Essential Role of Thioredoxin 2 in Mitigating Oxidative Stress in Retinal Epithelial Cells

The retina is constantly subjected to oxidative stress, which is countered by potent antioxidative systems present in retinal pigment epithelial (RPE) cells. Disruption of these systems leads to the development of age-related macular degeneration. Thioredoxin 2 (Trx2) is a potent antioxidant, which acts directly on mitochondria. In the present study, oxidative stress was induced in the human RPE cell line (ARPE-19) using 4-hydroxynonenal (4-HNE) or C2-ceramide. The protective effect of Trx2 against oxidative stress was investigated by assessing cell viability, the kinetics of cell death, mitochondrial metabolic activity, and expression of heat shock proteins (Hsps) in Trx2-overexpressing cell lines generated by transfecting ARPE cells with an adeno-associated virus vector encoding Trx2. We show that overexpression of Trx2 reduced cell death induced by both agents when they were present in low concentrations. Moreover, early after the induction of oxidative stress Trx2 played a key role in the maintenance of the cell viability through upregulation of mitochondrial metabolic activity and inhibition of Hsp70 expression

Measurement of Electroretinograms and Visually Evoked Potentials in Awake Moving Mice

<div><p>The development of new treatments for intractable retinal diseases requires reliable functional assessment tools for animal models. <i>In vivo</i> measurements of neural activity within visual pathways, including electroretinogram (ERG) and visually evoked potential (VEP) recordings, are commonly used for such purposes. In mice, the ERG and VEPs are usually recorded under general anesthesia, a state that may alter sensory transduction and neurotransmission, but seldom in awake freely moving mice. Therefore, it remains unknown whether the electrophysiological assessment of anesthetized mice accurately reflects the physiological function of the visual pathway. Herein, we describe a novel method to record the ERG and VEPs simultaneously in freely moving mice by immobilizing the head using a custom-built restraining device and placing a rotatable cylinder underneath to allow free running or walking during recording. Injection of the commonly used anesthetic mixture xylazine plus ketamine increased and delayed ERG oscillatory potentials by an average of 67.5% and 36.3%, respectively, compared to unanesthetized mice, while having minimal effects on the a-wave and b-wave. Similarly, components of the VEP were enhanced and delayed by up to 300.2% and 39.3%, respectively, in anesthetized mice. Our method for electrophysiological recording in conscious mice is a sensitive and robust means to assess visual function. It uses a conventional electrophysiological recording system and a simple platform that can be built in any laboratory at low cost. Measurements using this method provide objective indices of mouse visual function with high precision and stability, unaffected by anesthetics.</p></div

Comparison of VEPs recorded under awake and anesthetized conditions in the same mice.

<p>Representative traces of scotopic (top) and photopic (bottom) VEPs from the same mouse recorded in the awake (left) and anesthetized (right) condition. Amplitudes of scotopic (top) and photopic (bottom) VEPs from the same mice recorded in the awake (black) and anesthetized (red) condition. Implicit times of scotopic (top) and photopic (bottom) ERGs from the same mice recorded in the awake (black) and anesthetized (red) condition. * <i>P</i> < 0.05. Data from seven mice. The bars indicate mean ± S.E.M. anes: anesthetized.</p

Comparison of OPs extracted from ERGs acquired under awake and anesthetized conditions.

<p>Representative ERG traces before and after the extraction of OPs from a scotopic ERG (0 log cd s/m²). Schematic illustration showing the amplitude (a_n) and implicit time (t_n) of each OP wavelet (OP_n). C, D. Amplitudes (C) and implicit times (D) of individual OP wavelets (OP₁–OP₄) recorded in the awake (black) and anesthetized (red) condition from the same mice. E, F. Summed amplitudes (C) and implicit times (D) of all OP wavelets (OP₁–OP₄) from ERGs recorded under awake (black) and anesthetized (red) conditions from the same mice. * <i>P</i> < 0.05. Data from seven mice. The bars indicate mean ± S.E.M. anes: anesthetized.</p

Stability of the ERG and VEP recording.

<p>A, B, Amplitude time courses of ERG and VEP waveforms recorded in anesthetized mice (<i>N</i> = 4 for each time point). The flashes (0 log cd/m²) were given in the scotopic condition. The bars indicate mean ± S.E.M.</p

SIG-1451, a Novel, Non-Steroidal Anti-Inflammatory Compound, Attenuates Light-Induced Photoreceptor Degeneration by Affecting the Inflammatory Process

Age-related macular degeneration is a progressive retinal disease that is associated with factors such as oxidative stress and inflammation. In this study, we evaluated the protective effects of SIG-1451, a non-steroidal anti-inflammatory compound developed for treating atopic dermatitis and known to inhibit Toll-like receptor 4, in light-induced photoreceptor degeneration. SIG-1451 was intraperitoneally injected into rats once per day before exposure to 1000 lx light for 24 h; one day later, optical coherence tomography showed a decrease in retinal thickness, and electroretinogram (ERG) amplitude was also found to have decreased 3 d after light exposure. Moreover, SIG-1451 partially protected against this decrease in retinal thickness and increase in ERG amplitude. One day after light exposure, upregulation of inflammatory response-related genes was observed, and SIG-1451 was found to inhibit this upregulation. Iba-1, a microglial marker, was suppressed in SIG-1451-injected rats. To investigate the molecular mechanism underlying these effects, we used lipopolysaccharide (LPS)-stimulated rat immortalised Müller cells. The upregulation of C-C motif chemokine 2 by LPS stimulation was significantly inhibited by SIG-1451 treatment, and Western blot analysis revealed a decrease in phosphorylated I-κB levels. These results indicate that SIG-1451 indirectly protects photoreceptor cells by attenuating light damage progression, by affecting the inflammatory responses