the pathogenesis. The subsequent retinal activation of signal transducer and activator of transcription 3 (STAT3), at least partly, reduced the rhodopsin protein expression in a post-transcriptional manner and impaired rod photoreceptor function. Rapamycin also attenuated inflammatory leukocyte adhesion to the vessel walls, and increased potentially neuroprotective autophagy in the retina during inflammation.</p

The interaction between mTOR and NF-κB in the retina during inflammation.

<p>Immunoblot analyses (A-H). The protein level of phosphorylated and activated mTOR (pmTOR) was not increased in the retina 3 hours after the induction of inflammation by an intraperitoneal lipopolysaccharide (LPS) injection, although the level of pmTOR was suppressed by the rapamycin treatment (A, B). At 12 hours after the LPS injection, the pmTOR expression was increased in the retina, which was suppressed by the rapamycin treatment (C, D). The pmTOR level in the retina at 12 hours was also suppressed by the NF-κB inhibitor, DHMEQ (E, F). The ratio of the LC3II to LC3I protein levels (an autophagy marker) in the retina was not changed after LPS injection but was increased by rapamycin at 12 hours after LPS injection (G, H); n = 5. *p < 0.05, **p < 0.01.</p

Rapamycin suppressed inflammatory molecules in the retina during inflammation.

<p>Immunoblot analysis showed that phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells p65 (pNF-κBp65) was increased in the retina 3 hours after the induction of inflammation by an intraperitoneal injection of lipopolysaccharide (LPS), and that this was attenuated by the rapamycin treatment (A, B). ELISA assays showed that the protein levels of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) were increased in the retina 12 hours after the induction of inflammation, and that these changes were attenuated by the rapamycin treatment (C, D). Real-time PCR showed that the mRNA levels of both <i>IL-6</i> and <i>MCP-1</i> were increased 6 hours after the induction of inflammation, and that the mRNA level of <i>IL-6</i> (E), but not that of <i>MCP-1</i> (F), was attenuated by the rapamycin treatment. Immunoblot analyses showed that the expression level of phosphorylated and activated signal transducer and activator of transcription 3 (pSTAT3) was increased at 12 hours after inflammation, and that this increase was attenuated by the rapamycin treatment (G, H); n = 5. *p < 0.05, **p < 0.01.</p

Rapamycin suppressed leukocyte adhesion in the retina during inflammation.

<p>Analysis of leukocyte adhesion in flatmount retinas showed that adherent leukocytes were minimally observed in the vehicle-treated control mice (A), and were substantially increased in the vehicle-treated EIU mice (B). However, rapamycin attenuated the number of adherent leukocytes (C). Quantification of adherent retinal leukocytes (D). Scale bar, 100 μm; n = 5. **p < 0.01.</p

Rapamycin suppressed visual function impairment during inflammation.

<p>Representative waveforms of an individual mouse recorded by electroretinography 24 hours after the induction of inflammation by an intraperitoneal injection of lipopolysaccharide (LPS) (A). The amplitudes of both the a-wave (B) and b-wave (C) were reduced during inflammation, and were attenuated by the rapamycin treatment. The implicit times of the a-wave (D) and the b-wave (E) were not changed; n = 6. *p < 0.05, **p < 0.01.</p

Rapamycin suppressed rhodopsin reduction and OS shortening during inflammation in a post-transcriptional manner.

<p>Immunoblot analysis showed that both the monomer and dimer forms of rhodopsin were reduced in the retina 24 hours after inflammation induced by an intraperitoneal injection of lipopolysaccharide (LPS), and that this reduction was attenuated by rapamycin treatment (A, B; monomer, black bars; dimer, white bars). Real-time PCR showed that the <i>rhodopsin</i> mRNA level was not changed in the retina during inflammation in the presence or absence of rapamycin at the same time points (C). Immunohistochemical staining for rhodopsin (green) showed that reduction of the photoreceptor outer segment (OS) length in the retina during inflammation was suppressed by rapamycin (D, E). Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (A-C) n = 5; (D, E) n = 4. *p < 0.05.</p