Dynamic changes of inducible nitric oxide synthase expression in rat’s retina and its role on blood-retinal barrier injury after acute high intraocular pressure

Rationale: Acute high intraocular pressure (IOP) induces retinal ischemia/reperfusion (RI/R) that further initiates neuroinflammatory responses. This event can cause retinal tissue damage and neuronal death, ultimately resulting in irreversible blindness worldwide that lacks effective therapies, validated treatment targets and underlying mechanisms. We sought to explore the potential mechanisms on the causal link between the neuroinflammatory response and neurodegeneration following acute high IOP. Methods: A rat model of RI/R induced by acute high IOP was used to investigate the spatiotemporal profiles of blood-retinal barrier (BRB) disruption, peripheral immune cell infiltration, and innate immune cell response following acute glaucomatous injury. RNA sequencing and in vivo transfection with adeno-associated virus (AAV) were used to explore the pathogenic mechanisms of acute high IOP-induced neuroinflammation. Results: Disruption of the inner BRB and infiltration of macrophages and lymphocytes occurred during the early stage after acute high IOP. These events were accompanied by an innate immune response. RNA sequencing revealed that Lipocalin-2 (Lcn2) was one of the most significantly up-regulated inflammation-related genes. Lcn2 knockdown ameliorated inner BRB disruption, peripheral immune cell infiltration, and innate immune cell response, resulting in neuroprotective effects. Furthermore, we found that acute glaucomatous injury triggers high expression of LCN2 in the peripheral serum, which is strongly associated with the severity of the neuroinflammatory response in the retina. Conclusions: A "neuroinflammatory cascade" characterized by breakdown of inner BRB, peripheral immune cell infiltration, and innate immune cell response occurs during the initial stage following glaucomatous injury. We also identified a novel mechanism for LCN2 in acute high IOP-induced neuroinflammation. LCN2 has the potential to serve as a candidate biomarker for predicting the severity of the neuroinflammatory response following acute glaucoma, which may provide new evidence to retinal repair strategies for better visual function recovery at intervention time points and new targets.

To elucidate the role of vascular endothelial growth factor-165b (VEGF-165b) in blood-retinal barrier (BRB) injury in the rat acute glaucoma model. In this study, the rat acute high intraocular pressure (HIOP) model was established before and after intravitreous injection of anti-VEGF-165b antibody. The expression of VEGF-165b and zonula occludens-1 (ZO-1) in rat retina was detected by double immunofluorescence staining and Western blotting, and the breakdown of BRB was detected by Evans blue (EB) dye. The intact retina of rats expressed VEGF-165b and ZO-1 protein, which were mainly located in the retinal ganglion cell layer and the inner nuclear layer and were both co-expressed with vascular endothelial cell markers CD31. After acute HIOP, the expression of VEGF-165b was up-regulated; the expression of ZO-1 was down-regulated at 12h and then recovered at 3d; EB leakage increased, peaking at 12h. After intravitreous injection of anti-VEGF-165b antibody, the expression of VEGF-165b protein was no significantly changed; and the down-regulation of the expression of ZO-1 was more obvious; EB leakage became more serious, peaking at 3d. EB analysis also showed that EB leakage in the peripheral retina was greater than that in the central retina. The endogenous VEGF-165b protein may protect the BRB from acute HIOP by regulating the expression of ZO-1. The differential destruction of BRB after acute HIOP may be related to the selective loss of retinal ganglion cells.

Purpose: To detect changes in local blood supply to central, middle, and peripheral retina following acute high intraocular pressure and to investigate the effects of changes in local retinal blood supply on the selective loss of retinal ganglion cells.Methods: The acute high intraocular pressure model of Sprague-Dawley rats was established by increasing the anterior chamber pressure to 110 mmHg via a normal saline perfusion system. Blood supply to the central, middle, and peripheral retina at 3, 6, and 12 h, and 1, 3, 7, and 14 d following induction of acute high intraocular pressure was detected by using gelatin-ink perfusion and fluorescent microsphere injection. Retinal ganglion cell loss following acute high intraocular pressure was detected by fluorogold retrograde labeling. Finally, the relationship between changes in local retinal blood supply and loss of retinal ganglion cells was investigated.Results: The increased ratio of blood supply of peripheral retina was less than that of the central and middle retina at 3 h to 14 d following acute high intraocular pressure. The percent of retinal ganglion cell loss in the peripheral retina was clearly greater than that in the central and middle retina during the first 3 d following induction of acute high intraocular pressure (p < 0.05). Using either the gelatin-ink infusion method or the microsphere injection method (p < 0.05), a significantly negative correlation between the percent of retinal ganglion cell loss and the corresponding increased local blood supply after induction of acute high intraocular pressure (r = -0.57 or -0.72) was found. Moreover, a significant negative correlation in the peripheral retina (r = -0.80 or -0.91; p < 0.05) was observed.Conclusion: A disparity exists between changes in local blood supply to the central and middle retina, and the peripheral retina following induction of acute high intraocular pressure in rats. This inequality of changes in local blood supply in rat retina is related to the selective loss of retinal ganglion cells.

Background The progressive death of retinal ganglion cells (RGCs) is the primary pathological characteristics of visual defects in glaucomatous eye.Research showed that endoplasmic reticulum stress (ERS) is involved in this progression.Glucose-regulated protein 78 ( GRP78 ) is a special marker of ERS.To understand the change in expression of GRP78 in the retina under the pressure induced is very important for the protection of visual function. Objective The present study was to observe the expression of GRP78 in rat retina with acute high intraocular pressure (IOP) and investigate the possible effect of ERS in acute glaucoma damage. Methods Fiftysix Wistar rats were randomly assigned to normal control group,anterior chamber punctured group and 12 hours,1 day,3,7,14 days groups following acute IOP rising.The acute high IOP models were established in the right eyes of 4.0 Wistar rats by paracentesis of the anterior chamber and infusion of normal saline solution into the anterior chamber.The histopathology changes of the retina were examined by hemotoxylin and eosin staining.The expression of GRP78 protein and mRNA in the retina were detected by immunohistochemistry and real-time fluorescence quantitative PCR. Results The retinal layers and cells were clear with normal alignement in the rats of the normal control group and the anterior chamber punctured group.The edema and thickening of retinas appeared in 12 hours after molding and peaked in 1 day after molding.Then the retina decreased in thickness and atrophied from 3 days through 14 days.The expression levels of GRP78 protein (A value) were 0.195±0.006 in the nounal rats and gradually increased from 12 hours through 3 days (0.268±0.017) following molding with the peak value at 1 day (0.499±0.039 ),showing significant differences in comparison with the normal control group (t =0.098,0.304,0.073,P＜0.05),and the reduction in expression at 7 and 14 days were not significantly different from the normal control group ( t =0.002,0.001,P＞0.05 ).The relative value of GRP78 mRNA ( 2△△C1 ) in the retina was 1.011 ±0.013 in the normal control group and gradually up-regulated from 12 hours through 3 days with the peak value at Iday (2.141±0.171 ) and then down-regulated from the third day onwards.A significant difference was seen in 2-△△C1value between the normal control group and 12 hours group,1 day group or 3 group ( t =0.525,1.130,0.409,P＜0.05 ).However,the 2-△△C1 values of GRP78 mRNA at 7 and 14 days was similar to that of the normal control group (t=0.020,0.004,P＞0.05). Conclusions GRP78 participates in the process of RGCs damage following acute high IOP.The results suggest that interfering with ERS may be helpful for protecting the retina and optical nerve from pressure-induced damage. Key words: Glaucoma/acute ocular hypertension, animal model; Glucose-regulated protein; Retinal ganglion cell; Endoplasmic reticulum stress

The purpose of this investigation was to elucidate the regulation of inducible nitric oxide synthase (iNOS) expression by vascular endothelial growth factor (VEGF) in a muscle flap ischemia model in rats. The gracilis muscle flap model was chosen. Sixty adult male Sprague Dawley rats were randomly divided into two groups (n = 30). After 4 hours ischemia, the experimental group received VEGF treatment, and the control group was given saline in the same fashion. At time intervals of 0, 2, 6, 12, and 18 hours (n = 6 for each time interval) after injection, tissue samples were biopsied for reverse transcriptase polymerase chain reaction, routine hematoxylin-eosin staining, and CD31 immunohistochemical staining. The result showed that iNOS expression is increased in the gracilis muscle flap ischemia model in rats compared with the control group within 6 hours after ischemia (p < 0.05). In the VEGF group, iNOS expression increased rapidly over the first 2 hours, but no statistically significant difference was observed at 12 and 18 hours between the two groups (p > 0.05). We concluded that the application of VEGF could maintain the structure of capillaries and upregulate iNOS expression during the first 6 hours after ischemia in the ischemic muscle flap of a rat model. These findings may provide the evidence to study the mechanism of VEGF in improving flap survival.

We constructed brain-derived neurotrophic factor (BDNF) expressing rat retinal pigment epithelial (RPE) cells by stable transfection of BDNF cDNA, and the RPE cells were cultured on a cross-linked collagen sheet (Coll-RPE-BDNF). BDNF expression of the Coll-RPE-BDNF was confirmed by western blot, and the Coll-RPE-BDNF was transplanted into the rabbit sclera. In vivo BDNF expression was confirmed by His expression that was linked to the expressing BDNF. The effect of the released BDNF was examined in a rabbit acute high intraocular pressure system by electroretinogram and histological examination. Statistically significant preservation of ERG b wave amplitude was observed in the rabbits treated by Coll-RPE-BDNF when compared to that of no treatment. Statistically significant preservation of the thickness of the inner nuclear layer at the transplanted area was observed in the rabbits treated by Coll-RPE-BDNF compared to that of no treatment. Intra-scleral Coll-RPE-BDNF transplantation may partially rescue retinal cells from acute high intraocular pressure.

Acute high intraocular pressure (HIOP) can induce plastic changes of retinal synapses during which the expression of the presynaptic marker synaptophysin (SYN) has a distinct spatiotemporal pattern from the inner plexiform layer to the outer plexiform layer. We identified the types of neurotransmitters in the retina that participated in this process and determined the response of these neurotransmitters to HIOP induction. The model of acute HIOP was established by injecting normal saline into the anterior chamber of the rat eye. We found that the number of glutamate-positive cells increased successively from the inner part to the outer part of the retina (from the ganglion cell layer to the inner nuclear layer to the outer nuclear layer) after HIOP, which was similar to the spatiotemporal pattern of SYN expression (internally to externally) following HIOP. However, the distribution and intensity of GABA immunoreactivity in the retina did not change significantly at different survival time post injury and had no direct correlation with SYN expression. Our results suggested that the excitatory neurotransmitter glutamate might participate in the plastic process of retinal synapses following acute HIOP, but no evidence was found for the role of the inhibitory neurotransmitter GABA.

To describe different patterns of diabetic macular oedema (DMO) using a computerized alignment and averaging of sequences in optical coherence tomography (OCT) B-scans and to show the correlation of these patterns with the pathophysiology of the condition. We carried out a prospective, uncontrolled study, including 46 human subjects with untreated DMO. Enhanced OCT images were produced. We correlated different OCT patterns of DMO with ETDRS visual acuity and with the thickness of the central 1-mm of the macula. We also correlated these patterns with theories of the pathophysiology of DMO. Enhanced OCT images enabled us to examine how different layers of the macula were affected. The external limiting membrane (ELM) was clearly seen in all stages, including advanced stages. The sequence of DMO events in different macular layers can be divided into five patterns. Morphologically, DMO starts at the outer nuclear layer/Henle's layer. As the oedema progresses, cysts are seen in the fovea and the DMO spreads further into the inner nuclear layer. The ELM seems to act as a barrier for proteins and plays an important role in the development of DMO. Enhanced OCT revealed new details of DMO pathophysiology. The different morphological patterns of DMO seen in enhanced OCT may represent different levels of severity of the disease.

Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. The signals that regulate neurogenesis in the dentate gyrus following ischemic stroke insult are not well known. We have previously reported that inducible nitric oxide synthase (iNOS) expression is necessary for ischemia-stimulated neurogenesis in the adult dentate gyrus. Here, we show that mice subjected to 90 min of middle cerebral artery occlusion (MCAO) significantly increased the number of new neurons and up-regulated iNOS expression in the dentate gyrus. Blockade of the L-type voltage-gated Ca(2+) channel (L-VGCC) prevented neurogenesis in the dentate gyrus and subventricular zone (SVZ), and down-regulated iNOS expression in the dentate gyrus after cerebral ischemia. This study suggests that Ca(2+) influx through L-VGCC is involved in ischemia-induced neurogenesis by up-regulating iNOS expression.

Purpose: To investigate whether retinal impairment was affected by age of rats in acute glaucoma model.Methods: Young adult and aged rats were randomly divided into normal control, 45 mmHg, 60 mmHg and 90 mmHg groups. Intraocular pressures (IOP) of rats were acutely elevated to 45 mmHg, 60 mmHg and 90 mmHg, respectively. Neuron loss in ganglion cell layer (GCL) and activation of retinal macrolgia and microglia 3 days after high IOP treatment were detected by immunofluorescence and further quantitatively analyzed.Results: Compared with normal control, significant loss of neurons at GCL of young adult retina wasn’t detected until IOP treatment of 90 mmHg. In contrast, obvious loss of neurons at GCL of aged retina was detected at IOP of 45 mmHg (p = 0.002 for central; p = 0.001 for peripheral). The loss level of neurons of aged retina was significantly higher than that of young adult retina at different IOP treatments. Compared with the young adult retina, high IOP induced more significant increase at area percentage of microglia and microglia number in inner part of aged retina. Activation of microglia and macroglia was either in parallel to or earlier than neuron loss of GCL of aged and young adult retina.Conclusion: Our data suggest there exists an age–related susceptibility of rat retina to the increased IOP. Therefore, the effect of ages should be considered at glaucoma study of rat models.

Human inducible nitric oxide synthase (iNOS) expression is regulated by transcriptional and post-transcriptional mechanisms. We have recently shown that the multifunctional RNA-binding proteins KH-type splicing regulatory protein and tristetraprolin are critically involved in the post-transcriptional regulation of human iNOS expression. Several reports have shown that KH-type splicing regulatory protein colocalizes with the polypyrimidine tract-binding protein (PTB), and both RNA-binding proteins seem to interact with the same mRNAs. Therefore we analyzed the involvement of PTB in human iNOS expression. In human DLD-1 cells, cytokine incubation necessary to induce iNOS expression did not change PTB localization or expression. However, intracellular binding of PTB to the human iNOS mRNA increased after cytokine stimulation. Overexpression of PTB resulted in enhanced cytokine-induced iNOS expression. Accordingly, small interfering RNA-mediated knock down of PTB reduced cytokine-dependent iNOS expression. Recombinant PTB displayed binding to an UC-rich sequence in the 3'-untranslated region of the human iNOS mRNA. Transfection experiments showed that PTB mediates its effect on iNOS expression via binding to this region. The underlying mechanism is based on a modulation of iNOS mRNA stability. In summary, human iNOS is the first example of a human pro-inflammatory gene regulated by PTB on the level of mRNA stability.

Ikaros is known as a critical regulator of lymphocyte development. We examined the regulatory role of Ikaros in LPS/IFN-gamma-induced inducible nitric oxide synthase (iNOS) expression by macrophages. Our results showed that IK6 (Ikaros dominant negative isoform) induction increases the iNOS expression. Ikaros DNA binding activity on the iNOS promoter was decreased, and a mutation of the Ikaros-binding site on the iNOS promoter resulted in an increase in LPS/IFN-gamma-induced iNOS expression. LPS/IFN-gamma increased the histone (H3) acetylation on the Ikaros DNA binding site. These results suggest that Ikaros acts as a negative regulator on iNOS expression. Treatment with a casein kinase 2 (CK2) inhibitor reversed LPS/IFN-gamma-induced decrease in Ikaros DNA binding activity. Moreover, overexpression of kinase-inactive CK2 decreased iNOS expression and a significant amount of CK2alpha1 translocated into the nucleus in LPS/IFN-gamma-treated cells. Overall, these data indicate that LPS/IFN-gamma decreases the Ikaros DNA binding activity via the CK2 pathway, resulting in an increase of iNOS expression.

Purpose: To assess the effect of electric acupuncture on the retinal ganglion cells (RGCs) in rabbits with acute high intraocular pressure (IOP). Methods: This comparative experimental study included 32 rabbits assigned into 3 groups: group 1 (n=5), group 2 (n=10) and group 3 (n=17). The rabbits in groups 2 and 3, which were anesthetized with an intravenous injection of 30% sodium urethane, were subjected to high pressure perfusion to anterior chambers by increasing the IOP to 30, 50 and 70 mmHg for 2 hours. After 2 weeks of electric acupuncture treatment with G6805-2 electron treatment device on acupoints Housanli and Jingming of rabbits in group 3, the rabbit’s eyes of all three groups were enucleated and its RGCs were observed with transmission electron microscope. Results: In group 1, that’s the normal control, its ultrastructures appeared normally while different changes of RGCs were happened in the other two groups. Ultrastructurally, in group 2, under different degrees of high pressure perfusion, its RGCs showed the light colour of cytoplasm, less organelles, irregular rough endoplasmic reticulum (RER) and Golgi apparatus, and vacuolated mitochondria with broken cristae etc. And in group 3, a few relatively intact RGCs were noticed especially under 30 mmHg and 50 mmHg perfusion but no obviously improved under 70 mmHg perfusion. Conclusion: Electric acupuncture is more helpful to RGCs with high pressure perfusion from 30 mmHg to 50 mmHg. Electric acupuncture might be used for vision injury of glaucoma with moderate high IOP.

In response to injury, synapse alteration may occur earlier than the changes in the cell body of neurons. Although retinal ganglion cell death and thinning of the inner part of retina were found after acute high intraocular pressure (HIOP), the structural and functional changes of synapses in the retina remain unknown. In the present study, we investigated the protein and mRNA expression of synaptophysin (SYN), an important molecule closely related to synaptic activities, synaptogenesis and synaptic plasticity. In addition, we also studied the ultrastructural changes of the retinal synapses. We found that (1) synaptophysin was upregulated transiently at both protein and mRNA level following HIOP; (2) broadened distribution of synaptophysin protein was present within the outer nuclear layer at the early stage following HIOP; (3) in the outer nuclear layer bouton-like vesicle-containing structures were observed by electron microscopy. This data suggested that, besides degeneration, synapses in rat retina may undergo regenerative events following HIOP.

Bacterial lipopolysaccharide (LPS) is known to induce endotoxic shock with inducible nitric oxide (NO) synthase (iNOS) expression and NO production. However, the major place for NO production in shock remains unclear. Although there is some literature about p38 mitogen-activated protein kinase (MAPK) in regulating LPS-induced iNOS expression, the results are contradictory. To interpret the precise cell mechanism and the role of p38 MAPK in the expression of iNOS during endotoxic shock, we carried out the following investigations. A severe endotoxic shock model was reproduced in mice 6 h after LPS injection. The plasma NO level was increased in a dose- and time-dependent manner after LPS stimulation and was suppressed by administration of SB203580 [4-(4-fluorophenyl)-2-4-methylsulfonylphenyl-5-(4-pyridyl) imidazole], a highly specific inhibitor of p38 MAPK. The iNOS expression was increased in many organs, including heart, liver, spleen, lung, gut, and kidney in endotoxic shock. Among them, the highest expression of iNOS mRNA and protein was in the lung, moderate expression was in the spleen and kidney, and the lowest expression was in the heart, gut, and liver. The level of expression in lung was 5.5 times that of iNOS mRNA and was 3.1 times that of iNOS protein than in heart, and 1.6 and 1.8 times that of iNOS mRNA and 1.7 and 1.4 times that of iNOS protein than in spleen and kidney, respectively. The p38 MAPK activity increased after LPS injection, and SB203580 markedly reduced LPS-induced expressions of iNOS protein and mRNA in the lung. The results indicates that lung, spleen, and kidney are the major places for iNOS expression in endotoxic shock and are important therapeutic target organs for attenuating NO production in shock treatment.