Mitochondrial Inhibition in Rat Retinal Cell Cultures as a Model of Metabolic Compromise: Mechanisms of Injury and Neuroprotection

Abstract

Purpose.: Ourstudy aimed to establish a model of energetic and metabolic dysfunction to cultured retinal cells by chemically inhibiting the mitochondrial electron transport chain with sodium azide (NaN₃), and subsequently investigating toxic mechanisms and potential neuroprotective strategies.

Methods.: Mixed rat retinal cultures comprising neurons and glia were treated with a range of NaN₃ concentrations for up to 24 hours and toxicity levels were determined by immunologic methods. Detailed pathologic mechanisms were investigated by assessing apoptosis (TUNEL assay), mitochondrial membrane potential, reactive oxygen species (ROS), and levels of adenosine triphosphate (ATP). Finally, a number of pharmacologic agents were tested to determine whether they could abrogate the effects of NaN₃ to retinal cells.

Results.: Neurons and glia were killed by NaN₃ in a concentration- and time-dependent manner, with neurons being relatively more susceptible. Cell loss was via apoptosis for glia but not for neurons. Cell death generally involved a loss of mitochondrial membrane potential, a reduction in cellular ATP, and an increase in intracellular ROS levels. Glucose was partially able to prevent neuron death, as were the antioxidants trolox and pyruvate, calpain inhibitor III, the ryanodine receptor blocker dantrolene, and the nitric oxide synthase inhibitor L-NAME.

Conclusions.: Mitochondrial respiratory inhibition via NaN₃ treatment, with delineated mechanisms of toxicity and neuroprotection, represents a valid and reproducible metabolic challenge to cultured retinal cells.

Introduction

It is believed that localized metabolic compromise, either at the perikaryon or within the portion of the axon traversing the optic nerve head, comprises one of the initial insults to retinal ganglion cells, which eventually leads to their death in diseases, such as glaucoma. ^1–3 Such a decrease in metabolic functioning is likely related to underperformance of mitochondria, which could result from localized anoxia, hypoxia, or ischemia (likely due to a vascular deficit); mechanical tissue compression (caused, for example, by elevated intraocular pressure); aging; or an inherent mitochondrial mutation or dysfunction. ^2–5 Whatever the cause of the metabolic compromise, the result is a localized decline in intracellular adenosine triphosphate (ATP) levels and the subsequent failure of homeostatic mechanisms. ⁶ This results, for example, in increased intracellular levels of calcium, ⁵ activation of proteolytic calcium-dependent neutral protease (calpain) enzymes, ^7,8 elevations in cellular reactive oxygen or nitrogen species, ⁹ and stimulation of apoptotic pathways. ¹⁰ Furthermore, there also is an uncontrolled release of neurotransmitters from affected neurons leading to the persistent stimulation of ionotropic glutamate receptors on neighboring cells. ¹¹ All of these processes will contribute to neuronal pathology. 

One way to investigate the effects of metabolic insults to retinal neurons is to subject in vitro tissue preparations to simulated anoxia, hypoxia, aglycemia, or hypoglycemia. The most recognized means to subject such in vitro preparations to a simulated metabolic insult is to deprive cells of oxygen and glucose simultaneously, commonly referred to as oxygen-glucose deprivation (OGD). For example, retinal explants, ¹² mixed retinal cultures, ¹³ retinal-derived RGC-5 cells, ^14,15 and immunopurified retinal ganglion cells ¹⁶ all have been subjected to OGD and have all been shown to suffer different degrees of injury as a result. 

In practical terms, the means by which researchers experimentally reduce or remove the oxygen or glucose supply to an in vitro retinal preparation varies substantially. An alteration in the bathing concentration of glucose is easy to establish: prior known amounts of this sugar are dissolved in the incubation medium. Modifying the oxygen levels to an in vitro preparation, however, is more problematic. Usually, a gas mixture is used that consists of 5% CO₂ and varying proportions of oxygen (e.g., 0% for anoxia or 1–5% for hypoxia) balanced to 100% with N₂; thus replicating the normal culture incubator environment (5% CO₂), but with replacement of part or all of the atmospheric oxygen. The practical variation in this technique lies in the manner of application of such a gaseous milieu to the cell/tissue preparation. For accurate incubation of cells, medium must be bubbled in a sterile manner with the appropriate gas for a sufficient amount of time to remove the background oxygen, cells must be placed in a sealed and aseptic chamber for a suitable period of time to equilibrate the preparation with the new gas mixture and, upon terminating the experiment, no reoxygenation should be allowed. Despite the current use of a variety of specialist hypoxia/anoxia chambers, ^17,18 these criteria are very difficult to adhere to practically. Thus, results obtained from such experiments can vary considerably. 

To produce an experimental alternative to the effect of removing oxygen, which is used by the electron transport chain, researchers instead can use chemical inhibitors of mitochondrial respiratory reactions. These compounds will block specific mitochondrial reactions regardless of the basal level of oxygen, thus producing controllable levels of bioenergetic and metabolic compromise. In this manner researchers have used rotenone, which inhibits mitochondrial complex I reactions; ¹⁹ malonate, which inhibits complex II; ²⁰ antimycin A, which inhibits complex III; ²¹ and cyanide, which inhibits complex IV. ²² Since oxygen itself binds to complex IV, it would seem most appropriate to use an inhibitor of this component of the electron transport chain to mimic anoxia. A more practical alternative to cyanide as a cytochrome C oxidase or complex IV inhibitor, therefore, is sodium azide (NaN₃). ^23,24 Although this compound has been evaluated toxicologically in cortical ^25–27 and cerebellar ²⁸ brain neuron preparations, it has seen a more limited use in retinal studies. Osborne et al. have subjected retinal RGC-5 cells to NaN₃ and have shown that this agent kills cells via production of reactive oxygen species (ROS) and a caspase-dependent apoptotic process. ²⁹ Xiong et al. have shown retinal β-secretase (BACE1) expression and tissue stress in response to this compound in vivo. ³⁰  

Our study, therefore, was conducted to assess the characteristics and the reproducibility of using NaN₃ as a means to subject retinal cells to metabolic stress, as a more practical alternative to incubating such cells in an actual absence of gaseous oxygen. Mixed retinal cultures consisting of dissociated neurons and glia were used to test the effects of this metabolic inhibitor on different retinal cell types. Finally, we attempted to delineate the mechanisms of cell pathology using a range of pharmacologic agents to inhibit NaN₃-induced cell death. 

Materials and Methods

Materials

General cell culture media and reagents, including fetal bovine serum (FBS), were obtained from Invitrogen (Mulgrave, Victoria, Australia). Culture vessels (flasks and plates), polypropylene centrifuge tubes of all sizes, and CellPlus charge-coated 96-well plates were from Sarstedt Pty (Adelaide, Australia). All other general chemical reagents, except where noted, were from Sigma-Aldrich Chemical Company (Castle Hill, New South Wales, Australia). Antibodies used for immunologic detection of proteins and peptides of interest are detailed in Table 1. Finally, animals for culture were obtained from Adelaide University. 

Table 1. 

 

Antibodies Used in the Study

Target	Host	Clone/Cat. No.*	Dilution (ICC)	Dilution (WB)	Source
Neuronal
GABA	Rabbit	A2052	1:5000	-	Sigma
Tau	Rabbit	A0024	1:10,000	1:1000	Dako
PGP9.5	Mouse	31A3*	1:10,000	1:10,000	Cedarlane
Glial
Vimentin	Mouse	V9*	1:1000	1:1000	Dako
Nestin	Mouse	Rat 401*	1:1000	1:1000	BD Transduction
CD11b	Mouse	OX42*	1:500	-	Serotec
Other
HO-1 (Hsp32)	Rabbit	SPA-895	1:5000	-	Stressgen
Calpain I	Mouse	MAB3104	1:500	-	Millipore

*  Clone number, as delineated in column heading. ICC, immunocytochemistry; WB, Western blotting.

Cell Cultures

Sprague-Dawley rat litters (1–3 days post-partum, average of 10–12 pups per litter) were obtained along with their mothers and housed according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes 2004, and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Rat retinal cell cultures comprising glia, photoreceptors, and neurons were prepared from the pups essentially as described previously, via a trypsin- and mechanical-digest procedure. ¹³ After tissue dissociation, cells were dispensed either onto 13 mm diameter borosilicate glass coverslips coated previously with poly-D-lysine (5 μg/mL, 2 hours) in 24-well culture plates for immunocytochemical, fluorescent dye-labeling, or apoptotic analyses, or into 96-well plates (0.1 mL/well; CellPlus, charged-coat plates; Sarstedt) for ATP determination, or into poly-D-lysine-coated 6-well plates for immunoblotting analysis. Mean cell density at seeding was approximately 0.5 × 10⁶ cells/ml. Cultures subsequently were grown at 37°C in a humidified incubator with 5% CO₂ in growth medium (MEM containing 10% FBS, 91 mg/L gentamicin sulfate, 2.3 mg/L amphotericin B, and 25 mM glucose). 

Treatment of Cultured Cells

After 7 days in vitro, culture medium was changed and treatments begun. Incubations for up to 24 hours (as defined) were carried out in the presence or absence of NaN₃ (range of concentrations 1 μM–50 mM). Previous work has delineated that completely depriving retinal cultures of glucose (and the alternative energy substrates, pyruvate or lactate) leads to a massive loss of cells by 24 to 28 hours. ¹³ Therefore, since the effect of additional energy substrates alongside NaN₃ was to be determined, all experiments were performed in the presence of a reduced amount of glucose (1 mM) compared to the initial culture conditions (25 mM). However, this concentration of glucose was able to maintain cell viability for (at least) 24 hours, ¹³ which equated to the longest experimental treatment duration in the study. Where experiments were performed to determine potential abrogation of induced effects, test compounds (along with their appropriate vehicle formulations as control) were applied to cultures 1 hour before NaN₃ to allow sufficient time for target interaction before induction of metabolic injury. After treatment, cells were harvested in the appropriate manner for each type of analysis, as described below. 

Immunocytochemical Analysis of Cultures

Cells on coverslips were fixed with neutral-buffered formalin containing 1% methanol for 15 minutes and then washed in PBS (137 mM NaCl, 5.4 mM KCl, 1.28 mM NaH₂PO₄, 7 mM Na₂HPO₄; pH 7.4). Cells were permeabilized with PBS containing 0.1% (vol/vol) Triton X-100 (PBS-T), followed by further washing in PBS and then blocking in horse serum (3.3%, vol/vol in PBS [PBS-HS]). Test antisera (Table 1), diluted in PBS-HS, were applied overnight at room temperature, after which coverslips were washed in PBS and labeled by consecutive incubations with appropriate biotinylated secondary antibodies (Vector Laboratories, Abacus ALS, Brisbane, Australia; 1:250 in PBS-HS; 30 minutes) and streptavidin-AlexaFluor 488 or streptavidin-AlexaFluor 594 (Molecular Probes, Invitrogen; 1:500 in PBS-HS; 1 hour). Finally, cells on coverslips were mounted using anti-fade mounting medium (DAKO, Botany, New South Wales, Australia) and examined under a confocal fluorescence microscope. 

To quantify immunocytochemistry in cultures, two different techniques were used. The first was applied to antibodies that labeled clearly separate cells (e.g., GABA, cd11b [OX42], PGP9.5, βIII-tubulin); such cells were counted manually in 5 random fields per coverslip and on 6–8 distinct coverslips, and counts expressed as percentages of untreated control numbers. The second method, which was applied to antibodies that either labeled large numbers of overlapping cells (e.g., tau) or that could not distinguish cellular boundaries (e.g., nestin, vimentin), was to acquire five separate, representative images from each coverslip, for 6 to 8 coverslips, and use Image J (available online at http://rsb.info.nih.gov/ij/index.html) to quantify the amount of positive staining per field, relative to untreated control cultures. 

Western Immunoblotting of Retinal Culture Extracts

Cells were sonicated in homogenization buffer (20 mM Tris-HCl, pH 7.4, 25°C; containing 2 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 50 μg/mL leupeptin, 50 μg/mL pepstatin A, 50 μg/mL aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride). An equal volume of sample buffer (62.5 mM Tris-HCl, pH 7.4, containing 4% SDS, 10% glycerol, 10% β-mercaptoethanol, and 0.002% bromophenol blue) then was added and samples were boiled for 3 minutes; protein concentrations in each sample were equalized according to the method of Bradford. ³¹  

Electrophoresis was performed using a Mini-PROTEAN TGX precast gel system (Bio-Rad Laboratories Pty Ltd., Gladesville, New South Wales, Australia). Denaturing polyacrylamide gels (7.5%, 10%, or 12%, as appropriate) were used for protein separation, which was undertaken as described previously. ¹³ After separation, proteins were transferred to polyvinylidine fluoride (PVDF) membranes for immunoprobing (Bio-Rad Laboratories Pty Ltd.). Membranes were incubated with the appropriate antisera (as detailed in Table 1), overnight, and labeling carried out using a two-step detection procedure. First, appropriate biotinylated secondary antibodies (Vector Laboratories; 1:500; 30 minutes) were reacted with membranes and then streptavidin-peroxidase conjugates were applied (Pierce, Rockford, IL). Positive antibody labeling was detected as described previously, ¹³ and quantification of detected proteins was achieved using the program, Adobe PhotoShop CS2 (Adobe Systems Inc., San Jose, CA). Detection of β-actin was assessed in all samples as a positive gel-loading control. 

Determination of Cellular ROS

Incubations were performed as described except that for the final 30 minutes dihydroethidium (DHE, 5 μM) was added to wells, as described previously. ³² DHE is a non-fluorescent, reduced form of ethidium that can cross passively plasma membranes of live cells. When DHE is oxidized to ethidium by ROS, it can bind to DNA and yield “red” fluorescence (excitation 475 nm/emission 610 nm). ³³ After incubation, cells either were fixed in neutral-buffered formalin containing 1% methanol for 15 minutes and then washed in PBS for direct visualization by fluorescent microscopy or extracted into PBS, and then any increase in labeling was quantified in cell extracts by fluorescence spectroscopy using a fluorometer (Fluostar Optima; BMG Labtech, Mornington, Victoria, Australia). For quantification, fluorescence readings from 6 individual coverslips were averaged. 

Assessment of DNA Breakdown by TUNEL

For the TUNEL procedure, treated cells on coverslips were fixed as described for immunocytochemistry, washed in PBS containing PBS-T for 10 minutes and immersed in PBS. The labeling procedure was performed as described previously ³⁴ using the enzyme TdT to add the d-UTP label. Final cell labeling was visualized in our study, however, using streptavidin AlexaFluor 594. Some cells were treated after fixation but before TUNEL staining with DNAse I (0.1 mg/mL) for 15 minutes at 37°C to determine that the labeling procedure correctly identified DNA breakdown in nuclei. ³⁴ Cells subsequently were immuno-labeled for tau-immunoreactivity as described previously, to determine whether putative TUNEL-positive cells were neurons. To quantify the numbers of TUNEL-labeled nuclei, counts were obtained and averaged from 5 different randomly-selected fields of at least four coverslips from six separate cultures. 

Mitochondrial Transmembrane Potential

The dye, 5,5′,6,6′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to assess mitochondrial transmembrane electrical potential in treated cultures. ²⁵ Basically, this compound is taken up equally into all cells; when the intracellular milieu is healthy and a physiologic mitochondrial transmembrane potential exists, the dye labels fluorescently in the red area of the spectrum (i.e., red fluorescent mitochondrial labeling can be detected; excitation 550 nm and emission 600 nm). When cells no longer maintain a healthy mitochondrial membrane potential JC-1 cannot accumulate in mitochondria and the cytoplasm is flooded with the dye, which labels in the green area of the spectrum (excitation 485 nm and emission 535 nm). Methodologically, towards the end of the test incubation, a stock solution of JC-1 (Molecular Probes) in DMSO was added to treated cultures grown on glass coverslips at a final concentration of 1.0 μg/mL and plates were incubated in the dark for a further 20 minutes. Each coverslip then was mounted on a glass slide with anti-fade mounting medium, and photographed for red and green fluorescence. Obtained images were overlaid. In some cases, cultures treated experimentally and, subsequently, with JC-1 were washed with PBS, and then appropriate labeling was quantified in cell extracts by fluorescence spectroscopy (green wavelength excitation 485 nm, emission 535 nm; red excitation 550 nm, emission 600 nm) using a fluorometer (Fluostar Optima). Data obtained (n = 6 in each case) were calculated as the ratio of red-to-green fluorescence, such that as the green labeling increased, the ratio decreased. Note that cells were treated with NaN₃ for 6 hours to determine changes in mitochondrial membrane permeability. It was inferred from previous experiments that treatment for 24 hours, in line with the majority of the other tests, would give rise mostly to dead cells and, therefore, it would have been impossible to assess this parameter accurately. 

Measurement of Cellular ATP Content

This measurement was performed using a bioluminescence assay kit obtained from Sigma-Aldrich Chemical Company. Basically, cell samples were prepared by removing culture medium and extracting cellular contents, including ATP, into hot (65°C) distilled water for 5 minutes to denature ATP-metabolizing enzymes. ATP levels in each sample were determined in comparison with a standard curve using a standard luciferin-luciferase assay on a luminometer (Fluostar Optima). Note that incubations were carried out only for up to 6 hours, since as with the JC-1 analyses, it was inferred from previous experiments that a 24-hour treatment would give rise mostly to dead cells and, therefore, it would have been impossible to assess this parameter accurately. 

Statistics

All experiments were done with appropriate controls performed in the same plates, with at least 6 to 8 coverslips/wells used to generate each data point. Data were analyzed for significance using a one-way ANOVA followed by a Tukey multiple-comparison test. Data were expressed as mean percentage of control value plus SEM. A P value of <0.05 was considered significant. 

Results

Immunocytochemical Labeling of Cultures

To determine the effect of NaN₃ on cultured retinal cells, three antibodies were selected for neurons (anti-PGP9.5, anti-tau, and anti-GABA) and three for glia (anti-vimentin, anti-nestin, and anti-OX42). These respectively label three, nonmutually exclusive populations of retinal neurons and three, non-mutually exclusive populations of retinal glia. The reason that three markers were used in each case was to determine whether individual classes of neurons or glia responded differently to the insults. These labeling data also enabled us to ascertain that the effects seen when analyzing one labeled group of neurons or glia in the cultures were not unusual or erroneous when compared to the others. 

Of the neuron-labeling antibodies used, anti-GABA ³⁵ and anti-PGP9.5 ³⁶ are known to label subsets of retinal neurons in culture and anti-tau labels all retinal neurons (Fig. 1). For later quantitative studies, PGP9.5-positive neurons were selected for counting, since this antibody-labeling produced the greatest signal-to-noise ratio of the three tested. Of the glial markers used (Fig. 2), anti-vimentin labeled Müller cells and astrocytes, anti-nestin labeled astrocytes and Müller cells that had been “stressed” by the culture procedure, ³⁷ and anti-OX42 labeled cd11b-positive microglial cells. In latter studies, vimentin-IR glial cells were selected as representing quantitative effects to glial cells, since this labeling produced the greatest signal-to-noise ratio of those glial markers tested. Photoreceptors are not labeled by anti-vimentin or anti-PGP9.5 antibodies, and so their presence in the culture was not relevant to any of the quantitative investigations performed. 

Figure 1. 

 

The effect of 24-hour incubations with NaN₃ on neurons in retinal cultures, as demonstrated by immunocytochemistry. Three antibodies were used to assess the influence on three distinct antigens in neurons: tau (A–D), PGP9.5 (E–H), and GABA (I–L). Control labeling is seen for the requisite antibodies in (A), (E), and (I), and the effects of increasing concentrations of NaN₃ (100 μM B, F, J; 1 mM C, G, K; 10 mM D, H, L) also are shown. It is evident that as the concentration of NaN₃ was increased, then immunolabeling for each of the three antigens was diminished. Scale bar: 20 μm.

Figure 2. 

 

The effect of 24-hour incubations with NaN₃ on glial cells in retinal cultures, as demonstrated by immunocytochemistry. Three antibodies were used to assess the influence on three distinct antigens in glial cells: vimentin (A–D), nestin (E–H), and cd11b (using clone OX42; I–L). Control labeling is seen for the requisite antibodies in (A), (E), and (I), and the effects of increasing concentrations of NaN₃ (100 μM B, F, J; 1 mM C, G, K; 10 mM D, H, L) also are shown. It is evident that as the concentration of NaN₃ was increased, then immunolabeling for each of the three antibodies was diminished. Scale bar: 20 μm.

Effect of NaN₃ on Neurons and Glia in Mixed Rat Retinal Cultures

Treatment with 100 μM–10 mM NaN₃ led to a dose-dependent loss of immunoreactivity for tau, PGP9.5, and GABA in the cultures (Fig. 1). This was measured by quantifying changes in neuron-labeling as detailed in the Methods section (Fig. 3A). There was a close agreement in the loss of labeling for all three types of labeled neurons. Furthermore, by analyzing the temporal loss of PGP9.5-IR neurons alone (see above), it was determined that 50% of cells were no longer detectable after just over 6 hours when cultures were treated with 1 mM NaN₃ and after approximately 45 minutes when treated with 10 mM NaN₃ (Fig. 4). The Western immunoblot data shown in Figure 5 confirmed these findings by demonstrating the time-dependence of the influence of NaN₃ on loss of protein markers for neurons. Table 2 provides a summary of the overall findings by demonstrating that the concentration of NaN₃ required to elicit a 50% loss of PGP9.5-IR neurons decreased as time of incubation increased. 

Figure 3. 

 

Quantification of the effects of a 24-hour incubation with NaN₃ on neurons (A) and glial cells (B) in mixed rat retinal cultures. Immuno-labeled cells were quantified as described in the Methods section. Three antibodies were used to immuno-label cultures in each case (neurons tau, GABA, and PGP9.5; glial cells vimentin, nestin, and OX42) and the response to each is shown on the graphs relative to concentration of NaN₃ applied. All labeled cells declined in number in a concentration-dependent manner except for OX42-immunoreactive/cd11b-positive (micro) glial cells, which initially seemed to proliferate up to 100 μM NaN₃ before their decrease. Data are expressed as remaining labeled cells as percentages of untreated (vehicle-treated) control cells (n = 6–8 for each treatment).

Figure 4. 

 

Quantification of the increasing effect of NaN₃ on PGP9.5-positive neurons (A) and vimentin-positive glial cells (B), as time increases. PGP9.5-positive neurons and vimentin-positive glial cells were selected as being typical of their general cell-type (i.e., neurons and glia, respectively) for the purposes of this quantification. After the outlined treatments, cells were labeled appropriately by immunocytochemistry and then quantified as detailed in the Methods section. Data are expressed as remaining labeled cells as percentages of untreated (vehicle-treated) control cells (n = 6–8 for each treatment). *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA test followed by a Tukey multiple-comparison test.

Figure 5. 

 

Western immunoblot analysis of changes in detectable antigens in mixed rat retinal cultures after treatment with 500 μM NaN₃ for either 6 or 24 hours. (A) Example immunoblots (n = 3 samples shown for clarity) showing detected molecular masses; antigens expressed by neurons (tau, PGP9.5) and glial cells (vimentin, nestin) were analyzed. (B) Quantification of effects as exemplified in (A). Black bars: control untreated cells. Grey bars: cells treated for 6 hours. White bars: cells treated for 24 hours. Data were normalized to the untreated control culture levels (as 100%) and all were corrected for the level of actin detected in each sample to ensure that any potential differences in sample loading levels were negated. Data represent n = 6 samples for each treatment. *P < 0.05, **P < 0.01. One-way ANOVA test followed by a Tukey multiple-comparison test.

Table 2. 

 

EC50 Values for NaN₃ to Produce Cell Loss in Mixed Retinal Cultures at Defined Times

Cell-Type	1 Hour	6 Hours	24 Hours
PGP9.5-positive neurons	4.636	1.237	0.515
Vimentin-positive glial cells	21.412	14.592	1.751

 NB.PGP9.5-positive neurons and vimentin-positive glial cells were selected as being typical of their general cell-type for the purposes of this quantification. Cells were treated with various concentrations of NaN₃ for 3 different time periods and appropriately immunocytochemically labeled. After quantification, results were plotted and the EC₅₀ values for the requisite general cell-type calculated from plots. Data are derived from n = 6 individual determinations and concentration values are expressed in mM.

Figure 2 shows that NaN₃ influenced the number of glial cells detectable in retinal cultures after treatment for 24 hours. There was a concentration-dependent loss of nestin-IR and vimentin-IR cells, which decreased to less than 10% of control labeling by 24 hours when cultures were treated with greater than 5 mM NaN₃ (Fig. 3B). Conversely, there was an increase in the number of (micro)glial cells labeling for cd11b (OX42) after treatment with 100 μM NaN₃ for 24 hours, compared to untreated control cells. This was quantified and represented approximately 250% of the basal number of positively-labeling cells at its peak (Fig. 3B). When the NaN₃ concentration was increased further above 100 μM, the number of detectable cd11b-positive microglia decreased, such that none of these cells could be detected when 5 mM was administered. The influence of NaN₃ on glial cells not only was concentration-dependent, but also time-dependent (Fig. 4B). Western immunoblot experiments verified the findings for vimentin-IR and for nestin-IR positive labeling (Fig. 5), but also showed that neurons were relatively more susceptible to the insult than glia, as levels of neuron-specific proteins were reduced significantly 6 hours after treatment with 500 μM NaN₃, whereas levels of glial-specific proteins were not. These findings are confirmed in Table 2, which shows that the concentration of NaN₃ required to elicit 50% loss of vimentin-IR glial cells was much greater than for PGP9.5-IR neurons at 1, 6, and 24 hours. 

Mechanisms of Toxicity

Investigations subsequently were undertaken into putative mechanisms of NaN₃-induced retinal cell toxicity. Table 3 shows that NaN₃ toxicity in retinal cultures compared to that induced by the alternative mitochondrial toxins, oligomycin A, and antimycin A. All of these compounds were toxic to retinal cells and, indeed, were more toxic preferentially to neurons than glia. At the highest concentration tested, for example, antimycin A was responsible for the loss of all PGP9.5 immunolabeling but no loss of vimentin-IR cells. An approximate 1000-fold greater concentration of NaN₃ was required to elicit a similar effect on cells, however. 

Table 3. 

 

Comparison of Detrimental Influences of NaN₃ with Other Respiratory Inhibitors

Treatment	PGP9.5-Positive Neurons	Vimentin-Positive Glial Cells
NaN3 500 μM	56.2 ± 9.9*	84.4 ± 9.2
NaN3 1 mM	8.6 ± 4.1‡	61.7 ± 9.1*
Antimycin A 5 nM	75.3 ± 7.4*	104.6 ± 11.8
Antimycin A 50 nM	29.3 ± 6.1†	96.3 ± 8.1
Antimycin A 500 nM	0‡	99.8 ± 11.3
Oligomycin A 500 nM	84.5 ± 8.2*	107.2 ± 12.0
Oligomycin A 5 μM	12.7 ± 4.2‡	87.4 ± 11.6

 Treatments were all for 24 hours. PGP9.5-positive neurons and vimentin-positive glial cells were selected as being typical of their general cell-type for the purposes of this quantification. After the outlined treatments, cells were labeled appropriately by immunocytochemistry and then quantified as detailed in the Methods section. Data are expressed as remaining labeled cells as percentages of untreated (vehicle-treated) control cells (n = 6 for each treatment, except for NaN₃, where n = 10–12 determinations).

*   P < 0.05 by two-way ANOVA test followed by a Bonferroni correction.

†   P < 0.01 by two-way ANOVA test followed by a Bonferroni correction.

‡   P < 0.001 by two-way ANOVA test followed by a Bonferroni correction.

Figure 6A reveals that treatment with NaN₃ for 24 hours gave rise to an increase in detectable TUNEL-positive cell nuclei in retinal cultures. This was not colocalized with the neuron marker, tau, but did colocalize with the glial cell marker, vimentin, and was widely present even when the concentration was 1 mM, where many cells already had become undetectable (Fig. 1C). The incidences of detectable TUNEL-positive nuclei in relation to loss of cells coincided with the death of glia (Figs. 2, 3). 

Figure 6. 

 

Determination of some putative toxicologic mechanisms of action of NaN₃ on mixed rat retinal cultures after 24 hours of treatment. (A) TUNEL labeling for apoptotic cell death (red) does not colocalize with tau-immunoreactive neurons (green), but did colocalize with vimentin-positive glial cells (lower bank of figures; green). (B) HO-1 immunoreactivity. (C) immunolabeling for active μ-calpain. (D) detection of ROS with DHE. In each case, representative photomicrographs are shown for control, untreated cells, and cells treated with either 100 μM or 1 mM NaN₃ for 24 hours. Furthermore, quantification of results obtained is indicated by accompanying graph (n = 6). *P < 0.05 by one-way ANOVA test followed by a Tukey multiple-comparison test. Scale bar: 20 μm.

Haem oxygenase-1 (HO-1, Fig. 6B) and active μ-calpain (Fig. 6C) immunoreactivities increased as the concentration of NaN₃ was elevated. Levels of ROS as detected with DHE labeling also were increased as the concentration of NaN₃ was increased (Fig. 6D). Increased fluorescence was observed in all cells and was not confined specifically to either neurons or glia. 

Data shown in Figure 7 demonstrate that NaN₃ treatment caused a decrease in mitochondrial membrane potential for cells in the retinal cultures and that this effect was concentration-dependent. Control cells contained fluorescently-labeled mitochondria (emission 600 nm), which demonstrated that such organelles retained their membrane integrity (Fig. 7A). These cells contrasted with those treated with the apoptosis-inducing agent, staurosporine (Fig. 7D), which causes mitochondria in treated cells to lose their membrane potential. ³⁸ In the latter case all cells fluoresced in the green region of the spectrum (emission, 535 nm) and little red mitochondrial labeling could be seen, indicating loss of membrane potential as all JC-1 dye had leaked into the cytoplasm (Fig. 7D). Cells treated with NaN₃ demonstrated labeling that was intermediate between control and staurosporine-treated cells: when 1 mM NaN₃ was applied, red-labeled mitochondria still could be seen, even though some cellular cytoplasm was labeled green. When 10 mM NaN₃ was applied, there was little red-labeling of mitochondria, and cells mainly fluoresced in the green region of the spectrum. Quantification of this effect in Figure 7E showed that there was a linear relationship between concentration of NaN₃ and decrease in the red-to-green fluorescence labeling ratio. 

Figure 7. 

 

Determination of changes in mitochondrial membrane potential using the dye, JC-1. Red-labeled mitochondria represent those organelles that retain an intact membrane potential. When mitochondrial membrane potential is lost, green fluorescence results in the cellular cytoplasm. (A) Control, untreated cells. (B) Cells treated with 1 mM NaN_3. (C) Cells treated with 10 mM NaN_3. (D) Cells treated with 100 μM staurosporine (STSN) as a positive control for loss of mitochondrial membrane potential. Note, all incubations were performed for 6 hours. It is obvious that control cells fluoresce in the red region of the electromagnetic spectrum and as the concentration of NaN₃ is increased, cells take on a greater green fluorescence such that at 10 mM NaN₃, no red fluorescence appears to remain. Cells treated with STSN show no red fluorescence at all. Scale bar: 20 μm. (E) Quantification of results, as described in the Methods section. Labeling was determined by fluorescence spectroscopy (green wavelength excitation 485 nm, emission 535 nm; red excitation 550 nm, emission 600 nm) and the ratio of red-to-green fluorescence calculated. Loss of mitochondrial membrane potential was demonstrated by a reduction in the red-to-green ratio, and was seen clearly after treatment with STSN and with increasing concentrations of NaN₃. Data represent n = 6 samples for each treatment. *P < 0.05, **P < 0.01 by one-way ANOVA test followed by a Tukey multiple-comparison test.

NaN₃ treatment also led to a concentration- and time-dependent decrease in the level of cellular ATP in cultures (Figs. 8A, 8B, respectively). When incubations were done for one hour (Fig. 8A), total ATP in culture wells was decreased significantly relative to the untreated control level when the concentration of NaN₃ was at or above 100 μM (P < 0.05 for 100 μM, and P < 0.01 for 1 mM and 10 mM). By six hours after treatment (Fig. 8B) ATP levels had decreased to below 20% of control levels for 100 μM NaN₃, while for 10 mM the level of detectable ATP was negligible. 

Figure 8. 

 

Analysis of cellular ATP levels after treatment of mixed rat retinal cultures with NaN₃. (A) One-hour treatments delineated that there was a dose-dependent relationship between concentration of NaN3 used and loss of cellular ATP content. (B) The temporal relationship of NaN₃ concentration and ATP decrease is shown more clearly here; by six hours after treatment detectable ATP levels had decreased to below 20% of control levels in all cases; for 10 mM NaN₃ the level of detectable ATP was negligible. Data are expressed as percentages of values obtained untreated control cells and represent n = 6 samples for each treatment. *P < 0.05, **P < 0.01 by one-way ANOVA test followed by a Tukey multiple-comparison test.

Protection of Cultures from NaN₃-Induced Toxicity

Several pharmacologic agents were tested alongside NaN₃ treatments to determine whether they could abrogate the toxic responses of this compound to PGP9.5-IR neurons (Table 4). These data also served to provide additional information as to the mechanisms of toxicity of NaN₃ to rat retinal cultures. 

Table 4. 

 

Influence of Additional Agents on NaN₃-Induced Loss of Retinal Neurons

Compound	Action	500 μM NaN3	1 mM NaN3
Control	–	56 ± 9	8 ± 4
+ 25 mM glucose	Energy substrate	94 ± 7†	72 ± 9*
+ 5 mM pyruvate	Energy/antioxidant	74 ± 6*	45 ± 2*
+ 10 μM L-NAME	Nonselective NOS inhibitor	80 ± 11*	13 ± 5
+ 10 μM Trolox	Antioxidant	83 ± 5*	43 ± 11*
+ 5 μM MK801	NMDA-R inhibitor	49 ± 4	6 ± 2
+ 25 μg/mL calpain inhibitor III	Calpain inhibitor	75 ± 4*	39 ± 4*
+ 5 μM thapsigargin	Blocks ER Ca2+ takeup	19 ± 8*,‡	0*,‡
+ 10 μM SN50	NF-κB inhibitor	42 ± 11	12 ± 7
+ 100 μM olomoucine	Cdk2 inhibitor (blocks astrogliosis)	52 ± 6	21 ± 13
+ 10 μM dantrolene	RyR blocker (decreases cellular Ca2+ levels)	76 ± 5*	50 ± 11*

 All treatments were for 24 hours in the presence of 1 mM glucose. Data were determined by quantifying alterations in numbers of PGP9.5-positive neurons per field; this class of neurons was selected as representing a typical example of the general cell-type for the purposes of this quantification. Data are expressed as percentage of remaining labeled cells as related to untreated (vehicle-treated) control cells (n = 6 determinations for each treatment). Data were compared to treatment with NaN₃ alone (for statistical purposes) to determine whether each additional test compound was able to ameliorate or exacerbate the effects of this toxin. RyR, ryanodine receptor.

*   P < 0.05 compared to NaN₃-alone-treated by two-way ANOVA test followed by a Bonferroni correction.

†   P < 0.01 compared to NaN₃-alone-treated by two-way ANOVA test followed by a Bonferroni correction.

‡  Exacerbated the effects of NaN₃ (P < 0.05).

Treatment of retinal cultures with NaN₃ led to significant decreases in numbers of detectable PGP9.5-IR neurons to 56 ± 9% (500 μM NaN₃) and 8 ± 4% (1 mM NaN₃) of untreated, control cell counts (Table 4). Increasing the background level of glucose to 25 mM served to provide the greatest protection to neurons that were treated with 500 μM NaN₃ (94 ± 7% relative to control cells, P < 0.01 compared to cells without additional glucose) or 1 mM NaN₃ (72 ± 9%, P < 0.05). Inclusion of pyruvate, which can act as an energy substrate or an antioxidant, ³⁹ also offered a significant degree of protection (500 μM NaN₃ 74 ± 6%, P < 0.05; 1 mM NaN₃ 45 ± 2%, P < 0.05), as did the specific antioxidant, trolox (500 μM NaN₃ 83 ± 5%, P < 0.05; 1 mM NaN₃ 43 ± 11%, P < 0.05), calpain inhibitor III (500 μM NaN₃ 75 ± 4%, P < 0.05; 1 mM NaN₃ 39 ± 4%, P < 0.05), and the ryanodine receptor blocker, dantrolene (500 μM NaN₃ 76 ± 5%, P < 0.05; 1 mM NaN₃ 50 ± 11%, P < 0.05). The non-specific nitric oxide synthase (NOS) inhibitor, L-nitro-L-arginine methyl ester (L-NAME) was able to protect significantly neurons from 500 μM NaN₃ (80 ± 11, P < 0.05), but not 1 mM NaN₃. The endoplasmic reticulum (ER) calcium ATPase blocker, thapsigargin, conversely, enhanced the toxic response of cells to NaN₃ (500 μM NaN₃ 19 ± 8%, P < 0.05; 1 mM NaN₃ 0%, P < 0.05). The N-methyl-D-aspartate (NMDA) receptor antagonist, MK801; the cyclic dependent kinase (Cdk) inhibitor, olomoucine; and the NF-kB blocker, SN-50, all were without effect. 

In the case of vimentin-positive glial cells, only 1 mM NaN₃ was tested (because 500 μM did not have a significant detrimental effect on glial cells after 24 hours). Results are shown in Table 5. Treatment with 1 mM NaN₃ led to a reduction to 62 ± 9% of control levels of vimentin-positive cells. Only glucose (25 mM; 95 ± 4%, P < 0.05), trolox (10 μM; 86 ± 4%, P < 0.05), and dantrolene (10 μM; 89 ± 6%, P < 0.05) significantly reduced the toxic effect of 1 mM NaN₃ to vimentin-positive glial cells. As for neurons, thapsigargin, significantly enhanced the toxic influence of NaN₃: only 23 ± 8% of vimentin-positive cells remained after treatment (P < 0.05). 

Table 5. 

 

Influence of Additional Agents on NaN₃-Induced Loss of Retinal Glia

Compound	1 mM NaN3
Control	62 ± 9
+ 25 mM glucose	95 ± 4*
+ 5 mM pyruvate	65 ± 7
+ 10 μM L-NAME	55 ± 11
+ 10 μM Trolox	86 ± 4*
+ 5 μM MK801	54 ± 9
+ 25 μg/mL calpain inhibitor III	70 ± 5
+ 5 μM thapsigargin	23 ± 8*,†
+ 10 μM SN50	64 ± 5
+ 100 μM olomoucine	52 ± 12
+ 10 μM dantrolene	89 ± 6*

 All treatments were for 24 hours in the presence of 1 mM glucose. Data were determined by quantifying alterations in numbers of vimentin-positive glial cells per field; this class of glial cell was selected as representing a typical example of the general cell-type for the purposes of this quantification. Data are expressed as percentage of remaining labeled cells as related to untreated (vehicle-treated) control cells (n = 6 determinations for each treatment). Data were compared to treatment with NaN₃ alone (for statistical purposes) to determine whether each additional test compound was able to ameliorate or exacerbate the effects of this toxin.

*   P < 0.05 compared to NaN₃-alone-treated by two-way ANOVA test followed by a Bonferroni correction.

†  Exacerbated the effects of NaN₃ (P < 0.05).

Discussion

Our study described an experimental paradigm for treatment of cultured retinal cells, which, by inhibiting mitochondrial respiratory function with NaN₃, represents a model of cellular metabolic dysfunction useful for assessment of damage mechanisms and putative protective strategies. Therefore, the present paradigm can act as a convenient, reproducible, and, indeed, intrinsically calibrated means to assess retinal cellular responses to injury. Furthermore, by using NaN₃ to stifle mitochondrial activity concurrently with reducing or removing glucose and/or other nutrients (e.g., serum growth factors) in the bathing medium, related but distinct and equally useful models of metabolic compromise can be produced. 

Our data showed by immunocytochemistry and Western immunoblotting that NaN₃ causes a reproducible degree of toxicity to cells in mixed rat retinal cultures. This is in agreement with previous studies showing that this agent induces non-specific cell death in cortical, ^25,40–43 cerebellar, ²⁸ and motor neuron cultures. ⁴⁴ In our study, however, the specific effects of NaN₃ on neurons and glial cells were delineated, rather than just those on all cells in a non-specific manner. The most obvious conclusion to be drawn from such analyses is that higher concentrations of NaN₃ were required to elicit the same toxic effect to glial cells as to neurons (Table 2, for example). It is known that the major action of NaN₃ is to inhibit cytochrome C oxidase (complex IV), in effect inhibiting the process of mitochondrial oxidative phosphorylation. ²⁴ Thus, cells that rely more upon this process to meet their ATP demands likely would be more susceptible to mitochondrial respiratory inhibition. This is, of course, the case in the retina, where it is known that neurons produce much of their energy through mitochondrial reactions, whereas photoreceptors and glial cells produce much of their ATP through glycolysis. ^13,45 It is interesting to note, however, that the retina as a whole, readily undergoes compensatory metabolic alterations to become a predominantly glycolytic tissue when challenged with mitochondrial inhibition or anoxia/hypoxia. ^12,46,47 This is the Pasteur effect and it describes that in the intact state, the retina can use glucose catabolism alone to meet its metabolic needs in times of low oxygen availability. Our data indicated that this is, indeed, the case even when the tissue has been dissociated, thus validating the use of the current culture model for these studies. Obviously, for in vitro investigation of retinal metabolism in an intact system, whole retinal explants (e.g., Bull et al. ⁴⁸ ) also would prove useful. 

A number of distinct retinal neuronal and glial markers were used in investigating the effects of NaN₃ on retinal cultures. In the case of neurons, tau-positive, GABA-positive, and PGP9.5-positive cells were analyzed. Although GABA-positive and PGP9.5-positive neurons represented different (but overlapping) cell populations, all such neurons also labeled for the microtubule-associated protein, tau. Indeed, as far as can be ascertained, all neurons (except photoreceptors) were immuno-labeled positively for tau. Furthermore, the antibodies for GABA ^49,50 and PGP9.5 marked cell bodies and neurites of positively-labeling cells enabling their clear identification, whereas in the case of tau, labeling was present mainly in dendrites and only marginally in the perikaryal periphery. Overall, however, it was clear that the relative gross effect of NaN₃ on the three identified classes of neurons was the same: a time- and concentration-dependent loss of each cell-type with similar relative kinetic properties. This being the case, only one class of neuron was selected for the detailed analyses that followed. Due to the low signal-to-noise ratio of PGP9.5-positive labeling, compared to the other two antibodies used for neuronal identification, this class of cell was selected for subsequent investigations. 

Similarly, three distinct classes of glial cells were identified in the cultures by using three different glial-specific antibodies: vimentin-IR cells, nestin-IR cells, and cd11b-IR cells were detected. Vimentin has been described as a Müller cell-specific marker in the retina, ⁵¹ but, in fact, this intermediate filament protein also is expressed by astrocytes. ⁵² Nestin is not expressed in untreated rat retinas, but is induced in astrocytes and Müller cells upon tissue injury similar to the significant increase seen in expression of glial fibrillary acidic protein (GFAP). ⁵¹ Anti-GFAP was not used itself for glial characterization in our study because use of this antiserum gave a lower signal-to-noise ratio and greater background labeling than nestin-IR, which therefore was chosen in preference. In contrast to vimentin and nestin, cd11b is labeled by the OX42 antibody in microglial cells. ⁵³ Also in contrast to vimentin-IR and nestin-IR cells, the initial response of the cd11b-IR microglia to NaN₃ (up to 100 μM; 24 hours) in the cultures is an increased labeling: a greater number of microglia are detected. This could be due to a proliferation of existing microglia or the novel expression of cd11b in microglia that lack this antigen before treatment. It is likely, however, to be the former since proliferation of cd11b-positive microglia is known to occur in the retina, in vivo, in response to experimentally-induced injury. ⁵³ Regardless of the cause of the increased labeling, when concentrations of more than 100 μM NaN₃ were applied to cultures, then the number of detected cd11b-positive microglia declined in a concentration-dependent manner. It is interesting to speculate as to whether the NaN₃ acted directly on the microglia or whether these cells proliferated/altered cd11b expression in response to factors released by dying cells (neurons). Furthermore, the response of the microglia to mitochondrial challenge of the cultures is an interesting phenomenon: do they subsequently attempt to phagocytose dead material or are they merely dying themselves? 

In our study, toxicity of NaN₃ to neurons and glia was shown to be mediated through a variety of mechanisms, namely a reduction of ATP, an increase in apoptosis (as detected by the TUNEL reaction), an increase in production of ROS, an increase in expression of active μ-calpain, an increase in expression of HO-1, and a loss of mitochondrial membrane potential (Figs. 6–8). All of these events are likely to be associated with suboptimal mitochondrial functioning and consequent general metabolic stress to a cell. The fact that mitochondrial inhibition will readily kill retinal cells (glia and neurons) also is shown by using the alternative mitochondrial poisons antimycin A (complex III inhibitor) and oligomycin A (mitochondrial ATP synthase inhibitor, Table 3). It should be noted, however, that the TUNEL-positive cells that were detected were not colocalized with tau-IR, but were colocalized with vimentin-IR (Fig. 6A) so, therefore, they must have been glial in origin. This is in agreement with a previous study showing that NaN₃ killed neurons by a nonapoptotic process. ²⁵ Furthermore, Osborne et al. showed that NaN₃ killed undifferentiated RGC-5 ganglion cells via apoptosis, ²⁹ but the recent doubt thrown onto the phenotype of such cells, that is that they actually behave like glial cells before differentiation, ^54,55 also is in agreement with the present result that neurons are killed rapidly by nonapoptotic means and that glial cells die by apoptosis. 

If, however, the toxicity profile of NaN₃ is related solely to its inhibition of cytochrome C oxidase, and hence to a failure of the incumbent cell to synthesize sufficient ATP for maintenance of cellular and particularly membrane homeostasis, then it may be expected that addition of saturating levels of glucose alone would protect cultures. This is the case particularly with retinal cells, which, as mentioned previously, have a propensity to convert predominant energy production to glycolysis under situations of mitochondrial stress. ^12,45,46 In fact, glucose does offer significant protection for neurons in this study, but this NaN₃-induced toxicity prevention is not complete: there still is a small amount of neuron loss, even when the glucose concentration is elevated to 25 mM. This implies that NaN₃ exerts its toxicologic effects via more than one mechanism in neurons. The presence of glucose is able to prevent the toxic effects of NaN₃ on glial cells completely, however, meaning that these cells can survive on the energy derived solely from metabolism of this sugar. 

The definitive toxicologic reactivity of NaN₃ in biologic systems remains incompletely understood. Aside from inhibiting electron transfer between cytochrome C oxidase (mitochondrial complex IV) and oxygen, and thereby asphyxiating treated cells by binding to the haem groups of cytochrome C, ²⁴ NaN₃ is known to have other detrimental cellular effects. These include direct inhibition of mitochondrial ATPase, ⁵⁶ superoxide dismutase, ⁵⁷ and DNA synthesis. ⁵⁸ Furthermore, it is well established that NaN₃ acts as a potent vasodilator and it has become clear that this action is due to its ability to mimic nitric oxide (NO), either due to its intracellular conversion to the latter compound ⁵⁹ or to its structural capacity to compete at NO-interacting sites. ^24,60 In our study, the nonselective NOS inhibitor, L-NAME, also showed a partial protective effect against NaN₃ toxicity in retinal neurons. This is in agreement with there being a role for nitric oxide in the NaN₃–induced retinal cell death seen herein. Furthermore, the protective effects of the ryanodine receptor blocker dantrolene and calpain inhibitor III suggest a role for increased intracellular calcium in the toxic process. This is reinforced further by the exacerbation of neuronal death when the ER calcium influx inhibitor thapsigargin is co-applied. This agent will maintain cytosolic calcium levels and prevent takeup of this ion into the ER, thus intensifying the injurious process. Calcium has been shown previously to be involved in the toxic effects of NaN₃, ^40,41,61,62 as has an increased intracellular level of ROS. ^24,25,60,63 The latter role for NaN₃ also is in agreement with our study where the antioxidants trolox and pyruvate partially prevented the induced cell death of neurons and glial cells. Interestingly, the NMDA receptor antagonist, MK801 has been shown to protect cortical neurons from NaN₃-induced toxicity, ²⁵ but had no protective effect in our study. This would indicate that cortical neurons express functional NMDA-receptors, but that the cells in the present retinal cultures do not; an explanation for this is that the retinal neuron class that predominantly expresses the NMDA-type of ionotropic glutamate receptor is the ganglion cell, ^64,65 which is present only in low numbers in the present culture model. ¹³  

In conclusion, our study has demonstrated that NaN₃ can be used to produce a reproducible metabolic injury to retinal cells in culture by inhibiting mitochondrial activity. Therefore, it is suggested that this paradigm represents a useful means to screen potential retinal neuroprotective compounds, particularly where putative bioenergetic action is suggested. 

Footnotes

 Supported by the Ophthalmic Research Institute of Australia, Glaucoma Australia, and the National Health and Medical Research Council (Grants 565202, 626964, and 508123).

Footnotes

 Disclosure: J.P.M. Wood, None; T. Mammone, None; G. Chidlow, None; T. Greenwell, None; R.J. Casson, None