Purpose.: Valproic acid (VPA) has been demonstrated to have neuroprotective effects in neurodegenerative conditions. VPA inhibits histone-deacetylases (HDAC) and delays apoptosis in degenerating neurons. The authors investigated whether VPA delays retinal ganglion cell (RGC) death and enhances axonal regeneration after optic nerve crush (ONC). Furthermore, potential molecular targets involved in VPA-mediated protection were analyzed. Methods.: ONC was performed on the left eye of rats, which received VPA or Ringer's solution subcutaneously (SC; 300 mg/kg twice daily) or intravitreally (single postlesional injection). Densities of fluorogold-labeled RGC were analyzed in retinal flatmounts after 5 or 8 days. Retinal tissue was also harvested and processed to quantify axon growth in retinal explants; evaluate caspase-3 activity; analyze transcription factor cAMP response element binding protein (CREB); and determine acetylated histone 3 and 4, as well as phosphorylated extracellular signal-regulated kinase (pERK) 1/2. Results.: Five and 8 days after ONC, 93% and 58% RGC survived after subcutaneous VPA treatment in comparison to Ringer's solution (62% and 37% viable RGC), respectively (P < 0.001). Likewise, a single intravitreal injection of VPA immediately after injury significantly delayed apoptosis in RGC (P = 0.0016). Injured RGC treated with VPA showed better regeneration of their axons in culture (196 axons/explant) than the crushed controls receiving Ringer (115 axons/explant). RGC axons of the right control eyes regenerated more after VPA treatment. VPA-mediated neuroprotection and neuroregeneration were accompanied by decreased caspase-3 activity, CREB induction, pERK1/2 activation, but not by altered histone-acetylation. Conclusions.: VPA provided neuroprotection and axonal regrowth after ONC. Alterations were observed in several pathways; however, the precise mechanism of VPA-mediated protection is not yet fully understood.

To measure the ciliary sulcus-to-sulcus (STS) diameter in 4 axes in emmetropic and myopic eyes using ultrasound biomicroscopy (UBM) and compare the measurements with automated horizontal white-to-white (WTW) diameter measurements. University Eye Hospital Freiburg, Freiburg im Breisgau, Germany. Evaluation of diagnostic technology. The STS diameter was measured at 0, 45, 90, and 135 degrees using 35 MHz UBM. The 0-degree WTW diameters were obtained using scanning-slit topography (Orbscan) and partial coherence interferometry (PCI) biometry (IOLMaster). The 0-degree STS and 90-degree STS were compared using the paired t test; the Pearson correlation was used to assess whether the 0-degree STS diameter could be predicted from the 0-degree WTW diameter. The mean SE refraction was -0.48 diopter (D) ± 0.35 (SD) in emmetropic eyes and -9.55 ± 3.70 D in myopic eyes. In 35 of 37 eyes, 90-degree STS was greater than 0-degree STS. The mean 90-degree STS was 12.51 ± 0.43 mm. The mean 0-degree STS was 12.19 ± 0.47 mm (P<.01). The mean 0-degree WTW diameters were 11.73 ± 0.37 mm (scanning-slit topography) and 12.20 ± 0.42 mm (PCI biometry). The correlations were good between 0-degree STS and 0-degree WTW with PCI biometry (r(2) = 0.82) and scanning-slit topography (r(2) = 0.86) in emmetropic eyes but weak between 0-degree STS and 0-degree WTW in myopic eyes (r(2) = 0.36 and r(2) = 0.40, respectively). Sulcus diameter measurements were most precise using UBM. The ciliary sulcus is vertically oval. The WTW diameter is not suitable for calculating a PC pIOL diameter, particularly in myopic eyes.

Purpose. Retinal ischemia/reperfusion (I/R) injury damages retinal neurons. Carbon monoxide (CO) recently attracted attention as cytoprotective because of its anti-inflammatory and antiapoptotic effects. Rapid preconditioning of retinal neurons by inhaled CO before I/R injury may reduce inflammation and apoptosis in retinal ganglion cells (RGCs). Methods. I/R injury was performed on the left eyes of rats (n = 8) with or without inhaled CO preconditioning (250 ppm) for 1 hour before ischemia. Densities of fluorogold-prelabeled RGCs were analyzed 7 days after injury in whole-mounts. Retinal tissue was further harvested to analyze protein expression of TNF-α, HSP-70, and mitogen-activated protein kinases (MAPKs) pERK1/2 and p-p38. DNA-binding activities of the transcription factors NF-κB, AP-1, CREB, and HSF-1 were determined to elucidate a possible pathway of neuroprotection. Results. Seven days after I/R injury, RGC death decreased by 52% in the CO preconditioning group compared with controls receiving room air (P < 0.001). Similarly, CO inhalation resulted in attenuated caspase-3 activity and TNF-α protein expression. In contrast, HSP-70 protein expression was elevated in the retina after CO. CREB and HSF-1 showed CO-dependent regulation and p-p38 MAPK. Conclusions. Rapid preconditioning with CO mediates anti-inflammatory and antiapoptotic effects in retinal I/R injury, thus making it neuroprotective. Further studies are needed to evaluate whether CO posttreatment may represent a therapeutic option counteracting ischemic neuronal injury.

Retinal ischemia/reperfusion (I/R) injury plays an important role in the pathophysiology of various ocular diseases. Retinal ganglion cells (RGCs) are particularly vulnerable to ischemia. Hydrogen sulfide (H2S) was recently shown to be neuroprotective in the brain and retina due to its antiapoptotic effects. Rapid preconditioning of retinal neurons by inhaled H2S before I/R injury may reduce apoptosis in the rat retina. I/R injury was created on the left eye of rats (n=8) with or without inhaled H2S preconditioning (80 ppm) for one hour before ischemia. Densities of fluorogold prelabeled RGCs were analyzed 7 days after injury in retinal whole mounts. Retinal tissue was harvested to analyze protein expression of heat shock protein (HSP)-90 and the mitogen-activated protein kinases (MAPKs) c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)1/2 and p38 to elucidate a possible pathway of neuroprotection. DNA binding activity of the transcription factors nuclear factor-kappa-light-chain-enhancer of activated B-cells (NF-κB), cyclic adenosine monophosphate response element binding protein (CREB), and heat shock element (HSE), as well as caspase-3 cleavage and activity, were determined. Retinal sections were further assessed using anti–glial fibrillary acidic protein staining. RGC death after I/R injury decreased by 41.5% after H2S preconditioning compared to room air (p<0.001). H2S inhalation before ischemia reduced caspase-3 cleavage (p<0.001) and attenuated caspase-3 activity (p<0.001). Furthermore, HSP-90 expression was significantly elevated in the retina after H2S preconditioning. NF-κB but not CREB or HSE showed specific, H2S-dependent regulation, as well as the MAPKs ERK1/2 and JNK but not p38. H2S preconditioning mediates antiapoptotic effects in retinal I/R injury, thus exhibiting neuroprotection. Based on these observations, H2S could represent a novel and promising therapeutic agent to counteract neuronal injuries in the eye. Further studies are needed to prove H2S’s neuroprotective propensity using a postconditioning approach.

Histone deacetylase inhibitors (HDACi) have neuroprotective effects under various neurodegenerative conditions, e.g., after optic nerve crush (ONC). HDACi-mediated protection of central neurons by increased histone acetylation has not previously been demonstrated in rat retinal ganglion cells (RGCs), although epigenetic changes were shown to be associated with cell death after ONC. We investigated whether HDACi can delay spontaneous cell death in purified rat RGCs and analyzed concomitant histone acetylation levels. RGCs were purified from newborn (postnatal day [P] 0–P2) rat retinas by immunopanning with antibodies against Thy-1.1 and culturing in serum-free medium for 2 days. RGCs were treated with HDACi, each at several different concentrations: 0.1–10 mM sodium butyrate (SB), 0.1–2 mM valproic acid (VPA), or 0.5–10 nM trichostatin A (TSA). Negative controls were incubated in media alone, while positive controls were incubated in 0.05–0.4 IU/µl erythropoietin. Survival was quantified by counting viable cells using phase-contrast microscopy. The expression of acetylated histone proteins (AcH) 3 and 4 was analyzed in RGCs by immunohistochemistry. SB and VPA enhanced RGC survival in culture, with both showing a maximum effect at 0.1 mM (increase in survival to 188% and 163%, respectively). Their neuroprotective effect was comparable to that of erythropoietin at 0.05 IU/µl. TSA 0.5–1.0 nM showed no effect on RGC survival, and concentrations ≥5 nM increased RGC death. AcH3 and AcH4 levels were only significantly increased in RGCs treated with 0.1 mM SB. VPA 0.1 mM produced only a slight effect on histone acetylation. Millimolar concentrations of SB and VPA delayed spontaneous cell death in purified RGCs; however, significantly increased histone acetylation levels were only detectable in RGCs after SB treatment. As the potent HDACi TSA was not neuroprotective, mechanisms other than histone acetylation may be the basis on which SB and VPA are acting in this model. Additional studies are necessary to identify HDACi-targeted genes and pathways involved in RGC protection.