Oligodendrocyte death, neuroinflammation, and the effects of minocycline in a rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION).

Purpose: Optic nerve (ON) damage following nonarteritic anterior ischemic optic neuropathy (NAION) and its models is associated with neurodegenerative inflammation. Minocycline is a tetracycline derivative antibiotic believed to exert a neuroprotective effect by selective alteration and activation of the neuroinflammatory response. We evaluated minocycline's post-induction ability to modify early and late post-ischemic inflammatory responses and its retinal ganglion cell (RGC)-neuroprotective ability. Methods: We used the rodent NAION (rNAION) model in male Sprague-Dawley rats. Animals received either vehicle or minocycline (33 mg/kg) daily intraperitoneally for 28 days. Early (3 days) ON-cytokine responses were evaluated, and oligodendrocyte death was temporally evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. Cellular inflammation was evaluated with immunohistochemistry, and RGC preservation was compared with stereology of Brn3a-positive cells in flat mounted retinas. Results: Post-rNAION, oligodendrocytes exhibit a delayed pattern of apoptosis extending over a month, with extrinsic monocyte infiltration occurring only in the primary rNAION lesion and progressive distal microglial activation. Post-induction minocycline failed to improve retinal ganglion cell survival compared with the vehicle treated (893.14 vs. 920.72; p>0.9). Cytokine analysis of the rNAION lesion 3 days post-induction revealed that minocycline exert general inflammatory suppression without selective upregulation of cytokines associated with the proposed alternative or neuroprotective M2 inflammatory pathway. Conclusions: The pattern of cytokine release, extended temporal window of oligodendrocyte death, and progressive microglial activation suggests that selective neuroimmunomodulation, rather than general inflammatory suppression, may be required for effective repair strategies in ischemic optic neuropathies.

Unstable oxygen supply and glaucoma.

The pathogenesis of the glaucomatous optic neuropathy (GON) is an ongoing bone of contention. While the role of intraocular pressure (IOP) is well known, it is also clear that a variety of other factors, particularly those of a vascular nature, are involved as well. In contrast to other eye diseases, it is an unstable oxygen supply, as opposed to chronic hypoxia, that contributes to GON. The major cause of fluctuations in the local oxygen tension is an unstable ocular blood flow (OBF). OBF, in turn, fluctuates if the IOP spikes, blood pressure drops, or OBF autoregulation is defective. The main reason for disturbed autoregulation is a primary vascular dysregulation (PVD), particularly in the context of the so-called Flammer syndrome. Unstable oxygen tension leads to local oxidative stress with many detrimental effects, such as the activation of glial cells, which alters their morphology and gene expression. As a consequence, the local concentrations of nitric oxide and the metalloproteinases increase. The metalloproteinases digest extracellular matrix and thereby contribute to tissue remodelling. The short-lived nitric oxide easily diffuses into the neighbouring neuronal axons, allowing a fusion with the superoxide anion and thereby generating the cell-damaging peroxynitrite. Both this tissue remodelling and damage of the axons contribute to the development and progression of GON.

Selective up-regulation of the soluble pattern-recognition receptor pentraxin 3 and of vascular endothelial growth factor in giant cell arteritis: relevance for recent optic nerve ischemia.

OBJECTIVE: To assess local expression and plasma levels of pentraxin 3 (PTX3) in patients with giant cell arteritis (GCA). METHODS: Plasma and serum samples were obtained from 75 patients with GCA (20 of whom had experienced optic nerve ischemia in the previous 3 weeks and 24 of whom had experienced symptom onset in the previous 6 months and had no history of optic nerve ischemia) and 63 controls (35 age-matched healthy subjects, 15 patients with rheumatoid arthritis, and 13 patients with chronic stable angina). In 9 patients in whom GCA was recently diagnosed, circulating levels of interleukin-1ß (IL-1ß), IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12p70, CCL2/monocyte chemotactic protein 1, CCL3/macrophage inflammatory protein 1α (MIP-1α), CCL4/MIP-1ß, CCL11/eotaxin, CXCL9/monokine induced by interferon-γ, CXCL10/interferon-γ-inducible 10-kd protein, tumor necrosis factor α (TNFα), interferon-γ, vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor, and FasL were measured via a multiplexed cytometric assay. PTX3 and VEGF concentrations were assessed by enzyme-linked immunosorbent assay. PTX3 and CD68 expression were determined by immunohistochemistry and immunofluorescence on temporal artery samples. RESULTS: GCA patients with very recent optic nerve ischemia had significantly higher PTX3 and VEGF levels compared to other GCA patients and controls. GCA patients with a disease duration of <6 months had significantly higher PTX3 levels compared to other GCA patients and controls. Immunohistochemistry revealed selective PTX3 expression in the wall of inflamed arteries. CONCLUSION: Our findings indicate that local expression of PTX3 is a feature of vascular inflammation in GCA; elevated circulating levels of PTX3 identify patients with very recent optic nerve ischemia or a recent diagnosis. Optic nerve ischemia is also associated with increased circulating VEGF levels.

Sectoral loss of myelin and axons in anterior ischemic optic neuropathy.

PURPOSE: We report a case of focal myelinated nerve fiber (MNF) loss caused by anterior ischemic optic neuropathy. CASE REPORT: A 62-year-old woman with segmental MNF presented with anterior ischemic optic neuropathy. One year later, MNF lost superiorly, with preservation of inferior segment MNF. Spectral-domain optical coherence tomography of the optic nerve head and posterior pole retinal thickness showed localized thinning of superior nerve fibers (axons) and retinal thickness. CONCLUSIONS: This case highlights the focal nature of anterior ischemic optic neuropathy and may be the first patient in whom sectoral loss of myelinated nerve fiber has been photographically documented.

Increased expression of oxyproteins in the optic nerve head of an in vivo model of optic nerve ischemia.

BACKGROUND: To investigate the effects of microvascular compromise on the expression of oxidative proteins in the optic nerve head. METHODS: Endothelin-1 (0.1 µg/day) was delivered to the perineural region of the anterior optic nerve by osmotically driven minipumps for two, four, and eight weeks in ten rabbits, respectively. As a control, a balanced salt solution was delivered for two and eight weeks in five rabbits, respectively. Expression of oxyproteins in the cornea, vitreous, retina, and optic nerve head for each time period was determined using the OxyBlot protein oxidation detection kit. Retina was stained with H&E and TUNEL for histological examination. RESULTS: There was a significant increase in the expression of oxyproteins in the optic nerve head after two weeks of endothelin-1 administration (p < 0.001, Mann Whitney U test). In contrast, there was no expression of oxyproteins in the cornea, retina, or vitreous. The number of cells in the retinal ganglion cell layer, inner nuclear layer, and outer nuclear layer decreased remarkably with time in the endothelin-1-treated group. Furthermore, the inner and outer nuclear layers, as well as the inner and outer plexiform layers, became thinner over time. CONCLUSIONS: Administration of endothelin-1 to the microvasculature of the optic nerve leads to increased expression of oxyproteins in the optic nerve head and loss of retinal ganglion cells.

Biomarkers of lesion severity in a rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION).

The rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION) is similar in many of its pathophysiological responses to clinical NAION. Like human NAION, there is significant variability in the severity of the lesion produced, and little is known of the parameters associated with rNAION induction severity or if pre- or early post-induction biomarkers can be identified that enable prediction of lesion severity and ultimate loss of retinal ganglion cells (RGCs). Adult male Sprague-Dawley outbred rats were evaluated for various parameters including physiological characteristics (heart rate, respiratory rate, temperature, hematocrit [Hct]), optic nerve head (ONH) appearance, pre- and post-induction mean diameter, and intravenous fluorescein and indocyanine green angiographic patterns of vascular leakage at 5 hours post-induction, performed using a spectral domain-optical coherence tomography (SD-OCT) instrument. Early changes were correlated with ultimate RGC loss by Brn3a (+) immunohistology. RGC loss also was correlated with the relative level of laser exposure. The severity of ONH edema 2d, but not 5hr, post induction was most closely associated with the degree of RGC loss, revealing a threshold effect, and consistent with a compartment syndrome where a minimum level of capillary compression within a tight space is responsible for damage. RGC loss increased dramatically as the degree of laser exposure increased. Neither physiological parameters nor the degree of capillary leakage 5hr post induction were informative as to the ultimate degree of RGC loss. Similar to human NAION, the rNAION model exhibits marked variability in lesion severity. Unlike clinical NAION, pre-induction ONH diameter likely does not contribute to ultimate lesion severity; however, cross-sectional ONH edema can be used as a biomarker 2d post-induction to determine randomization of subjects prior to inclusion in specific neuroprotection or neuroregeneration studies.

Sigma 1 Receptor Modulates Optic Nerve Head Astrocyte Reactivity.

Purpose: Stimulation of Sigma 1 Receptor (S1R) is neuroprotective in retina and optic nerve. S1R is expressed in both neurons and glia. The purpose of this work is to evaluate the ability of S1R to modulate reactivity responses of optic nerve head astrocytes (ONHAs) by investigating the extent to which S1R activation alters ONHA reactivity under conditions of ischemic cellular stress. Methods: Wild type (WT) and S1R knockout (KO) ONHAs were derived and treated with vehicle or S1R agonist, (+)-pentazocine ((+)-PTZ). Cells were subjected to six hours of oxygen glucose deprivation (OGD) followed by 18 hours of re-oxygenation (OGD/R). Astrocyte reactivity responses were measured. Molecules that regulate ONHA reactivity, signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa B (NF-kB), were evaluated. Results: Baseline glial fibrillary acidic protein (GFAP) levels were increased in nonstressed KO ONHAs compared with WT cultures. Baseline cellular migration was also increased in nonstressed KO ONHAs compared with WT. Treatment with (+)-PTZ increased cellular migration in nonstressed WT ONHAs but not in KO ONHAs. Exposure of both WT and KO ONHAs to ischemia (OGD/R), increased GFAP levels and cellular proliferation. However, (+)-PTZ treatment of OGD/R-exposed ONHAs enhanced GFAP levels, cellular proliferation, and cellular migration in WT but not KO cultures. The (+)-PTZ treatment of WT ONHAs also enhanced the OGD/R-induced increase in cellular pSTAT3 levels. However, treatment of WT ONHAs with (+)-PTZ abrogated the OGD/R-induced rise in NF-kB(p65) activation. Conclusions: Under ischemic stress conditions, S1R activation enhanced ONHA reactivity characteristics. Future studies should address effects of these responses on RGC survival.

Inhibition of Retinal Ganglion Cell Loss By a Novel ROCK Inhibitor (E212) in Ischemic Optic Nerve Injury Via Antioxidative and Anti-Inflammatory Actions.

Purpose: This study investigated the neuroprotective effects of administration of ROCK inhibitor E212 on ischemic optic neuropathy. Methods: Rats received an intravitreal injection of either E212 or PBS immediately after optic nerve infarct. The oxidative stress in the retina was detected by performing superoxide dismutase activity and CellROX assays. The integrity of retinal pigment epithelium was determined by staining of zona occludens 1. The visual function, retinal ganglion cell (RGC) density, and RGC apoptosis were determined by using flash visual-evoked potential analysis, retrograde FluoroGold labeling, and TdT-dUTP nick end-labeling assay. Macrophage infiltration was detected by staining for ED1. The protein levels of TNF-α, p-CRMP, p-AKT1, p-STAT3, and CD206 were evaluated using Western blotting. Results: Administration of E212 resulted in a 1.23-fold increase in the superoxide dismutase activity of the retina and 2.28-fold decrease in RGC-produced reactive oxygen species as compared to the levels observed upon treatment with PBS (P < 0.05). Moreover, E212 prevented the disruption of the blood-retinal barrier (BRB) in contrast to PBS. The P1-N2 amplitude and RGC density in the E212-treated group were 1.75- and 2.05-fold higher, respectively, than those in the PBS-treated group (P < 0.05). The numbers of apoptotic RGCs and macrophages were reduced by 2.93- and 2.54-fold, respectively, in the E212-treated group compared with those in the PBS-treated group (P < 0.05). The levels of p-AKT1, p-STAT3, and CD206 were increased, whereas those of p-PTEN, p-CRMP2, and TNF-α were decreased after treatment with E212 (P < 0.05). Conclusions: Treatment with E212 suppresses oxidative stress, BRB disruption, and neuroinflammation to protect the visual function in ischemic optic neuropathy.

P2X7 receptors mediate ischemic damage to oligodendrocytes.

Brain ischemia leading to stroke is a major cause of disability in developed countries. Therapeutic strategies have most commonly focused on protecting neurons from ischemic damage. However, ischemic damage to white matter causes oligodendrocyte death, myelin disruption, and axon dysfunction, and it is partially mediated by glutamate excitotoxicity. We have previously demonstrated that oligodendrocytes express ionotropic purinergic receptors. The objective of this study was to investigate the role of purinergic signaling in white matter ischemia. We show that, in addition to glutamate, enhanced ATP signaling during ischemia is also deleterious to oligodendrocytes and myelin, and impairs white matter function. Thus, ischemic oligodendrocytes in culture display an inward current and cytosolic Ca(2+) overload, which is partially mediated by P2X7 receptors. Indeed, oligodendrocytes release ATP after oxygen and glucose deprivation through the opening of pannexin hemichannels. Consistently, ischemia-induced mitochondrial depolarization as well as oxidative stress culminating in cell death are partially reversed by P2X7 receptor antagonists, by the ATP degrading enzyme apyrase and by blockers of pannexin hemichannels. In turn, ischemic damage in isolated optic nerves, which share the properties of brain white matter, is greatly attenuated by all these drugs. Ultrastructural analysis and electrophysiological recordings demonstrated that P2X7 antagonists prevent ischemic damage to oligodendrocytes and myelin, and improved action potential recovery after ischemia. These data indicate that ATP released during ischemia and the subsequent activation of P2X7 receptor is critical to white matter demise during stroke and point to this receptor type as a therapeutic target to limit tissue damage in cerebrovascular diseases.

Increased expression of the endothelin system in arterial lesions from patients with giant-cell arteritis: association between elevated plasma endothelin levels and the development of ischaemic events.

OBJECTIVE: Approximately 15-20% of patients with giant-cell arteritis (GCA) develop ischaemic complications often preceded by transient ischaemia. The expression of the endothelin (ET) system in GCA lesions was investigated to assess its relationship with the development of ischaemic complications. METHODS: Plasma ET-1 was quantified by immunoassay in 61 patients with biopsy-confirmed GCA and 16 healthy donors. ET-1, endothelin-converting enzyme (ECE-1) and endothelin receptor (ET(A)R and ET(B)R) messenger RNA were measured by real-time quantitative reverse transcriptase-PCR in temporal arteries from 35 of these patients and 19 control arteries. Proteins were measured by immunoassay and Western blot. RESULTS: ET-1 concentration was increased at the protein level in temporal artery samples from GCA patients compared with controls (0.98 (SEM 0.32) vs 0.28 (SEM 0.098) fmol/mg, p = 0.028). ECE-1, ET(A)R and ET(B)R/actin ratios (Western blot) were also significantly higher in GCA patients. Intriguingly, mRNA expression of ET-1, ECE-1 and both receptors was significantly reduced in GCA lesions compared with control arteries. When investigating mechanisms underlying these results, platelet-derived growth factor and IL-1beta, present in GCA lesions, were found to downregulate ET-1 mRNA in cultured human temporal artery-derived smooth muscle cells. Glucocorticoid treatment for 8 days did not result in significantly decreased endothelin tissue concentration (0.87 (SEM 0.2) vs 0.52 (SEM 0.08); p = 0.6). Plasma endothelin concentrations were higher in patients with ischaemic complications (1.049 (SEM 0.48) vs 1.205 (SEM 0.63) pg/ml, p = 0.032). CONCLUSIONS: The endothelin system is increased at the protein level in GCA lesions creating a microenvironment prone to the development of ischaemic complications. Recovery induced by glucocorticoids is delayed, indicating persistent exposure to endothelin during initial treatment.

Axonal degeneration, regeneration and ganglion cell death in a rodent model of anterior ischemic optic neuropathy (rAION).

Using laser-induced photoactivation of intravenously administered rose Bengal in rats, we generated an ischemic infarction of the intrascleral portion of the optic nerve (ON) comparable to that which occurs in humans to investigate optic nerve axon degenerative events following optic nerve infarct and the potential for axon re-growth. Animals were euthanized at different times post infarct. Axon degeneration was evaluated with SMI312 immunolabeling, and GAP-43 immunostaining was used to identify axon regeneration. Terminal dUTP nick end labeling (TUNEL) was used to evaluate retinal ganglion cell (RGC) death. There was significant axon structural disruptinot ion at the anterior intrascleral portion of the ON by 3d post-infarct, extending to the posterior ON by 7d post-stroke. Destruction of normal axon structure and massive loss of axon fibers occurred by 2 weeks. GAP-43 immunoreactivity occurred in the anterior ON by 7d post-infarct, lasting 3-4 weeks, without extension past the primary ischemic lesion. TUNEL-positive cells in the RGC layer appeared by 7d post-insult. These results indicate that following induction of ischemic optic neuropathy, significant axon damage occurs by 3d post-infarct, with later neuronal death. Post-stroke adult rat retinal ganglion cells attempt to regenerate their axons, but this effort is restricted to the unmyelinated region of the anterior ON. These responses are important in understanding pathologic process that underlies human non-arteritic anterior ischemic optic neuropathy (NAION) and may guide both the appropriate treatment of NAION and the window of opportunity for such treatment.

Thrombophilic risk factors in the pathogenesis of non-arteritic anterior ischemic optic neuropathy patients.

BACKGROUND: Non-arteritic anterior ischemic optic neuropathy (N-AION) is caused by acute ischemic infarction of the optic nerve head, supplied by the posterior ciliary arteries. Thrombophilia is the tendency/predisposition to vascular thromboses of arteries and veins, and the existence of thrombophilic risk factors leads to blood hypercoagulability and potentially increased risk for thromboses. OBJECTIVES: To investigate whether there is an association between N-AION and a wide spectrum of thrombophilic risk factors. PATIENTS AND METHODS: Seventy-seven consecutive cases of confirmed N-AION and 60 age- and sex-matched consecutive controls constituted the study group. Fibrinogen levels, deficiency of proteins C, S, ATIII, lupus anticoagulant, activated protein C resistance, factor V Leiden, factor V H1299R, factor II G20210A, MTHFR C677T, MTHFR A1298C, GPIIIa A1/A2, and ACE I/D polymorphisms were analysed. RESULTS: Statistical analysis of the plasma proteins in our study demonstrated that the only significant difference was the one concerning protein S levels. In particular, the mean value for N-AION patients was 78.8% +/- 21.2, and for the control group the mean value was 88% +/- 21.2 (p = 0.013). Despite the above-mentioned result, there was not any statistical difference between the two subgroups regarding actual protein S deficiency, as 9/77 (11.7%) patients and 4/60 (6.7%) controls had protein S levels below 60% (p = 0.32). In our study sample, homozygosity for MTHFR C677T polymorphism in the study group as a whole, and the presence of at least one A2 allele of GPIIIa in the subgroup of male patients as compared to healthy male controls, proved to be the most significant thrombophilic risk factors, with odds ratios of 16.78 (95% C.I 0.96-294.42, p = 0.054) and 4.6 (95% C.I 1.52-13.88, p = 0.007) respectively. CONCLUSION: Screening for these polymorphisms would probably constitute a valuable procedure in N-AION patients, as they may have an important contribution to the pathogenesis of the disease.

Pharmacologic intervention targeting glycolytic-related pathways protects against retinal injury due to ischemia and reperfusion.

Retinal ischemia contributes to multiple ocular diseases while aminoguanidine (AMG) treatment significantly inhibits the neuronal and vascular degeneration due to acute retinal ischemia and reperfusion (I/R) injury. In the present study, 2-D DIGE was applied to profile global protein expression changes due to retinal I/R injury, and the protection effects mediated by AMG. Retinal ischemia was induced by elevated intraocular pressure to 80-90 mmHg for 2 h, and reperfusion was established afterward. Retinal tissues were collected 2 days after I/R injury. After 2-D DIGE analysis, a total of 96 proteins were identified. Among them, 28 proteins were identified within gel spots whose intensities were normalized by AMG pretreatment, pathway analysis indicated that most were involved in glycolysis and carbohydrate metabolism. Selected enzymes identified by MS/MS within these pathways, including transketolase, triosephosphate isomerase 1, aldolase C, total enolase, and pyruvate kinase were validated by quantitative Western blots. Glycolytic enzymes and other differentially regulated proteins likely play previously unrecognized roles in retinal degeneration after I/R injury, and inhibition of the resulting metabolic changes, using pharmacologically agents such as AMG, serve to inhibit the changes in metabolism and mitigate retinal degeneration. Select glycolytic enzymes may provide novel therapeutic targets for inhibiting the neuronal and vascular degeneration after retinal I/R injury.

White matter vulnerability to ischemic injury increases with age because of enhanced excitotoxicity.

Stroke incidence increases with age and this has been attributed to vascular factors. We show here that CNS white matter (WM) is intrinsically more vulnerable to ischemic injury in older animals and that the mechanisms of WM injury change as a function of age. The mouse optic nerve was used to study WM function. WM function in older animals (12 months) was not protected from ischemic injury by removal of extracellular Ca2+ or by blockade of reverse Na+/Ca2+ exchange, as is the case with young adults. Ischemic WM injury in older mice is predominately mediated by glutamate release and activation of AMPA/kainate-type glutamate receptors. Glutamate release, attributable to reverse glutamate transport, occurs earlier and is more robust in older mice that show greater expression of the glutamate transporter. The observation that WM vulnerability to ischemic injury is age dependent has possible implications for the pathogenesis of other age-related CNS conditions.

Chromatic pupil responses: preferential activation of the melanopsin-mediated versus outer photoreceptor-mediated pupil light reflex.

OBJECTIVE: To weight the rod-, cone-, and melanopsin-mediated activation of the retinal ganglion cells, which drive the pupil light reflex by varying the light stimulus wavelength, intensity, and duration. DESIGN: Experimental study. PARTICIPANTS: Forty-three subjects with normal eyes and 3 patients with neuroretinal visual loss. METHODS: A novel stimulus paradigm was developed using either a long wavelength (red) or short wavelength (blue) light given as a continuous Ganzfeld stimulus with stepwise increases over a 2 log-unit range. The pupillary movement before, during, and after the light stimulus was recorded in real time with an infrared illuminated video camera. MAIN OUTCOME MEASURES: The percent pupil contraction of the transient and sustained pupil response to a low- (1 cd/m(2)), medium- (10 cd/m(2)), and high-intensity (100 cd/m(2)) red- and blue-light stimulus was calculated for 1 eye of each subject. From the 43 normal eyes, median and 25th, 75th, 5th, and 95th percentile values were obtained for each stimulus condition. RESULTS: In normal eyes at lower intensities, blue light evoked much greater pupil responses compared with red light when matched for photopic luminance. The transient pupil contraction was generally greater than the sustained contraction, and this disparity was greatest at the lowest light intensity and least apparent with bright (100 cd/m(2)) blue light. A patient with primarily rod dysfunction (nonrecordable scotopic electroretinogram) showed significantly reduced pupil responses to blue light at lower intensities. A patient with achromatopsia and an almost normal visual field showed selective reduction of the pupil response to red-light stimulation. A patient with ganglion cell dysfunction owing to anterior ischemic optic neuropathy demonstrated global loss of pupil responses to red and blue light in the affected eye. CONCLUSIONS: Pupil responses that differ as a function of light intensity and wavelength support the hypothesis that selected stimulus conditions can produce pupil responses that reflect phototransduction primarily mediated by rods, cones, or melanopsin. Use of chromatic pupil responses may be a novel way to diagnose and monitor diseases affecting either the outer or inner retina.

Therapeutic Effects of Puerarin Against Anterior Ischemic Optic Neuropathy Through Antiapoptotic and Anti-Inflammatory Actions.

Purpose: This study investigated the therapeutic effects of puerarin (PR) on a rat model of anterior ischemic optic neuropathy (rAION). Methods: The neuroprotective effects of PR on rAION were evaluated using flash visual-evoked potentials (FVEP), retrograde labeling of retinal ganglion cells (RGCs), TUNEL assay of the retina, optical coherence tomography (OCT) images of optic nerve width, and ED1 staining of the optic nerve (ON). The inflammatory response of ON and Akt signaling pathways were analyzed through Western blot. M2 polarization was determined by immunostaining and immunoblotting in ONs. Results: In FVEP analysis, the amplitude of P1-N2 and the RGC density in the PR-treated group were 2.3- and 1.6-fold higher than those in the PBS-treated group, respectively (P < 0.05). The number of apoptotic RGC in the PR-treated group was 2.8-fold lower than that in the PBS-treated group. OCT images demonstrated that PR treatment-reduced ON edema in the acute phase compared to PBS treatment (P < 0.05). Macrophage infiltration was reduced by 5.2-fold by PR treatment compared with the PBS treatment (P < 0.05). PR treatment inhibited the levels of iNOS, IL-1ß, and TNF-α, induced the levels of IL-10, Arg1, and Fizz1 in the rAION model. The levels of p-Akt1 and C/EBPß in the PR-treated group increased by 3.4-fold and 5.89-fold compared with those in the PBS-treated group (P < 0.05). Inhibition of Akt activation reduced the number of M2 macrophage in the PR-treated group (P < 0.05). Conclusions: PR treatment provided the neuroprotective effects in the rAION model, which may lead to new clinical applications.

Neuron stress and loss following rodent anterior ischemic optic neuropathy in double-reporter transgenic mice.

PURPOSE: Nonarteritic anterior ischemic optic neuropathy (NAION) is an optic nerve infarct involving axons of retinal ganglion cell (RGC) neurons. The rodent NAION model (rAION) can use transgenic mouse strains to reveal unique characteristics about the effects of sudden optic nerve ischemia on RGCs and their axons. The impact of rAION on RGC stress patterns, RGC loss, and their axons after axonal infarct were evaluated. METHODS: A double-transgenic mouse strain was used, containing a construct with cyan fluorescent protein (CFP) under Thy-1 promoter control, and a construct with beta-galactosidase (lacZ) linked to the stress gene c-fos promoter. Thy-1 in the retina is expressed predominantly in RGCs, enabling stereologic analysis of CFP(+) RGC numbers and loss post-rAION-using confocal microscopy. RGC loss was correlated with axonal counts using transmission electron microscopy (TEM). LacZ immunohistochemistry was used to evaluate retinal cell stress after rAION. RESULTS: The 45,000 CFP(+) cells in the RGC layer of control animals compared with previous RGC quantitative estimates. rAION produced RGC stress, defined as lacZ expression, in patterns corresponding with later RGC loss. rAION-associated RGC loss correlated with regional nerve fiber layer loss. Axonal loss correlates with stereologically determined RGC loss estimates in transgenic mice retinas. CONCLUSIONS: Post-ON infarct RGC stress patterns correlate with regional RGC loss. Cellular lacZ levels in most RGCs are low, suggesting rAION-affected RGCs express c-fos only transiently. CFP(+) cell loss correlates closely with quantitative axonal loss, suggesting that the Thy-1 (CFP) transgenic mouse strain is appropriate for RGC stereologic analyses.

Aqueous Humor Cytokines in Patients With Acute Nonarteritic Anterior Ischemic Optic Neuropathy.

PURPOSE: To measure and compare the cytokine concentrations in the aqueous humor of patients with acute nonarteritic anterior ischemic optic neuropathy (NAION) and normal age-related cataract controls. DESIGN: Prospective, comparative observational study. METHODS: Aqueous humor samples were obtained in 10 patients with acute NAION (within 14 days of symptom onset) and 15 control patients with age-related cataract. The levels of 6 cytokines-vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-2, IL-6, and IL-8-were determined using a multiplex bead immunoassay. The clinical characteristics of patients were also collected for correlation analysis. RESULTS: The mean concentration of VEGF (94.1 ± 40.4 pg/mL) was significantly higher in the NAION group compared to the cataract controls (52.2 ± 20.8 pg/mL; P = .010) and the mean concentration of IL-2 (5.56 ± 1.27 pg/mL) was significantly lower in the NAION group than in the cataract controls (16.6 ± 14.0 pg/mL; P = .002). There were no differences in the concentration of TNF-α, IL-1ß, IL-6, and IL-8. There was a strong negative correlation between the VEGF concentration and the peripapillary retinal nerve fiber layer (RNFL) thickness at presentation (r = -0.657, P = .055). There was no significant correlation between the RNFL thickness and any other cytokines, the mean deviation on 24-2 Humphrey visual fields, or the duration of vision loss. CONCLUSIONS: Acute NAION is associated with higher VEGF and lower IL-2 concentrations without a change in other inflammatory cytokines. This has implications for future therapeutic interventions and diagnostic testing in patients with this acute optic neuropathy.

Experimental Anterior Ischemic Optic Neuropathy in Diabetic Mice Exhibited Severe Retinal Swelling Associated With VEGF Elevation.

Purpose: Diabetes mellitus (DM) is one of the most important risk factors for nonarteritic anterior ischemic optic neuropathy (AION). In this study, we investigated for the first time the impact of experimental AION in a DM model. Methods: We induced a photochemical thrombosis model of AION after streptozotocin-induced DM and performed serial optical coherence tomography (OCT), morphometric analyses, and VEGF levels in the retina and sera. Results: Compared with non-DM animals, experimental AION in DM mice led to significantly greater retinal swelling on day 1 and worse thinning at week 3 on OCT measurements. Greater retinal swelling on OCT in DM-AION eyes was associated with significantly increased loss of brain-specific homeobox/POU domain protein 3A (Brn3A+) retinal ganglion cells at week 3. In acute AION, there was greater inflammation as seen by an increase in ionized calcium-binding adapter molecule 1 (Iba1+)-activated microglia. On day 1, there was increase in vascular endothelial growth factor (VEGF) level in nondiabetic AION retinae and sera, but the VEGF level was the highest in the diabetic AION group, which decreased to nondiabetic levels after insulin treatment. The decrease in retinal and serum VEGF levels after insulin treatment correlated with a reduction in retinal swelling. Conclusions: In the setting of hyperglycemia, AION led to greater acute, postischemic microglial activation and elevation of VEGF levels, which likely contributed to greater retinal swelling acutely and worse retinal thinning and loss of retinal ganglion cells chronically. Treatment of hyperglycemia with insulin reduced VEGF levels and retinal swelling, consistent with the idea that VEGF is an important factor in postischemic swelling and that good glycemic control following AION may lead to better visual outcome.

[Study on electroacupuncture along the visual conductive pathway for ocular cell apoptosis in anterior ischemic optic neuropathy].

OBJECTIVE: To study the protective effect of electroacupuncture (EA) along the visual conductive pathway for the optic nerve tissue of anterior ischemic optic neuropathy (AION) in terms of the structure and apoptosis. METHODS: The AION model of right eye was established with laser in 48 New Zealand white ear rabbits. All rabbits were randomly divided into a model group, an acupuncture group and an acupuncture combined with EA group, 16 rabbits in each one. Other 16 normal left eyes were selected as a blank group. Acupuncture and EA of 30 min were used in the corresponding groups for 3 days at the right "Cuanzhu" (BL 2), "Yuyao" (EX-HN 4) and "Qiaomingxue" (Extra), once a day. There was no intervention in the model group and the blank group. The morphological structure and retinal thickness of lining of the optic nerve were detected with HE stain. The expressions of the Bcl-2 and Bax in the retina were detected with immunohistochemistry. And the concentration of tumor necrosis factor-α (TNF-α) in the retina was detected with enzyme linked immunosorbent assay (ELISA). RESULTS: In the model group, the ganglion cell layer revealed hyperplasia and disorder, and the retina ganglion cells revealed loose arrangements, thin inner nuclear layers, and reduction of cell amounts, some long parts missing cells. However, the above structural damages were much weaker in the acupuncture group and acupuncture combined with EA group. The inner layer of the retina in the model group was thinner than that in the blank group (P<0.05). That in the acupuncture combined with EA group showed significant better results than those in the acupuncture and model groups (both P<0.05), which was not statistically different from that in the normal group (P>0.05). The Bcl-2 count in the model group was lower than that in the blank group (P<0.05), and that in the acupuncture combined with EA group was better than those in the acupuncture and model groups (both P<0.05), not significantly different from that in the blank group (P>0.05). The number of Bax in the model group was higher than that in the blank group (P<0.05), and that in the acupuncture combined with EA group was lower than those in the acupuncture and model groups (both P<0.05), and was similar to that in the blank group (P>0.05). Bcl-2/Bax in the model group was lower than that in the blank group (P<0.05). The value in the acupuncture combined with EA group presented better than those in the acupuncture and model groups (both P<0.05), which had no difference from that in the blank group (P>0.05). TNF-α in the model group was higher than that in the blank group (P<0.05), and no such differences were detected between other groups and the model group (bothP>0.05). CONCLUSIONS: EA along the visual conductive pathway is protective to some extent for optic nerve tissue, which can increase the expression of Bcl-2 and reduce the expression of Bax so as to restrain ganglion cell apoptosis.