Bio-energetic preservation of cones in a mouse model of retinitis pigmentosa

ARVO Annual Meeting AbstractDaniel Narayan, Glyn Chidlow, John P M Wood, Robert James Casso

Expression of inducible heat shock proteins Hsp27 and Hsp70 in the visual pathway of rats subjected to various models of retinal ganglion cell injury

Inducible heat shock proteins (Hsps) are upregulated in the central nervous system in response to a wide variety of injuries. Surprisingly, however, no coherent picture has emerged regarding the magnitude, duration and cellular distribution of inducible Hsps in the visual system following injury to retinal ganglion cells (RGCs). The current study sought, therefore, to achieve the following two objectives. The first aim of this study was to systematically characterise the patterns of Hsp27 and -70 expression in the retina and optic nerve in four discrete models of retinal ganglion cell (RGC) degeneration: axonal injury (ON crush), somato-dendritic injury (NMDA-induced excitotoxicity), chronic hypoperfusion (bilateral occlusion of the carotid arteris) and experimental glaucoma. The second aim was to document Hsp27 and -70 expression in the optic tract, the subcortical retinorecipient areas of the brain, and the visual cortex during Wallerian degeneration of RGC axons. Hsp27 was robustly upregulated in the retina in each injury paradigm, with the chronic models, 2VO and experimental glaucoma, displaying a more persistent Hsp27 transcriptional response than the acute models. Hsp27 expression was always associated with astrocytes and with a subset of RGCs in each of the models excluding NMDA. Hsp27 was present within astrocytes of the optic nerve/optic tract in control rats. During Wallerian degeneration, Hsp27 was upregulated in the optic nerve/optic tract and expressed de novo by astrocytes in the lateral geniculate nucleus and the stratum opticum of the superior colliculus. Conversely, the results of our study indicate Hsp70 was minimally induced in any of the models of injury, either in the retina, or in the optic nerve/optic tract, or in the subcortical, retinorecipient areas of the brain. The findings of the present study augment our understanding of the involvement of Hsp27 and Hsp70 in the response of the visual system to RGC degeneration.Glyn Chidlow, John P. M. Wood, Robert J. Casso

Comment on 'A method to quantify regional axonal transport blockade at the optic nerve head after short term intraocular pressure elevation in mice by A. Korneva et al.' (Exp. Eye Res. doi: https://doi.org/10.1016/j.exer.2020.108035)

Letter to the EditorRobert J. Casson, Glyn Chidlow, John Woo

The responses of cone photoreceptors to retinal detachment in the rat

ARVO Annual Meeting AbstractGlyn Chidlow, W. Chan, J. P. Wood, R. J. Casso

Creatine is neuroprotective to retinal neurons in vitro but not in vivo

Purpose: To investigate the neuroprotective properties of creatine in the retina using in vitro and in vivo models of injury. Methods: Two different rat retinal culture systems (one containing retinal ganglion cells [RGC] and one not) were subjected to either metabolic stress, via treatments with the mitochondrial complex IV inhibitor sodium azide, or excitotoxic stress, via treatment with N-methyl-D-aspartate for 24 hours, in the presence or absence of creatine (0.5, 1.0, and 5.0 mM). Neuronal survival was assessed by immunolabeling for cell-specific antigens. Putative mechanisms of creatine action were investigated in vitro. Expression of creatine kinase (CK) isoenzymes in the rat retina was examined using Western blotting and immunohistochemistry. The effect of oral creatine supplementation (2%, wt/wt) on retinal and blood creatine levels was determined as well as RGC survival in rats treated with N-methyl-D-aspartate (NMDA; 10 nmol) or high IOP-induced ischemia reperfusion. Results: Creatine significantly prevented neuronal death induced by sodium azide and NMDA in both culture systems. Creatine administration did not alter cellular adenosine triphosphate (ATP). Inhibition of CK blocked the protective effect of creatine. Retinal neurons, including RGCs, expressed predominantly mitochondrial CK isoforms, while glial cells expressed exclusively cytoplasmic CKs. In vivo, NMDA and ischemia reperfusion caused substantial loss of RGCs. Creatine supplementation led to elevated blood and retinal levels of this compound but did not significantly augment RGC survival in either model. Conclusions: Creatine increased neuronal survival in retinal cultures; however, no significant protection of RGCs was evident in vivo, despite elevated levels of this compound being present in the retina after oral supplementation.Paul Ikgan Sia, John P. M. Wood, Glyn Chidlow, Robert Casso

Glucose protects cultured retinal cells from oxidative injury via pentose phosphate pathway activation and maintenance of reduced glutathione

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.Purpose : Oxidative injury has been implicated to play a role in a range of retinal neurodegenerative conditions. Thus, protecting retinal cells in vivo from such an insult is extremely beneficial. We therefore sought to investigate whether glucose, acting via the pentose phosphate pathway (PPP) was able to counteract oxidative stress to retinal cells in culture. Methods : Mixed retinal neuron-glial cultures were prepared from 2 day old rat pups and used at 7 days in vitro. Neuron-only and primary Muller cell cultures were prepared from mixed cultures, by treatment with cytosine arabinoside to kill dividing cells, or, by regular medium changes for 30 days, respectively. At appropriate stages, cultures were treated with t-butyl hydroperoxide (tbH; 100nM-10mM) in energy substrate-free DMEM to induce oxidative stress. Some cultures were co-treated with glucose (100µM-25mM) or other metabolic substrates (5mM; pyruvate, lactate, glutamine, fructose-1,6-bisphosphate). Glycolysis was inhibited with iodoacetate (IOA; 10µM) or 2-deoxy-D-glucose (2-DG; 1mM). PPP was inhibited with 6-aminonicotinamide (6NA; 500µM) and glutathione biosynthesis with buthionine sulphoxamine (BSO; 100µM). Cell viability, immunocytochemistry and Western blot were employed to assess cellular outcomes after treatments. An antioxidant assay identified which, if any, of the metabolic substrates tested had intrinsic antioxidant properties. Results : Oxidative stress resulted in loss of viability to mixed retinal cells and primary Muller cells: the EC50 for tbH was approximately 35µM in each case. Glucose dose-dependently reduced the toxicity of tbH with a maximal effect at 5mM (EC50 of tbH elevated to approximately 250µM). Pyruvate was also partially protective, but had intrinsic antioxidant properties. Glycolytic blockade had no effect on the protective effect of glucose but both 6NA and BSO inhibited the protective response. When cultured alone, neurons were equally susceptible to tbH-toxicity but could not be protected by glucose. Conclusions : Glucose prevented oxidative stress to retinal cells via the PPP and the consequent generation of reduced glutathione. Neurons were not subjected to glucose-induced protection except when glial cells were present, implying the passage of a transmissible protective factor between the two cell types.John P M Wood, Glyn Chidlow, Teresa Mammone, Robert James Casso

Glucose metabolism in mammalian photoreceptor inner and outer segments

Photoreceptors are the first-order neurons of the visual pathway, converting light into electrical signals. Rods and cones are the two main types of photoreceptors in the mammalian retina. Rods are specialized for sensitivity at the expense of resolution and are responsible for vision in dimly lit conditions. Cones are responsible for high acuity central vision and colour vision. Many human retinal diseases are characterized by a progressive loss of photoreceptors. Photoreceptors consist of four primary regions: outer segments, inner segments, cell bodies and synaptic terminals. Photoreceptors consume large amounts of energy, and therefore, energy metabolism may be a critical juncture that links photoreceptor function and survival. Cones require more energy than rods, and cone degeneration is the main cause of clinically significant vision loss in retinal diseases. Photoreceptor segments are capable of utilizing various energy substrates, including glucose, to meet their large energy demands. The pathways by which photoreceptor segments meet their energy demands remain incompletely understood. Improvements in the understanding of glucose metabolism in photoreceptor segments may provide insight into the reasons why photoreceptors degenerate due to energy failure. This may, in turn, assist in developing bio-energetic therapies aimed at protecting photoreceptors.Daniel S Narayan, Glyn Chidlow, John PM Wood and Robert J Casso

Expression and activation of SAPK/JNK in the ONH in a rat model of ocular hypertension

Teresa Mammone, Glyn Chidlow, Robert J Casson, John PM Woo

Neuroprotection in glaucoma: recent advances and clinical translation

Intraocular pressure (IOP) reduction is currently the only evidence-based treatment strategy for glaucoma. However, IOP control in some individuals is challenging. Despite optimal treatment, a significant proportion of individuals will progress, with loss of visual field, loss of driving vision and impaired quality of life. A new modality that could augment current treatment and reduce the rate of neurodegeneration to preserve vision throughout life would be a major breakthrough. A vast number of studies have reported effective neuroprotection in animal models of glaucoma; however, translation to the clinic remains a major hurdle. Herein, we explore the therapeutic advancements in non-IOP-dependent neuroprotection research based upon potential pathogenic mechanisms and propose strategies to improve the clinical translation of neuroprotective research in glaucoma.Chelsea Guymer, John PM Wood, Glyn Chidlow and Robert J Casso

Distribution and activity of mitochondrial proteins in vascular and avascular retinas: implications for retinal metabolism

Purpose:Understanding the energetics of retinal neurons and glia is crucial for developing therapies for diseases that feature deficits in nutrient or oxygen availability. Herein, we performed a detailed characterization of the distribution and activity of mitochondrial proteins in the vascularized retinas of rat and marmoset, and the avascular retinas of rabbit and guinea pig. Further, we delineated expression of ubiquitous mitochondrial creatine kinase (uMtCK). Methods:Expression of eight mitochondrial proteins was investigated using Western blotting, single- and double-labeling immunohistochemistry. Activities of cytochrome c oxidase, succinate dehydrgogenase, and isocitrate dehydrogenase were determined by enzyme histochemistry using unfixed tissue sections. Results:In vascularized retinas, immunoreactivities were characterized by strong, punctate labeling in the plexiform layers, photoreceptor inner segments, somas of various cell types, notably retinal ganglion cells (RGCs), and the basolateral surface of the retinal pigment epithelium. In avascular retinas, immunoreactivities featured intense labeling of inner segments, together with weak, but unambiguous, staining of both plexiform layers. RGCs were relatively enriched. In Müller cells of avascular retinas, mitochondria were restricted to scleral-end processes. For each species, enzyme activity assays yielded similar results to the protein distributions. Labeling for uMtCK in vascular and avascular retinas was fundamentally similar, being restricted to neuronal populations, most notably inner segments and RGCs. Of all of the mitochondrial proteins, uMtCK displayed the strongest labeling in avascular retinas. uMtCK was not detectable in Müller cells in any species. Conclusions:The current findings advance our understanding of the metabolic similarities and differences between vascular and avascular retinas.Glyn Chidlow, John P. M. Wood, Paul I. Sia, Robert J. Casso