Retinal laser photocoagulation was first described 50 years ago and remains the standard of care for many retinal diseases, including extrafoveal choroidal neovascularization, retinal tears, diabetic retinopathy, and retinal vein occlusions.1 Panretinal photocoagulation (PRP) for proliferative retinopathies involves the purposeful destruction of a significant fraction of the outer retina including photoreceptors.2 The exact mechanism by which laser therapy achieves its therapeutic end point is an area of active investigation. Several mechanisms have been proposed to underlie the efficacy of retinal laser therapy, including improved retinal oxygenation, reduction in metabolic activity through destruction of photoreceptors, inhibition of angiogenic stimulators, increased production of angio-inhibitory factors, and reduction in oxidative stress.3–6 Selective retinal therapy using microsecond pulses to selectively destroy RPE by vaporization of melanosomes while sparing the photoreceptors induces RPE proliferation and migration.7,8
Subvisible and sublethal laser therapy has also been shown to result in a constellation of molecular changes in cells, including upregulation of heat shock protein (hsp)-70 and other inflammatory markers locally.9–11 Photocoagulation creates a surge in free radicals.6,12 Matrix metalloproteinases (MMP-9 and alpha-2-macroglobulin)5 and transforming growth factor beta 2 increase dramatically after PRP.3 Angiostatin, vascular endothelial growth factors (VEGF), and fibroblasts are thought to influence the therapeutic effects of PRP.4,13 The retinal pigment epithelium (RPE) modulates local cytokines, including VEGF, pigment epithelial-derived factor (PEDF), MMP, and tissue inhibitor of matrix metalloproteinases (TIMP). Rat models of hypoxia have shown that photoreceptors are a repository of pro-angiogenic growth factors (such as VEGF, nitric oxide synthases, HIF-1, and insulin-like growth factor-1), which have been generated in other cells in hypoxic conditions. Thus, destruction of photoreceptors could reduce the pro-angiogenic growth factors by reducing this repository.14
A similar inflammatory cascade has been implicated in numerous degenerative ocular conditions, including age-related macular degeneration (AMD). AMD is the leading cause of irreversible blindness in adults older than 65 years in the developed world, affecting more than 1.75 million Americans, and is expected to increase by 54% in the next 15 years.15 The role of single nucleotide polymorphisms and inflammation in AMD has been well-reviewed.16 Complement factor H is strongly associated with AMD.17 Serum antiglial fibrillary acidic protein and carboxyethylpyrrole antibodies are elevated in patients with AMD.18,19 Alpha-enolase, a glycolytic enzyme, has been documented in patients with wet AMD and cancer-associated retinopathy.20,21 Pyruvate kinase M2 (PKM2) has been shown to be targeted in both dry and wet AMD, and higher levels of anti-PKM2 antibody are noted in wet AMD compared with dry AMD.22
The eye is an immune-privileged site, and thus sympathetic ophthalmia can develop after penetrating ocular injury or, rarely, laser treatment.23 Intense laser burns, particularly those that rupture Bruch’s membrane, disrupt this immune privilege and expose the eye to the systemic circulation and immune system. Rupture of Bruch’s membrane by laser remains the most established animal model of choroidal neovascularization for AMD.24 This study seeks to characterize the cellular, immunological, and inflammatory response to rupture laser lesions as a model of retinal degenerative diseases by assessing the changes in serum inflammatory markers using a microarray chip consisting of antigens from a proteomic analysis of constituents of drusen, retina, and choroidal membranes.
Materials and Methods
Retinal Laser Application
Seven C57BL/6 mice (8 weeks old) were used in accordance with the Association for Research in Vision and Ophthalmology Statement Regarding the Use of Animals in Ophthalmic and Vision Research, after approval from the Stanford University animal institutional review board. The mice were anesthetized using ketamine hydrochloride (80 mg/kg, intraperitoneal) and xylazine (16 mg/kg, intraperitoneal) administered 5 minutes before the procedure. Pupillary dilation was achieved by one drop each of 1% tropicamide and 2.5% phenylephrine hydrochloride. Topical tetracaine 0.5% was instilled in the treated eye prior to treatment. Goniosol, or hydroxypropyl methylcellulose, was then placed over each eye as a contact solution and a glass cover slip gently placed over the goniosol to visualize the retina. The non-treated eye was covered with goniosol and taped to prevent corneal exposure keratopathy.
The genetic background of T lymphocytes influences the development of the T helper phenotype. The susceptibility or resistance of different mouse strains to infection and immunity is influenced by differential induction of Th1 and Th2-type responses. C57BL/6 and BALB/c mice display opposite T-cell subset polarizations. BALB/c mice produce a Th2 response to antigens, whereas C57BL/6 mice are Th1 response slanted. Th1-induced ocular inflammation demonstrates more chemokines and their receptor transcripts than Th2-induced disease.25 In experimental autoimmune uveoretinitis (EAU), the inflammatory mediator expression is similar to Th1-induced disease. Susceptible strains of mice immunized for induction of EAU with the uveitogenic protein inter-photoreceptor retinoid-binding protein demonstrate type 1 response during the time of symptoms.26 Autoimmunity to a pathogenic retinal antigen begins as a balanced cytokine response that polarizes toward type 1 in a disease-susceptible genotype and toward type 2 in a disease-resistant genotype. We are interested in studying inflammation and autoimmunity, so Th1-susceptible C57BL/6 mice were selected for use in this study.
The PASCAL laser system (Topcon, Tokyo, Japan) was used to perform 532-nm laser radiation. A rupture was assumed to occur if small bubbles with or without hemorrhage appeared at the lesion site. Seven C57BL/6 mice (8 weeks old) were irradiated using the PASCAL laser with beam diameter of 60 µm, power of 400 mW, and pulse duration of 50 ms to induce numerous laser burns that ruptured Bruch’s membrane. Ten laser burns were placed in the peripapillary region of each retina. After the laser, topical bacitracin/polymyxin 500/10,000 ophthalmic ointment was instilled in both eyes. At the completion of all experiments, the animals were euthanized with inhaled carbon dioxide, confirmed by cervical dislocation. While only some animals develop choroidal neovascularization after Bruch’s membrane rupture (60% within 2 weeks in the initial study describing this technique),27 all animals have a damaged blood-retina barrier, and thus the immune-privileged retina is exposed to the systemic immune system.
Blood was drawn from the saphenous mouse vein prior to and 2 months after laser treatment as described previously.28 The time point of 2 months was selected due to a previous rabbit laser model noting stable gliosis after 2 months, suggesting that the inflammatory and remodeling pathway resulting from laser injury had likely concluded by this time.29
For 10 minutes, EMLA or lidocaine-based cream was applied on the skin at the site of the venipuncture before collection. A heat lamp was used to induce vasodilation. Mice were encouraged to enter a perforated 50-mL centrifuge tube while the tail was held. A hind leg was extended, and the tarsal area of the hind leg shaved. Mild pressure was applied above the knee to occlude the saphenous vein. A 27-gauge needle and a capillary pipette flushed with EDTA was used to acquire 50 mL of blood. Manual pressure was then applied for hemostasis.
The whole blood was placed in a 1.5 mL Eppendorf tube and incubated at room temperature for 30 minutes. It was then centrifuged for 10 minutes in a Minispin centrifuge (Eppendorf, Hamburg, Germany) at 669 RCF or 3200 rpm for 10 minutes. The serum was then transferred to another Eppendorf tube using pipettes and stored at −80°C.
Antigen Microarray Analysis
The serum was run on an antigen microarray chip (Whatman Schleicher & Schleicher BioScience, Keene, NH) with 85 molecules associated with AMD and other retinal diseases, as described previously.30,31 The antigens were selected from proteomics analyses of constituents of drusen, retina, and choroidal membranes after performing western blot and mass spectrometer analysis of animals models of AMD. The antigen array was designed as described previously for systemic autoimmune diseases and included 18 intraocular autoantigens that are constituents of drusen and extracellular matrix. Collagen III, collagen IV, elastin, fibronectin, and histone H2B have been shown to be significantly elevated in the sera of patients with AMD compared to healthy controls30 and were included in our study.
Antigens printed on a slide are then incubated with the serum, and the inflammatory markers binding to the antigens (Cy3 labeled anti-immunoglobulin G) were detected using Genepix 4000B scanner (Molecular Devices, Sunnyvale, CA). The optimal printing concentration for all antigens was determined to be 1 mg/mL, which was tested previously and proved efficient for antibody binding. Antigen microarrays have been a useful tool for antibody profiling to identify autoantigens of many autoimmune diseases, including systemic lupus erythematosus, Sjögren’s syndrome, rheumatoid arthritis, multiple sclerosis, and autoimmune retinopathy.32
Each antigen was normalized to the average intensity of total immunoglobulin G printed on the array as an internal control to generate a normalized fluorescence reactivity intensity. Protein concentration was not standardized or measured between samples. The serum was tested at various dilutions, and then all samples were tested at the same dilution to achieve the best signal to give a normalized signal. Resulting two-dimensional images represent net fluorescence intensities minus background fluorescence. Heat maps of these fluorescent signals with clustering were generated using Cluster and TreeView software. Statistical analysis was performed in Microsoft Excel using a two-tailed, homoscedastic (two-sample equal variance) Student’s t test of the two data sets.
Rupture of Bruch’s membrane by laser results in statistically significant changes in the immunoglobulin reactivity of a variety of inflammatory markers 2 months after laser injury (Figure 1). Expression of most inflammatory markers changed, with approximately two-thirds increasing and one-third decreasing the expression. A variety of inflammatory markers were increased to some degree after laser injury, including the following: complement C3, C-reactive protein (CRP), PKM2, calreticulin, collagen V, Helicobacter pylori, collagen VI, complement factor B, haptoglobin, ceruloplasmin, beta2-microglobulin, factor X, superoxide dismutase 1, aldolase, annexin V, cyclic nucleotide phosphodiesterase, annexin II, creatine kinase BB, apolipoprotein B, retinol binding protein, transferin, L-glutamine synthetase, recoverin, and calmodulin (Figure 2). All of the changes in Figure 2 were statistically significant (P < .05) using a two-tailed Student’s t test.
Heat map of immunoglobulin G (IgG) reactivity after laser application. The average signal intensity of each antigen in each sample was normalized to the average intensity of total IgG printed on the arrays as an internal control. The normalized fluorescence intensity was used to generate the heat map. The reactivity intensities of each antigen are shown on a relative scale. Green, black, and red represent net fluorescence intensities below, equal to, and above the mean, respectively. This map demonstrates a distinctive pattern of IgG inflammatory markers 2 months after laser injury.
Average of relative signal intensities for immunoglobulin G inflammatory markers before and after laser application. Significance analysis of the microarray signals identified inflammatory markers with significant (P < .05) change in expression after laser injury, compared to control before laser application. Approximately two-thirds of the antigens increased expression and one-third decreased expression after laser. These include many inflammatory markers notable for their role in retinal degenerations. nfi = normalized fluorescence intensity.
The 24 notable inflammatory marker levels that significantly increased after rupture laser are summarized in the Table, showing the average pre- and post-laser levels, standard deviations, and P values. Complement C3 increased from 1004 nfi (normalized fluorescence intensity) before laser to 3655 nfi after laser (P = 1.5 *10−6). Mouse knockout models lacking the complement component C3 and the receptors for complement activation fragments C3a (C3aR) and C5a (C5aR) have been shown to result in caspase-3 upregulation, early retinal degeneration, and retinal dysfunction to light.33 CRP increased from 467 nfi before laser to 2,030 nfi after laser (P = .00048). CRP has been shown to be significantly higher in immunohistochemistry samples of patients with AMD at the retina/choroid interface.34 CRP is a nonspecific systemic inflammatory marker that can activate the classic complement pathway through C1q, thus playing an important pro-inflammatory role.
Aldolase increases from 111 nfi before laser to 281 nfi after laser (P = 8.11 *10−5). Aldolase has been found to be elevated in patients with diabetic retinopathy but not in patients with diabetes without diabetic retinopathy.35 Aldolase C is an enzyme associated with sugar metabolism whose expression is limited to the central nerve system, including brain and retina. Therefore its expression may be used as a marker for the breakdown of retinal blood barrier.
PKM2 is increased from 451 nfi before laser to 1,018 nfi after laser (P = .0021) and L-glutamine synthetase is increased from 3.4 nfi before laser to 23.7 nfi after laser (P = .015). Both of these antibodies are expressed in Alzheimer’s disease.36 Aldolase C and PKM2 were also found as inflammatory markers in rabbit serum with minimally visible and moderate (grade 2) laser lesions, although these were not found to be elevated previously in intense (grade 3) laser lesions.37
Several inflammatory markers were downregulated after rupture laser injury. One example is GAPDH (glyceraldehyde 3-phosphate dehydrogenase), which is an enzyme in the glycolytic pathways that catalyzes production of 1,2 bisphosphoglycerate from glyceraldehyde 3-phosphate. Prior studies have shown that increased oxidative stress impairs energy metabolism and causes GAPDH down-regulation and redistribution (from soluble cytoplasmic to insoluble aggregates in nucleus).38 This indicates that the complex cellular reaction to injury downregulates some inflammatory markers due to the increased stress shifting cellular metabolism.
Intense rupture burns that disrupt the blood retinal barrier and retinal immune privilege cause a statistically significant change in serum inflammatory markers after 2 months, including some retinal autoantibodies. Many of the affected inflammatory markers are identical to ones that have been implicated in AMD, diabetic retinopathy, Alzheimer’s, and retinal degenerations, including complement C3, CRP, aldolase, PKM2, and L-glutamine synthetase. Unexpectedly, the destruction of photoreceptors during irradiation resulted in the production of only one phototransduction-specific antibody, recoverin, which we are unable to explain at this time.
Neurodegenerative conditions are difficult to study in animal models because they are often slowly progressive, age-associated, and human-specific, thus necessitating the investigation of surrogate markers. The similarity of serum inflammatory markers in this animal model and neurodegenerative diseases can help to develop an improved model to monitor different stages of retinal degenerative conditions by checking the inflammatory marker levels in serum after rupture laser. Inflammatory markers may directly cause pathogenic retinal changes, or it could be a side effect of tissue damage, or autoantibody-induced pathogenesis may require previous tissue damage. This model of serum inflammatory marker formation through laser could be used as a surrogate end point to study the natural history of disease progression and clinical efficacy of pharmaceutical agents to treat retinal degenerations through analysis of the level of serum inflammatory marker levels. Additional natural history studies in humans correlating the expression of these antigens will be required to test the utility of this novel animal model of neurodegeneration using rupture laser burns, but our preliminary results suggest that this might be a potentially fruitful area for further investigation.
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Inflammatory Marker Levels Before and After Laser Injury
|Antibody||Average Pre-Laser||SD Pre-Laser||Average Post-Laser||SD Post-Laser||P Value|
Inflammatory Marker Levels Before and After Laser Injury