Research into retinal function can be identified as taking place on a broad range of fronts and involving diverse international research groups. Investigations seek to improve the understanding of cell dynamics within normal functional parameters and also the specific details of disease processes, such as wet and dry age-related macular degeneration and glaucoma.
Researchers are cautious about predicting the future potential outcomes of their research programmes, though the current ‘road map’ anticipates at some stage the availability of treatments to slow disease processes and even reverse loss of function.
This article will take a look at some of this research areas with a focus upon studies into the role of metals within retinal metabolism, and the regeneration of retinal ganglion cells following trauma to the optic nerve or disease processes associated with glaucoma.
It is characteristic of research into regeneration of retinal cells that such work overlaps with more general nerve regeneration research including, for example, spinal injuries.
Metals within the retina
It has been appreciated for some time that the involvement of metals within the human retina in processes of cell metabolism is of significant importance. In particular, it has been identified that the trace elements iron, zinc, and copper have significant roles in maintaining normal retinal function. Research within this field has the potential to provide more precise information about these roles and thus identify mechanisms of potential disease prevention and treatment.
It is generally assumed that excess iron triggers tissue damage in the retina and is linked with age-related macular degeneration (AMD). One mechanism associated with this process has been thought to involve the creation of hydroxyl radicals through the process of Fenton’s reaction via the presence of hydrogen peroxide.
Gelfand et al,1 however, suggest that the mechanism of damage is more likely to involve a specific immune response pathway. This more complex involvement of iron metabolism in the retina is also referenced by García-Castiñeiras.2 For some time there has been an assumed link between the conversion of vitamin A to retinal via the mediation of zinc, though Christian et al3 point to the lack of evidence to confirm this.
Mapping metal distribution in the retina
Ugarte et al4 have used the novel technique of x-ray fluorescence to map the distribution of metals within the rat retina. High intensity yet extremely small cross section beams of x-rays generated by the diamond light source at Didcott in Oxfordshire were used to determine metal distribution within the retinas of animal subjects.
In the diamond light source, electrons are initially created in an electron ‘gun’ then accelerated in a linear accelerator and subsequently in a booster synchrotron before being injected into the main outer storage ring.
This ring is essentially a series of straight sections which include ‘insertion devices’ where electromagnetic energy ‘wiggles’ the electrons and in so doing causes the electrons to generate intense beams of photons of precise energy/wavelength. The radiated photons are then directed along beam lines to experimental areas where samples are irradiated. The beam is then ‘bent’ by collimating magnets into the next linear section of the storage ring.
Figure 1 shows the various beam lines available with the diamond light source. Each beam line is associated with specific groups of researchers investigating the properties of a wide range of materials/substances. A range of ophthalmic research projects currently utilise beam lines provided by the facility. Figure 2 shows an aerial view of the diamond light source facility.
Figure 1: Summary of beam lines of the Diamond Light Source Synchrotron (Courtesy Diamond Light Source)
Beam line I18 of the Diamond Synchrotron which supports ‘micro-focus spectroscopy’ was used for the retinal tissue scanning project. This allowed an extremely small cross section beam to be scanned across the target sample. Elements could be identified by their characteristic narrow lines of x-ray radiation, which is associated with energy transitions of electrons within their orbiting shell structures.
The energy of photons of the beam line is typically in range 2.05 - 20.5 keV (keV = 1000 electron volts). Characteristic lines of radiation for iron, copper and zinc are respectively 0.1936 nm, 0.1541 nm and 0.1435 nm with corresponding photon energies 6.403 keV, 8.045 keV and 8.639 keV and which lie within the photon energy range of the I18 beam line.
Studies in zinc
The Age-Related Eye Disease Study (AREDS)5 was a study undertaken in the USA by the National Eye institute to investigate a combination of dietary supplements on the development of age-related macular degeneration (AMD) and cataracts.
The initial study utilised a specific formulation of vitamin C, vitamin E, vitamin A, zinc oxide and cupric oxide where the latter was included to prevent zinc administration triggering copper deficiency anaemia. Within the study group of 3,640 patients monitored between 1992 and 2001, there was a 25% reduction of patients within one sub-group category developing advanced AMD who took the supplement formulation compared to a control group.
Subsequently, the AREDS2 study6 was undertaken in 2006 where variations in formulations were investigated which included the addition of omega-3 fatty acids, the addition of lutein and zeaxanthin, the removal of beta-carotene and the reduction of zinc from 80mg to 25mg. No apparent loss of effectiveness, however, was associated with the reduction of zinc concentration, though there remains some uncertainty regarding the minimum effective dose.
Specific studies relating solely to administration of additional zinc, tend to have inconclusive findings. One study by Newsome et al7 involving 151 patients with daily administration of 81mg of elemental zinc showed a reduced loss of visual function for patients with dry AMD but with no corresponding effect for patients with wet AMD.
The authors, however, comment on the possible toxic effects and complications of oral zinc administration, implying that such oral medication would probably be potentially problematic.
The body does not have a system of metabolising zinc from reserve supplies and around 1% of available zinc has to be replaced on a daily basis. Some evidence exists to indicate that zinc deficiency can be associated with reduced dark adaptation where Mockizuki et al8 observed impaired electroretinogram function for scotopic and photopic responses in a patient with zinc deficiency associated with liver dysfunction. Following restoration of appropriate zinc intake/metabolism, night vision and scotopic electroretinogram function responses were restored.
There would appear to be no documented evidence of excess zinc oral uptake resulting in retinal abnormalities. Wood and Watson9 describe, however, in vitro observations of retinal photoreceptor cell cultures which, when exposed to high levels of zinc, indicate rapid cell damage.
In independent work, Sheline et al10 have investigated the role of zinc chelation agents on the viability of cultured human retinal cell lines and also in vivo in animal studies following toxic levels of light exposure. It was identified that light damage in in vivo studies was associated with zinc (Zn2+) toxicity and that reduced cellular damage was observed where mechanisms of zinc chelation were adopted.
The authors identify administration of zinc chelation products as a possible treatment for light induced damage to the retina which could be sustained through, for example, sun exposure, lasers and intense pulsed light sources or surgical procedures. The authors, however, indicate that excessive doses of zinc chelation products can also result in retinal detachment.11
There has been increasing interest in the possible role of cadmium in the development of diseases such as age related macular degeneration.
Wills et al12,13,14 has investigated the concentration of cadmium within a selection of donor eyes in order to investigate any link with age and gender. It was identified that cadmium was higher in all retinal tissues for subjects greater than age 55 and that levels were significantly higher in the neural retina and retinal pigment epithelium of older females.
This suggested that mechanisms of accumulation of cadmium within the retina with age are gender-related and cadmium could be a risk factor in relation to the development of age-related macular degeneration.
Zinc is associated with key roles within the retina and new lines of enquiry are being followed by researchers, for example, at Boston Children’s Hospital and Harvard Medical School who are investigating the removal of excess levels of zinc as a mechanism for protection of the optic nerve and to encourage nerve tissue regeneration.
Towards nerve regeneration
A significant breakthrough in research in nerve regeneration at the FM Kirby Neurobiology Center and Department of Neurosurgery at Boston Children’s Hospital, under Professor Benowitz, was the discovery (reported by Berry et al15 at the University of Birmingham) that, in animal studies, the implantation of a peripheral nerve graft in the retina was able to trigger the growth of retinal ganglion cells and develop axons into the optic nerve.
Ongoing research by Professor Benowitz’s team has been able to confirm that such effects could be stimulated by the introduction of an inflammatory reaction. It was later found that the regenerative activity was associated with the production of a growth factor called oncomodulin.16
In addition, Benowitz et al17 described how research at other centres has identified other factors such as transcription factors and suppressors of axon growth which can trigger the growth of retinal ganglion cells. This has now provided researchers with a range of options for further research. The extent of regeneration, however, has been limited, and other factors have been investigated which introduce inhibitory regenerative effects.
Current work,18 however, is able to demonstrate in animal studies some measure of retinal cell regeneration and re-establishment of visual processes. This represents significant progress in the establishment of retinal cell regeneration and where such work is being progressed on a broad international front.
Some of the factors so far identified for optic nerve regeneration, however, potentially apply to other clinical disciplines involving nerve fibre regeneration following trauma. While some regeneration had been observed in animal studies, the level of regrowth had been relatively limited, and numerous studies were unable to demonstrate techniques to ensure significant nerve regeneration.
Collaboration with key researchers including Paul Rosenberg, also at the FM Kirby Neurobiology Center at the Boston Children’s Hospital, has identified19 that processes of injury to the optic nerve can result in significantly raised levels of zinc. In animal studies, it was found that levels of zinc ions increase by a factor of seven within an hour of initial injury to the optic nerve and this is subsequently transferred to retinal ganglion cells.
Based on these observations, it is thought that the time frame for reduction of zinc by chelation mechanisms extends for several days after initial injury. This identifies an area of research as the evaluation of intravitreal injections of zinc ion chelators to manage or even reverse disease processes such as glaucoma.
Introducing ORBIT
ORBIT (Ocular Repair by Integrated Technology) is a recently formed consortium of European biomedical and clinical neuroscientists with expertise in specialist fields such as neuroprotection and regeneration, myelination, inflammation and scarring. While this is a newly formed body, it links research groups which have an extensive history of research within neuronal regeneration.
Expertise is also available within enabling technologies such as the delivery of novel genes, cells and drug therapies. ORBIT has identified the need for the development of therapies to combat, for example, progressive retinal neurodegenerative diseases which are associated with significant socioeconomic impact within an ageing population.
A key contributor to ORBIT is Professor Martin Berry, of Birmingham University, whose initial work with regeneration of the optic nerve is credited with significantly advancing associated regenerative techniques across diverse centres of associated research.
Professor Keith Martin, an ORBIT member based at Cambridge Neuroscience, an interdisciplinary research centre within Cambridge University, has a key research focus based on understanding mechanisms of retinal ganglion cell degeneration and death within the development of glaucoma. Initially the aims of research at Cambridge would be to slow the process of associated visual loss, though the ultimate aim of the group would be to restore lost vision through the regeneration of retinal ganglion cells.20,21
There are indications, however, of developments within the research teams of ORBIT progressing to stages of clinical investigation. The spin-off company Neuregenix from work at Birmingham University, for example, provides a commercial vehicle for co-operative developments with the pharmaceutical industry.
Within its European context, the Department of Experimental Neurology at Heinrich Heine University in Dusseldorf has an active team linked to ORBIT which is actively researching axonal regeneration and where key lines of approach involve identifying the molecular basis of neuronal degeneration and developing new gene therapies and pharmacological agents as lines of clinical intervention.22,23 Dr Laurence Fitzhenry of the Waterford Institute of Technology in Ireland, is linked to the ORBIT group through a research interest in pharmaceutical delivery systems associated in particular with ocular drug delivery.
Summary
Significant advances have been made in the understanding of disease processes involving the retina and, where in specific work relating to retinal cell regeneration, this overlaps with more general work in neuronal regeneration. Such work, however, indicates that treatment processes are likely to include multifactorial components developed across multidisciplinary research teams.
Dr Douglas Clarkson is development and quality manager at the department of clinical physics and bio-engineering, Coventry and Warwickshire University Hospital Trust.
References
1 Gelfand BD, Wright CB, Kim Y, Yasuma T, Yasuma R et al. Iron Toxicity in the Retina Requires Alu RNA and the NLRP3 Inflammasome. Cell Rep. 2015; 11(11):1686-93. doi: 10.1016/j.celrep.2015.05.023.
2 García-Castiñeiras, S. (2010). Iron, the retina and the lens: a focused review. Experimental Eye Research 2010; 90(6), 664–678. http://doi.org/10.1016/j.exer.2010.03.003
3 Christian P, West KP Jr. Interactions between zinc and vitamin A: an update. Am J Clin Nutr. 1998 Aug;68(2 Suppl):435S-441S. doi: 10.1093/ajcn/68.2.435S
4 Ugarte, M, Osborne, NN, Brown, LA, Bishop, PN. Iron, zinc, and copper in retinal physiology and disease. Survey of ophthalmology. 2013; 58(6): 585-609. DOI: 10.1016/j.survophthal.2012.12.0022
5 Age-Related Eye Disease Study Research Group. AREDS Report No. 8: A Randomized, Placebo-Controlled, Clinical Trial of High-Dose Supplementation With Vitamins C and E, Beta Carotene, and Zinc for Age-Related Macular Degeneration and Vision Loss. Archives of Ophthalmology. 2001; 119 (10): 1417–1436. doi:10.1001/archopht.119.10.1417.
6 Age-Related Eye Disease Study 2 (AREDS2). NIH study provides clarity on supplements for protection against blinding eye disease. May 2013. Archived from the original on 2013-06-07. Lutein/Zeaxanthin and Omega-3 Fatty Acids for Age-Related Macular Degeneration. The Age-Related Eye Disease Study 2 (AREDS2) Controlled Randomized Clinical Trial. JAMA. 2013.
7 Newsome DA, Swartz M, Leone NC, Elston RC, Miller E. Oral zinc in macular degeneration. Oral zinc in macular degeneration. Arch Ophthalmol. 1988;106(2):192-8.
8 Mochizuki K, Murase H, Imose M, Kawakami H, Sawada A. Improvement of scotopic electroretinograms and night blindness with recovery of serum zinc levels. Jpn J Ophthalmol. 2006;50(6):532-536. doi: 10.1007/s10384-006-0376-3.
9 Wood JP, Osborne NN. The influence of zinc on caspase-3 and DNA breakdown in cultured human retinal pigment epithelial cells. Arch Ophthalmol. 2001;119(1):81-8.
10 Sheline CT, Zhou Y, Bai S. Light-induced photoreceptor and RPE degeneration involve zinc toxicity and are attenuated by pyruvate, nicotinamide, or cyclic light. Mol Vis. 2010 ;16:2639-52.
11 Yoo MH, Lee JY, Lee SE, Koh JY, Yoon YH. Protection by pyruvate of rat retinal cells against zinc toxicity in vitro, and pressure-induced ischemia in vivo. Invest Ophthalmol Vis Sci. 2004;45(5):1523-3
12 Wills NK, Ramanujam VM, Kalariya N, Lewis JR, van Kuijk FJ. Copper and zinc distribution in the human retina: relationship to cadmium accumulation, age, and gender. Exp Eye Res. 2008; 87(2):80-8. doi: 10.1016/j.exer.2008.04.013.
13 Wills NK , Kalariya N, Sadagopa Ramanujam VM, Lewis JR, Haji Abdollahi S, Husain A et al, Retinal cadmium accumulation as a factor in the etiology of age-related macular degeneration. Exp Eye Res. 2009;89(1):79-87. doi: 10.1016/j.exer.2009.02.014.
14 Wills NK, Ramanujam VM, Chang J, Kalariya N, Lewis JR, Weng TX et al, Cadmium accumulation in the human retina: effects of age, gender, and cellular toxicity. Exp Eye Res. 2008 ;86(1):41-51.
15 Berry M, Carlile J, Hunter A. Peripheral nerve explants grafted into the vitreous body of the eye promote the regeneration of retinal ganglion cell axons severed in the optic nerve. J Neurocytol. 1996;25(2):147-70.
16 Yin, Y., Cui, Q., Gilbert, H., Yang, Y., Yang, Z., Berlinicke, C. et al . Oncomodulin links inflammation to optic nerve regeneration. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(46), 19587–19592. http://doi.org/10.1073/pnas.0907085106
17 Benowitz LI, He Z, Goldberg JL Reaching the brain: Advances in optic nerve regeneration. Exp Neurol. 2017;287(Pt 3):365-373. doi: 10.1016/j.expneurol.2015.12.015.
18 de Lima S, Koriyama Y, Kurimoto T, Oliveira JT, Yin Y, Li Y, Gilbert HY et al., Full-length axon regeneration in the adult mouse optic nerve and partial recovery of simple visual behaviors. Proc Natl Acad Sci U S A. 2012;109(23):9149-54. doi: 10.1073/pnas.1119449109. .
19 Li Y, Andereggen L, Yuki K, Omura K, Yin Y, Gilbert HY et al, Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration. Proc Natl Acad Sci U S A. 2017;114(2):E209-E218. doi: 10.1073/pnas.1616811114.
20 Johnson TV, Martin KR, Cell transplantation approaches to retinal ganglion cell neuroprotection in glaucoma. Cur Opin Pharmacol . 2013;13(1)78-82
21 Bull ND, Irvine KA, Franklin KR. Transplanted oligodendrocyte precursor cells reduce neurodegeneration in a model of glaucoma Invest Ophthal Vis Sci. 2009 ;50(9):4244-53
22 Fischer D, Harvey AR, Pernet V, Lemmon VP, Park KK. Optic nerve regeneration in mammals: regenerated or spared axons: Exp. Neurology. 2017; 296 : 83-88
23 Fischer D Hyper il-6: a potent and efficacious simulator of RGC regeneration, Eye. 2016; 10 1038/eye.2016.234