Nigel L Barnett

• DPhil (Oxon)
• Professor (Associate) at Bond University

Oxidative stress is a major contributor to progressive neurodegenerative disease and may be a key target for the development of novel preventative and therapeutic strategies. Nitroxides have been successfully utilised to study changes in redox status (biological probes) and modulate radical-induced oxidative stress. This study investigates the effi...

The importance of Müller glia for retinal homeostasis suggests that they may have vulnerabilities that lead to retinal disease. Here, we studied the effect of selectively knocking down key metabolic genes in Müller glia on photoreceptor health. Immunostaining indicated that murine Müller glia expressed insulin receptor (IR), hexokinase 2 (HK2) and...

The retinal pigment epithelium [RPE] is a multifunctional monolayer located at the back of the eye required for the survival and function of the light-sensing photoreceptors. In Age-related Macular Degeneration [AMD], the loss of RPE cells leads to photoreceptor death and permanent blindness. RPE cell transplantation aims to halt or reverse vision...

While limbal epithelial cells are used for treating ocular surface wounds, the therapeutic potential of mesenchymal cells cultivated from the limbal stroma (LMSC) is less clear. We have therefore examined the effects of LMSC when applied to acute ocular surface wounds. LMSC derived from male rabbits (RLMSC) were applied to the ocular surface of fem...

The intravitreal route faces many challenges in rapidly and effectively reaching posterior eye pathology, with administered therapeutics experiencing non-specific distribution around and premature clearance from ocular tissues. Nanobubbles and ultrasound may improve outcomes of intravitreally administered drugs by influencing the directionality of...

Research is currently under way to produce tissue engineered corneal endothelium transplants for therapeutic use in humans. This work requires the use of model animals, both for the supply of corneal endothelial cells (CECs) for experimentation, and to serve as recipients for test transplants. A variety of species can be used, however, a number of...

Purpose Systemic increases in reactive oxygen species, and their association with inflammation, have been proposed as an underlying mechanism linking obesity and age-related macular degeneration (AMD). Studies have found increased levels of oxidative stress biomarkers and inflammatory cytokines in obese individuals; however, the correlation between...

Here we describe new fluorescent probes based on fluorescein and rhodamine that provide reversible, real-time insight into cellular redox status. The new probes incorporate bio-imaging relevant fluorophores derived from fluorescein and rhodamine linked with stable nitroxide radicals such that they cannot be cleaved, either spontaneously or enzymati...

Dual-acting hybrid anti-oxidant/anti-inflammatory agents were developed employing the principle of pharmacophore hybridization. Hybrid agents were synthesized by combining stable anti-oxidant nitroxides with conventional non-steroidal anti-inflammatory drugs (NSAIDs). Several of the hybrid nitroxide-NSAID conjugates displayed promising anti-oxidant...

Purpose: Complement system dysregulation is strongly linked to the progression of age-related macular degeneration (AMD). Deposition of complement including C3 within the lesions in atrophic AMD is thought to contribute to lesion growth, although the contribution of local cellular sources remains unclear. We investigated the role of retinal microg...

Herein we showcase the potential of ultrasound-responsive nanobubbles in enhancing macromolecular permeation through layers of the retina, ultimately leading to significant and direct intracellular delivery; this being effectively demonstrated across three relevant and distinct retinal cell lines. Stably engineered nanobubbles of a highly homogenou...

Supplementary protocols. (DOCX)

Ataxia-telangiectasia (A-T), an autosomal recessive disease caused by mutations in the ATM gene is characterised by cerebellar atrophy and progressive neurodegeneration which has been poorly recapitulated in Atm mutant mice. Consequently, pathways leading to neurodegeneration in A-T are poorly understood. We describe here the generation of an Atm k...

Mutations in the ataxia-telangiectasia (A-T)-mutated (ATM) gene give rise to the human genetic disorder A-T, characterized by immunodeficiency, cancer predisposition, and neurodegeneration. Whereas a series of animal models recapitulate much of the A-T phenotype, they fail to present with ataxia or neurodegeneration. We describe here the generation...

Light–induced degeneration in rodent retinas is an established model for of retinal degeneration, including the roles of oxidative stress and neuroinflammatory activity. In these models, photoreceptor death is elicited via photo-oxidative stress, and is exacerbated by recruitment of subretinal macrophages and activation of immune pathways including...

The New Zealand White rabbit has been widely used as a model of limbal stem cell deficiency (LSCD). Current techniques for experimental induction of LSCD utilize caustic chemicals, or organic solvents applied in conjunction with a surgical limbectomy. While generally successful in depleting epithelial progenitors, the depth and severity of injury i...

Nitroxides have been exploited as profluorescent probes for the detection of oxidative stress. In addition, they deliver potent antioxidant action and attenuate reactive oxygen species (ROS) in various models of oxidative stress, with these results ascribed to superoxide dismutase or redox and radical-scavenging actions. Our laboratory has develope...

Changes to the redox status of biological systems have been implicated in the pathogenesis of a wide variety of disorders including cancer, Ischemia-reperfusion (I/R) injury and neurodegeneration. In times of metabolic stress e.g. ischaemia/reperfusion, reactive oxygen species (ROS) production overwhelms the intrinsic antioxidant capacity of the ce...

Introduction: With the ever-increasing global burden of retinal disease, there is an urgent need to vastly improve formulation strategies that enhance posterior eye delivery of therapeutics. Despite intravitreal administration having demonstrated notable superiority over other routes in enhancing retinal drug availability, there still exist variou...

Sericin and fibroin are the two major proteins in the silk fibre produced by the domesticated silkworm, Bombyx mori. Fibroin has been extensively investigated as a biomaterial. We have previously shown that fibroin can function successfully as a substratum for growing cells of the eye. Sericin has been so far neglected as a biomaterial because of s...

It is routinely stated in the literature that Excitatory Amino Acid Transporter 5 (EAAT5) is a retina-specific glutamate transporter. EAAT5 is expressed by retinal photoreceptors and bipolar cells, where it serves as a slow transporter and as an inhibitory glutamate receptor, the latter role is due to the gating of a large chloride conductance. The...

Müller cells are the major glia of the retina that serve numerous functions essential to retinal homeostasis, yet the contribution of Müller glial dysfunction to retinal diseases remains largely unknown. We have developed a transgenic model using a portion of the regulatory region of the retinaldehyde binding protein 1 gene for conditional Müller c...

Excitatory amino acid transporter 5 (EAAT5) is an unusual glutamate transporter that is expressed in the retina, where it is localised to two populations of glutamatergic neurons, namely the bipolar neurons and photoreceptors. EAAT5 exhibits two distinct properties, acting both as a slow glutamate transporter and as a glutamate-gated inhibitory rec...

GLAST (EAAT1) is an abundant glial glutamate transporter in the mammalian brain. It plays important roles in terminating excitatory transmission in grey matter, as well as pathophysiological roles, including protecting white matter from excitotoxic injury. In normal brain, alternative splicing of GLAST has been described: GLAST1a and GLAST1b arise...

To investigate retinal cell population changes under chronic elevated intraocular pressure in an inducible mouse model of glaucoma. Chronic unilateral ocular hypertension was induced in 40 C57BL6/J mice by ablation of the limbal episcleral veins. After 5, 20, 40 and 60 days of elevated intraocular pressure, specific retinal cell types were identifi...

We have examined brains of neonatal pigs that were rendered hypoxic. Glutamine synthetase (GS), a key enzyme in the detoxification of glutamate and ammonia, was rapidly lost from astrocytes in regions susceptible to damage, including the CA1 of hippocampus and various cortical regions. Conversely, resilient areas such as the dentate gyrus exhibited...

The protein phosphatase 2B inhibitor, FK506, is an immunomodulatory polypeptide that has neuroprotective properties, the mechanisms of which have not been elucidated. A possible mechanism may be phosphorylation-mediated regulation of glutamate transporter activity. In the present study, we investigated the effect of FK506 on glutamate transporter l...

The survival and function of retinal neurons is dependent on mitochondrial energy generation and its intracellular distribution by creatine kinase. Post ischemic disruption of retinal creatine synthesis, creatine kinase activity, or transport of creatine into neurons may impair retinal function. S-adenosyl-L-methionine (SAMe) is required for creati...

Purpose: To determine the efficacy of rAAV.sFlt-1-mediated gene therapy in a transgenic mouse model of retinal neovascularization (trVEGF029) and to assess whether rAAV.sFlt-1 administration generated any deleterious, long-lasting immune response that could affect efficacy. Methods: trVEGF029 mice were injected subretinally with rAAV.sFlt-1 or p...

To correlate ganglion cell function with defined parameters of the elevated intraocular pressure profile (IOP) in a mouse glaucoma model and to determine the temporal relationship of these functional changes with ganglion cell death. Unilateral chronic ocular hypertension was induced in C57BL6/J mice by laser ablation of the limbal episcleral veins...

To investigate the relationship between intraocular pressure (IOP), retinal glutamate transport and retinal hypoxia during acute IOP elevations of varying magnitude. Female Dark Agouti rats were anaesthetized by ketamine/xylazine/acepromazine (10/5/0.5 mg/kg i.p.). The anterior chamber was cannulated with a 30-gauge needle attached to a saline rese...

This study tests whether cones in the rhodopsin-mutant transgenic P23H-3 retina are damaged by ambient light and whether subsequent light restriction allows repair of damaged cones. P23H-3 rats were raised in scotopic cyclic (12 hours of 5 lux, 12 hours of dark) ambient light. At postnatal day 90 to 130, some were transferred to photopic conditions...

In mammals there are two ubiquitous, catalytically indistinguishable isoforms of inosine monophosphate dehydrogenase and mutations in the type I isoform, but not type II, cause retina-specific disorders. We have characterised the spatio-temporal expression of these proteins during development of the rat retina and performed functional investigation...

It has previously been shown that inhibitors of protein kinase C (PKC) attenuate retinal glutamate uptake in situ. The aim of the current study was to determine whether PKCdelta-mediated inhibition differentially reduces the transport of glutamate into retinal Müller cells when compared with retinal neurons. The influence of two different types of...

Molecular Therapy (2006) 13, S160|[ndash]|S161; doi: 10.1016/j.ymthe.2006.08.482 418. AAV-Mediated sFLT-1 Gene Therapy for Ocular Neovascularization: Longevity, Toxicity and Efficacy Results from Mice and Monkeys E.P. Rakoczy1,2, C.M. Lai1,2, W.Y. Shen1,2, M. Brankov1,2, N. Barnett2,3, S.Y. Lee4, S.G. Leaver2, I. Yeo4, C.L. Ang4 and I.J. Constable...

The regulation of extracellular glutamate levels in the retina, under physiological and pathophysiological conditions, is essential for the prevention of excitotoxic neurodegeneration. Glial and neuronal high-affinity glutamate transporters (excitatory amino acid transporters, EAATs) facilitate the rapid removal of glutamate from the extracellular...

Gene therapy (GT) can be described as the in vivo transfer of DNA for therapeutic purposes. In the case of congenital retinal dystrophies (RD), GT can only be considered a successful treatment option if the gene transfer results in the restoration of vision at some level. Thus, a critical component of developing GT treatments for RDs is assessing t...

Vascular endothelial growth factor (VEGF) is one of the major mediators of retinal ischemia-associated neovascularization. We have shown here that adeno-associated virus (AAV)-mediated expression of sFlt-1, a soluble form of the Flt-1 VEGF receptor, was maintained for up to 8 and 17 months postinjection in mice and in monkeys, respectively. The exp...

Elevated extracellular concentrations of the neurotransmitter glutamate are neurotoxic and directly contribute to CNS damage as a result of ischemic pathologies. However, the main contributors to this uncontrolled rise in glutamate are still unconfirmed. It has been reported that the reversal of high-affinity glutamate transporters is a significant...

To determine whether the localization of retinal glutamate transporters is affected by retinal ischaemia and whether their ability to transport glutamate decreases with the progression of ischemic retinal and optic nerve degeneration. Retinal ischemia was induced in rats by acutely increasing the intraocular pressure (IOP, 110 mmHg/60 min). Reperfu...

Background Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65-/- knockout mice to ass...

There have been several reports demonstrating the potential of virus-mediated gene delivery for use in treating congenital retinal degenerations (Bennett and Maguire, 2000; Acland et al., 2001; Campochiaro et al., 2002; Narfstrom et al., 2002). A recent report on the restoration of vision to Briard dogs marked the first successful gene therapy for...

Age-related macular degeneration (AMD) is the major cause of blindness in the developed world. Its pathomechanism is unknown and its late onset, complex genetics and strong environmental components have all hampered investigations. Here we demonstrate the development of an animal model for AMD that reproduces features associated with geographic atr...

Neuronal and glial high-affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by fo...

Glutamate is the major excitatory neurotransmitter in the retina and is removed from the extracellular space by an energy-dependent process involving neuronal and glial cell transporters. The radial glial Müller cells express the glutamate transporter, GLAST, and preferentially accumulate glutamate. However, during an ischaemic episode, extracellul...

To reevaluate the longevity and intraocular safety of recombinant adenovirus (rAd)-mediated gene delivery after subretinal injection, and to prolong transgene expression through the combination of 2 synergistic immunosuppressants. An rAd vector carrying green fluorescent protein (GFP) gene was delivered subretinally in the rat eye. The GFP expressi...

Previous studies have demonstrated that adeno-associated virus (AAV) efficiently transduced retinal pigmented epithelial (RPE) cells. The goal of this study was to further evaluate and characterize transgene expression within the RPE cells over time in vivo. Adeno-associated virus-mediated gene transfer was monitored and quantified by retinal photo...

Exposure of isolated retinas to 30 microM D-aspartate, which is a substrate for all high affinity glutamate transporters, for 30 min, resulted in the accumulation of such D-aspartate into Müller glial cells but not glutamatergic neurons as evinced by immunocytochemistry for D-aspartate. Further incubation of such loaded retinas in physiological med...

It is widely assumed that neurones have sufficient metabolic reserves to allow them to function independently of glial cells for extended periods. The present study investigates the length of time taken before retinal neurones no longer respond normally to light after the inhibition of glial enzymes that are involved in the synthesis of precursors...

Excitatory amino acid transporter 5 (EAAT5) is a retina-specific glutamate transporter which has an associated chloride conductance. Thus it is comparable in its functional properties to the glutamate transport systems previously described in photoreceptors and some bipolar cells. We have raised antibodies to the carboxyl- and amino-terminal region...

To elucidate the role of the glial glutamate transporter GLAST, in the regulation of retinal function. Antisense oligonucleotides to GLAST were injected intravitreally into the left eye of Wistar rats. Sense oligonucleotides (control) were injected into the right eye over a period of 3 days. Scotopic flash electroretinograms were recorded over a 20...

The mammalian retina, like the rest of the central nervous system, is highly stable and can maintain its structure and function for the full life of the individual, in humans for many decades. Photoreceptor dystrophies are instances of retinal instability. Many are precipitated by genetic mutations and scores of photoreceptor-lethal mutations have...

The patterns of expression of the glutamate transporter GLAST were compared with the patterns of uptake of exogenous D-aspartate, which is a substrate for all glutamate transporters. At postnatal day 0, fine radial processes and end feet of presumptive Müller cells were weakly immunoreactive for GLAST. At postnatal day 3, intense labelling was asso...

Purpose. To elucidate the role of glial glutamate transporters in the regulation of retinal function. Methods. We administered antisense oligonucleotides to GLAST, a glutamate transporter which is expressed in retinal Müller cells, into one eye of each rat. Sense oligonucleotides (control) were injected into the other eye over a period of 5 days, a...

To compare the effects of glutamate agonists and different types of ischemic insult on choline acetyltransferase (ChAT) immunoreactivity in the rat retina. Rat retinas were exposed to different glutamate agonists in vivo or in vitro for specific periods of time, and the retinas were then fixed and processed for the localization of ChAT immunoreacti...

Reduction of the retinal blood flow by occlusion of both common carotid arteries suppressed the b-wave of the rat's electroretinogram. Transient occlusion of the carotids for 45 min reduced the b-wave by 46% without affecting the amplitude of the a-wave. The normal ERG activity returned 30 min after restoration of blood flow. Prolonged carotid occl...

gamma-Aminobutyric acid (GABA) is normally primarily in amacrine cells in the rat retina. Immediately after an ischaemic insult, attained by occlusion of the central retinal artery for 60 min, GABA is then found to be associated with Müller cells. During subsequent reperfusion, the distribution of GABA immunoreactivity gradually reverts from the gl...

Serotonin (5-hydroxytryptamine, 5-HT) reduces forskolin-induced stimulation of cyclic AMP in rabbit iris-ciliary body (ICB) homogenates. The effect is dose dependent and can be mimicked by a number of 5-HT1 receptor agonists including 5-carboxamidotryptamine (5-CT) and RU 24969 [5-methoxy-3-(1,2,3,6, tetrahydro-4-pyridinyl)-1-indole]. The inhibitor...

The rabbit isolated iris sphincter muscle maintained in an isotonic state is unaffected by applied serotonin (5-hydroxytryptamine or 5-HT) whereas carbachol causes the muscle to contract. Serotonin does, however, produce a relaxation of the contracted muscle in a dose-dependent manner. This effect is also induced by the 5-HT receptor agonists 8-OH-...

Endothelin (ET)-1 (10 nanoM) is about six times more effective than ET-3 in contracting the isolated iris sphincter muscle; the ET-1-induced contraction is insensitive to indomethacin treatment. The effect of ET-2 is intermediatory between ET-1 and ET-3 in contracting the muscle. The relative potency of the ETs to stimulate inositol phosphates (Ins...

The distribution of the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase was examined histochemically in the retina, iris, ciliary processes, cornea and conjunctiva of the rabbit eye. The epithelial cells of the ciliary process, iris, conjunctiva and, to a lesser extent, the cornea all showed intense staining. In the retina, st...

Intraocular injection of kainate into the rabbit eye causes both a translocation and transport of the bipolar cell's alpha PKC 6 h later. Although this effect is similar to what occurs for the phorbol ester, phorbol 12,13-dibutyrate (PDbut), it shows specificity in that N-methyl-D-aspartate (NMDA), 5,7-dihydroxytryptamine and 2-amino-4-phosphonobut...

Endothelin-1 (0.5-10 nM) produced a concentration-related contraction of the isolated iris sphincter muscle. The contraction rate of the muscle was slower for endothelin than carbachol, but endothelin was also more potent than carbachol, although the maximum contraction size was greater for carbachol. The endothelin response was unaffected by atrop...

The localisation and immunochemical identification of 3 different forms of protein kinase C (PKC-alpha, PKC-beta and PKC-gamma) in retinas of different species were analysed by immunohistochemistry and SDS-PAGE-Western blotting, respectively. Only in some cases was there a correlation between the findings from each procedure. One reason for the lac...

Using a monoclonal antibody against protein kinase C (PKC) that recognises the isoenzymes alpha, beta I, and beta II, positive immunoreactivity was observed throughout the cytoplasm of bipolar cells in both rat and rabbit retinas. PKC immunoreactivity was also associated with the outer segment of photoreceptors in the rabbit retina and presumed ama...

Monoclonal antibodies to DARPP-32 recognise an antigen which is present in specific neurones in the snail (Helix aspersa). Consecutive sections 10-microns-thick processed for the localisation of DARPP-32 and tyrosine-hydroxylase immunoreactivity did not show a coexistence in any neuronal structures. DARPP-32 positive cells were, however, often morp...

When exposed to exogenous serotonin, specific neurones in retinas from different species (guinea pig, rabbit, frog, pigeon and goldfish) take the amine up; these same neurones do not reveal endogenous serotonin following immunocytochemistry for the localization of the amine. The use of autoradiography and immunocytochemistry to localize the uptake...

Analysis of the mammalian retina for serotonin immunoreactivity suggests an absence of the amine. However, following an intraocular injection of forskolin (1 microM) into a rabbit eye 1 h before analysis of the retina, serotonin immunoreactivity is associated with a subpopulation of amacrine cells. These cells correspond in size and position to the...

During the past decade there has been considerable debate about whether serotonin (5-hydroxytryptamine) plays a transmitter role in the vertebrate retina. There is now strong support for the idea that serotonin is a transmitter substance in non-mammalian species, i. e. that serotonin is present in significant amounts in these retinas and can be loc...