Dendrimer mediated targeted delivery of sinomenine for the treatment of acute neuroinflammation in traumatic brain injury.

Traumatic brain injury (TBI) is a significant medical problem with limited treatment options and is one of the main causes of life-long disability. Neuroinflammation orchestrated by activated microglia/macrophages at the site of injury plays a critical role in the onset of many pathological events following TBI, leading to blood brain barrier (BBB) dysfunction, neuronal damage and long term neuronal and behavioral deficits. Current treatment involves intravenous administration of anti-inflammatory drugs which have limited clinical outcomes only when dosed within the early time window after injury. Hence there is an urgent need to develop improved drug delivery systems which have potential to cross impaired BBB, target and deliver drugs selectively to activated microglia/macrophages at the sites of injury, and suppress the detrimental effects of acute inflammation. In this study, we have used Sinomenine (Sino), a potent anti-inflammatory and antioxidant drug conjugated to hydroxyl terminated generation-4 PAMAM dendrimer (D-Sino) as a potential therapy for attenuating early inflammation in TBI. D-Sino conjugates were synthesized using highly robust copper-catalyzed click reaction with high purity. D-Sino conjugates enhanced the intracellular availability of Sino due to their rapid cellular uptake, significantly attenuated early/acute inflammation by suppressing pro-inflammatory cytokines (TNF-α, IL-1ß, CCL-3 and IL-6), and reduced oxidative stress (iNOS and NO) in LPS activated murine macrophages (RAW 264.7) by inhibiting NF-κB activation and its nuclear translocation (the root cause for inflammation inception) significantly more as compared to the free drug. Upon systemic administration in a rabbit model of pediatric TBI, D-Sino conjugates specifically targeted activated microglia/macrophages at the site of injury in the brain. Single dose of D-Sino attenuated inflammation in the injured brain areas by suppressing inflammatory cytokines expression whereas free Sino treatment did not demonstrate a significant effect. Together, these results suggest that D-Sino conjugate may open up new avenues for increasing the therapeutic window in the treatment of early inflammation and for improving the efficacy of the drug in TBI. Moreover, this treatment can work in conjunction with current clinical practices such as therapeutic hypothermia and pharmacologically induced coma for many indications associated with TBI, where acute inflammation plays a critical role in disease progression.

Pathologic study of early manifestations of polypoidal choroidal vasculopathy and pathogenesis of choroidal neo-vascularization.

PURPOSE: To describe the histopathologic features of an early case of presumably bilateral polypoidal choroidal vasculopathy (PCV) in two eyes obtained at autopsy from a patient with no prior ocular therapy. OBSERVATIONS: The choroid of both eyes at the macular and peripapillary regions was greatly thickened with dilated, thin walled choroidal venules intertwining with arteriosclerotic arterioles in the Sattler's layer of the choroidal vasculature. At the temporal and nasal equatorial regions of both eyes many of these congested venular channels abruptly disappeared and were replaced by loose connective tissue with loss of the normal choroidal stromal tissue and uveal melanocytes. A few remaining venules showed intraluminal sloughing of endothelial cells and deposition of fibrinous material networks suggesting occlusion of these choroidal venules. At this equatorial location, serous detachment of retinal pigment epithelium (RPE) appeared and a thin neovascular membrane with cords of endothelial cell invaded into the sub-RPE space. Anteriorly, the neovascular membrane expanded and bulged into the sub-retinal space with dilated neovascular capillaries in a "grape like" or polypoidal configuration. CONCLUSION AND IMPORTANCE: Polypoidal Choroidal Vasculopathy is a disease of the dilated and multi-layered choroidal venules. Occlusion of these choroidal vascular channels might give rise to choroidal stasis and ischemia leading to serous RPE detachment and a sub-RPE neovascular membrane. Gross dilatation of the choroidal venules and capillaries in the sub-RPE neovascular membrane leads to the characteristic "grape like" structures, a unique clinical feature in this disease entity. These pathologic features of PCV are different from the pathologic changes of neovascular age-related macular degeneration (nAMD). Consequently, PCV and nAMD are two distinct diseases. However, in the late stage of both entities, choroidal ischemia in both diseases, lead to sub-RPE neovascularization and subsequent sub-RPE and/or sub-retinal hemorrhage. These results in both entities showed comparable clinical and pathologic features that are frequently mistaken PCV as a sub-type of Neovascular AMD.

A review of the immunologic findings observed in retinitis pigmentosa.

Most patients suffering from retinitis pigmentosa (RP) inherit the disorder; however, the immune-pathologic features associated with this disease have yet to be extensively studied. Six reports correlate antiretinal immune activity with vision deterioration in RP patients. Some of these patients have sporadic RP that occurs in excess of expected gene segregation during inheritance. The hypothesis that a primary immune-mediated disease process occurs in this sporadic group is supported by significant associations of RP with autoimmune endocrinopathies and other immune-related conditions or factors; however, no immunologic difference regarding RP family history is reported in the peripheral blood studies of RP patients. Twenty-one percent to 51% of RP patients display antiretinal antibodies, whereas 19-58% have antiretinal lymphocyte reactivity to retinal extract, and 60-85% have activated T cells. Mutations in animal models of RP have been shown to cause endoplasmic reticulum stress that may initiate immunopathology for genetic RP, but oxidative stress also encourages immune cytotoxicity. In addition, necrotic cell death is evident, which promotes inflammatory conditions. We review mechanisms and evidence for an occult inflammation in genetic RP and examine reports of efficacy in retarding RP progression with anti-inflammatory agents in clinical trials.

Role of endoplasmic reticulum stress in the loss of retinal ganglion cells in diabetic retinopathy.

Endoplasmic reticulum stress is closely involved in the early stage of diabetic retinopathy. In the present study, a streptozotocin-induced diabetic animal model was given an intraperitoneal injection of tauroursodeoxycholic acid. Results from immunofluorescent co-localization experiments showed that both caspase-12 protein and c-Jun N-terminal kinase 1 phosphorylation levels significantly in-creased, which was associated with retinal ganglion cell death in diabetic retinas. The C/ERB mologous protein pathway directly contributed to glial reactivity, and was subsequently responsible for neuronal loss and vascular abnormalities in diabetic retinopathy. Our experimental findings dicate that endoplasmic reticulum stress plays an important role in diabetes-induced retinal neu-ronal loss and vascular abnormalities, and that inhibiting the activation of the endoplasmic reticulum stress pathway provides effective protection against diabetic retinopathy.

The international council of ophthalmology: vision for ophthalmic education in an interdependent world.

PURPOSE: To describe the emerging strategic global perspective of the International Council of Ophthalmology (ICO) efforts in ophthalmic education. DESIGN: A global perspective describing how the development of sophisticated educational tools in tandem with information technology can revolutionize ophthalmic education worldwide. METHODS: Review of ICO educational tools, resources, and programs that are available to ophthalmic educators across the globe. RESULTS: With the explosive growth of the Internet, the ability to access medical information in the most isolated of locations is now possible. Through specific ICO initiatives, including the ICO curricula, the "Teaching the Teachers" program, and the launching of the new ICO Center for Ophthalmic Educators, the ICO is providing ophthalmic educators across the globe with access to standardized but customizable educational programs and tools to better train ophthalmologists and allied eye care professionals throughout the world. CONCLUSION: Access to educational tools and strengthening of global learning will help providers meet the goals of VISION 2020 and beyond in eliminating avoidable blindness. It is the intent of the ICO that its programs for ophthalmic educators, including conferences, courses, curricula, and online resources, result in better-trained ophthalmologists and eye care professionals worldwide.

Toxic effect of methamphetamine on the retina of CD1 mice.

PURPOSE: To investigate whether systemic administration of methamphetamine (METH) induces retinal damage in CD1 mice. MATERIALS AND METHODS: Eighteen male CD1 mice were randomly assigned to three groups, six mice per group: Group 1 receiving a single dose of 40 mg/kg METH, Group 2 receiving four doses of 10 mg/kg METH, and Group 3 (control) receiving 40 mg/kg 0.9% NaCl solution. METH and NaCl were administered by intraperitoneal injection. Immunostaining of glial fibrillary acidic protein (GFAP), S-100 for astrocytes and Muller cells, CD11b for microglia, and tyrosine hydroxylase (TH) and TUNEL labeling for apoptotic cell death were performed on the retinal sections on day 1 and day 7 post-exposure. RESULTS: GFAP and S-100 immunoreactivity was observed in Group 1 mice. CD11b+ cells in Group 1 mice showed more intensely stained shorter and thicker cellular processes than Groups 2 and 3, indicating activated microglia in the mice exposed to large-dose METH. No significant difference in TH level was seen among the three groups. TUNEL labeling did not reveal positive cells in the retinas of any of the 18 CD1 mice. CONCLUSIONS: A single large dose of METH induces an increase in short-term protein expression of GFAP and S-100 and in microglial activation. The results suggest that METH has a neurotoxic effect on CD1 mouse retina.

Autophagy, exosomes and drusen formation in age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of loss of vision in developed countries. AMD is characterized by a progressive degeneration of the macula of the retina, usually bilateral, leading to a severe decrease in central vision. An early sign of AMD is the appearance of drusen, which are extracellular deposits that accumulate on Bruch's membrane below the retinal pigment epithelium (RPE). Drusen are a risk factor for developing AMD. Some of the protein components of drusen are known, yet we know little about the processes that lead to formation of drusen. We have previously reported increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we used in vitro modeling of increased mtDNA damage. Under conditions of increased mtDNA damage, autophagy markers and exosome markers were upregulated. In addition, we found autophagy markers and exosome markers in the region of Bruch's membrane in the retinas of old mice. Furthermore, we found that drusen in AMD donor eyes contain markers for autophagy and for exosomes. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients.

Autophagy and exosomes in the aged retinal pigment epithelium: possible relevance to drusen formation and age-related macular degeneration.

Age-related macular degeneration (AMD) is a major cause of loss of central vision in the elderly. The formation of drusen, an extracellular, amorphous deposit of material on Bruch's membrane in the macula of the retina, occurs early in the course of the disease. Although some of the molecular components of drusen are known, there is no understanding of the cell biology that leads to the formation of drusen. We have previously demonstrated increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we found that drusen in AMD donor eyes contain markers for autophagy and exosomes. Furthermore, these markers are also found in the region of Bruch's membrane in old mice. By in vitro modeling increased mtDNA damage induced by rotenone, an inhibitor of mitochondrial complex I, in the RPE, we found that the phagocytic activity was not altered but that there were: 1) increased autophagic markers, 2) decreased lysosomal activity, 3) increased exocytotic activity and 4) release of chemoattractants. Exosomes released by the stressed RPE are coated with complement and can bind complement factor H, mutations of which are associated with AMD. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients and may be associated with the pathogenesis of AMD in the elderly.

Differentiation of rabbit bone marrow mesenchymal stem cells into corneal epithelial cells in vivo and ex vivo.

PURPOSE: To examine whether bone marrow mesenchymal stem cells (MSCs) could be differentiated into corneal epithelial cells in vivo and ex vivo. METHODS: In vivo, BrdU labeled rabbit MSCs (Rb-MSCs) were suspended in the fibrin gels and transplanted onto the surface of the damaged rabbit corneas. Histology and molecular phenotype were studied on postoperative day 28. In vitro, labeled Rb-MSCs were cultured for three days in two different systems: (1) Group A: Rb-MSCs were co-cultured with rabbit limbal stem cells (Rb-LSCs) by the Transwell culture system. A suspension of Rb-LSCs was added to the upper membrane surface, and the inserts were positioned in the culture wells, which were incubated with Rb-MSCs; (2) Group B: Supernatant medium that had first been used to culture Rb-LSCs and then filtered with a 0.45 mum filter was used to culture Rb-MSCs. For both groups, immunofluorescence and flow cytometric analysis were used to examine the expression of cytokeratin 3 (CK3) in differentiated Rb-MSCs. RESULTS: In vivo, the data showed that following transplantation of Rb-MSCs, the rabbit's damaged corneal surface was successfully reconstructed and that some Rb-MSCs participated in the healing of the injured corneal epithelium and expressed CK3. In vitro, the data showed that Rb-MSCs rapidly differentiated into cells with a morphological and molecular phenotype of corneal epithelial-like cells. For both groups, the differentiated Rb-MSCs were positive for corneal epithelial-specific marker CK3. In Group A, flow cytometry analysis showed that at day one, only 3.46+/-1.9% of cells expressed CK3. This increased to 7.24+/-3.80% at day two and decreased slightly (5.50+/-3.33%) at day three. The proportion of CK3 in Group B was 4.09+/-1.84% at day one, rising to 9.31+/-5.92% after 24 h, but falling (4.37+/-2.61%) at day three. The mean differences are significant between each group and the negative control, but was not significant between Group A and Group B. CONCLUSIONS: MSCs could differentiate into corneal epithelial-like cells in vivo and ex vivo.

Baicalein reduces inflammatory process in a rodent model of diabetic retinopathy.

PURPOSE: This study was designed to elucidate the role of inflammatory process in diabetic retinopathy and to investigate the effect of baicalein treatment on diabetic rat. METHODS: Retinal microglial cells were identified with CD11b antibody, and retinal Müller cells were identified with glial fibrillary acidic protein (GFAP). The gene expression of interleukin (IL)-18, tumor necrosis factor (TNF)-alpha, and IL-1beta was examined by quantitative real-time PCR. The expression of GFAP and vascular endothelial growth factor (VEGF) was examined by quantitative real-time PCR, immunohistochemistry, and Western blot analysis. Vascular permeability was measured in vivo by bovine serum albumin conjugated with FITC. Baicalein was given by oral administration (150 mg/kg/d) with an animal feeding needle beginning 5 days after streptozotocin (STZ) injection. RESULTS: By 24 weeks after onset of diabetes, microglial cells were activated and proliferated, and Müller cells upregulated their GFAP and VEGF expression. Pro-inflammatory factors, including IL-18, TNF-alpha, and IL-1beta, were significantly upregulated. Obvious vascular leakage and abnormality were demonstrated, and ganglion cell loss was significant. Baicalein treatment ameliorated diabetes-induced microglial activation and pro-inflammatory expression, reduced the GFAP and VEGF expression from Müller cells, and significantly reduced vascular abnormality and ganglion cell loss within the retina. CONCLUSIONS: Inflammatory process, characterized by microglial activation and Müller cells dysfunction, was implicated in STZ-induced diabetic retinopathy. Baicalein treatment ameliorated inflammatory process, and therefore inhibited vascular abnormality and neuron loss in diabetic retinas.

Early retinal vascular abnormalities in African-American cocaine users.

PURPOSE: To investigate whether cocaine use is associated with early retinal vascular abnormalities. DESIGN: Population-based cross-sectional study. SETTINGS: Inner-city neighborhoods in Baltimore, Maryland. STUDY POPULATION: Sixty-eight participants were recruited from an ongoing observational study, investigating cardiovascular complications of human immunodeficiency virus (HIV) infection and cocaine use in African Americans aged between 25 and 54 years. Those with hypertension and known cardiovascular/cerebrovascular diseases were excluded. OBSERVATION PROCEDURES: Ophthalmoscopic examinations and fundus photography of the retinas of these subjects were performed after pupillary dilation. The largest angle of arterial bifurcation (LAAB), central retinal artery equivalent (CRAE), and central retinal vein equivalent (CRVE) were measured by single-masked fundus image examiners. MAIN OUTCOME MEASURES: LAAB, CRAE, and CRVE. RESULTS: Among the 68 study subjects, 52 (76.5%) were chronic cocaine users and 16 (23.5%) were non-cocaine users. Univariate and multivariate analyses indicated that the LAAB was associated with age and duration of cocaine use of more than 10 years. The LAAB was also inversely associated with very low-density lipoproteins levels. Multivariate analysis indicated a positive association between CRVE and cocaine use. CRAE was also associated with intravenous injection. We confirmed that CRAE was inversely associated with age. HIV infection was not found to be associated with any retinal vascular parameters. CONCLUSIONS: Cocaine use is associated with increased retinal arterial branching angle and venular caliber. The retinal vascular changes provided the first evidence that cocaine use has an effect on the retinal vascular system.

Activation of nuclear factor-kappaB during retinal degeneration in rd mice.

PURPOSE: Transcription factors of the nuclear factor-kappa beta (NF-kappaB) family have been demonstrated to play an important role in the regulation of gene expression in the chronic neurodegenerative disorders. The aims of the current study were to investigate the alteration of NF-kappaB activity during retinal degeneration in rd mice and further explore its role in photoreceptor apoptosis. METHODS: Activation of NF-kappaB and its nuclear translocation in the retina of rd mice at postnatal days (P) 8, 10, 12, 14, 16, 18, and 28 were studied by immunohistochemical analysis using NF-kappaB P65 antibody. The amount of NF-kappaB P65 protein and NF-kappaB DNA-binding activity in the whole retina were assessed by western blot analysis and gel shift analysis, respectively. Expression of NF-kappaB in microglial cells labeled with CD11b was determined by double labeling. RESULTS: NF-kappaB P65 nuclear translocation and its DNA binding activity started to increase in the rd retina at P10 and reached a peak at P12. Expressions of P65 remained at high levels from P12 to P18. Double labeling of P65 with CD11 at P14 showed colocalization of P65 in the microglial cells in the outer nuclear layer. CONCLUSIONS: NF-kappaB was activated in the retinal degeneration of rd mice. NF-kappaB modulation may play a role in the retinal degeneration through microglial activation.

Microglial activation in human diabetic retinopathy.

OBJECTIVE: To investigate microglial activation in human diabetic retinopathy. METHODS: Paraffin sections from 21 eyes of 13 patients with diabetic background, preproliferative, or proliferative retinopathies and 10 normal eyes of 9 individuals were studied with immunolabeling of microglia with antibodies against HLA-DR antigen, CD45, or CD68. RESULTS: In the healthy human eyes, ramified microglial cells were scattered in the inner retinal layers. In eyes with diabetic retinopathy, the microglia were markedly increased in number and were hypertrophic at different stages of the disease. These cells clustered around the retinal vasculature, especially the dilated veins, microaneurysms, intraretinal hemorrhages, cotton-wool spots, optic nerve, and retinal and vitreal neovascularization. In some retinas with cystoid macular edema, microglia infiltrated the outer retina and subretinal space. Cells in the epiretinal membrane were also labeled with microglial markers. CONCLUSIONS: Microglia were activated at different stages of human diabetic retinopathy and optic neuropathy. Microglial perivasculitis was a prominent feature of the disease process. CLINICAL RELEVANCE: Activated microglia and microglial perivasculitis may play a role in vasculopathy and neuropathy in diabetic retinopathy.

Endoplasmic reticulum stress is activated in light-induced retinal degeneration.

Exposure to excessive levels of light induces photoreceptor apoptosis and has previously been used as a model for the study of retinal degeneration. During the light exposure, intracellular calcium levels increase, and reactive oxygen species (ROS) are generated, which have been shown to cause endoplasmic reticulum (ER) stress. In the present study, we investigated the role of ER stress in light-induced photoreceptor apoptosis. Our study demonstrated that, after light exposure, the ER stress sensors including glucose-regulated protein-78 (GRP78/BiP), caspase-12, phospho-eukaryotic initiation factor 2 alpha (eIF2 alpha), and phospho-pancreatic ER kinase (PERK) were significantly up-regulated in a time-dependent manner. The up-regulation of these proteins coincided with or preceded the photoreceptor apoptosis indicated by TUNEL. These data showed that ER stress played an important role in light-induced photoreceptor apoptosis. Therefore, ER stress modulators could be strong candidates as therapeutic agents in the treatment of retinal degenerative diseases.

A possible mechanism of microglia-photoreceptor crosstalk.

PURPOSE: The goal of this study was to explore the relationship between photoreceptor apoptosis and retinal microglial activation. METHODS: A murine photoreceptor cell line (661W cells) was exposed to LPS-treated microglial cell conditioned medium (MGCM), and cell viability was assessed by terminal dUTP transferase nick end labeling (TUNEL) and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, microglia were exposed to culture media from light-damaged 661W photoreceptor cells (PRCM), and microglial activation was assessed morphologically by phase contrast microscopy. Reverse transcription polymerase chain reaction was used to examine mRNA levels of several chemokines and noxious factors in the MGCM-treated photoreceptor cells and the PRCM-treated microgial. Western blotting was used to analyze NF-kappaB p65 subunit, phosphorylated MAPKs p38, p44/42 (Erk1/2), and c-Jun N-terminal kinase (JNK). RESULTS: Our results showed 37% of 661W cells underwent apoptosis following exposure to MGCM for 24 h. MGCM-induced death was associated with down-regulation of chemokine expression (i.e., eotaxin and RANTES), upregulation of inflammatory mediators (i.e., MIP-1alpha, MIP-1beta, IL-10, iNOS, and TNF-alpha), and increased phosphorylation of p38, p44/p42, and JNK. Retinal microglia acquired an activated phenotype after exposure to PRCM for 24 h. Microglial activation was accompanied by increased NF-kappaB p65 expression, increased phosphorylation of p38 and JNK, and upregulation of chemokines (i.e., eotaxin and RANTES) and inflammatory mediators (i.e., iNOS and IL-10). CONCLUSIONS: Light-damaged photoreceptors release immunological signaling molecules that attract microglia, resulting in microglial activation and subsequent further degeneration of remaining photoreceptors. These results also suggest that p38, p44/42, and JNK may regulate glial-induced photoreceptor death and that p38, JNK, and NF-kappaB may regulate photoreceptor-induced microglial activation.

Activation of endoplasmic reticulum stress in degenerating photoreceptors of the rd1 mouse.

PURPOSE: Endoplasmic reticulum (ER) stress has been implicated in a wide variety of neurodegenerative disorders of the central nervous system (CNS). This study was designed to elucidate the role of ER stress in photoreceptor apoptosis in the rd1 mouse. METHODS: Photoreceptor apoptosis in the rd1 mouse was detected by terminal dUTP transferase nick-end labeling (TUNEL). Protein expressions of ER stress sensors, including glucose-regulated protein-78 (GRP78/BiP), caspase-12, phospho-eukaryotic initiation factor 2alpha (eIF2alpha), and phospho-pancreatic ER kinase (PERK), were examined by immunofluorescence and Western blot assays. RESULTS: Accompanying photoreceptor apoptosis in the rd1 mouse, the protein expressions of GRP78/BiP, caspase-12, phospho-eIF2alpha, and phospho-PERK were upregulated in a time-dependent manner. The upregulation of these proteins coincided with or preceded photoreceptor apoptosis. At the peak of their expression, these proteins were primarily located in the photoreceptor inner segments, the outer nuclear layer, or both. CONCLUSIONS: ER stress plays an important role in photoreceptor apoptosis in the rd1 mouse. Therefore, ER stress modulators may be strong candidates as therapeutic agents in the treatment of retinal degenerative diseases.

Sinomenine inhibits activation of rat retinal microglia induced by advanced glycation end products.

Diabetic retinopathy involves an inflammatory response in the retina characterized by an increase in inflammatory cytokines and activation of microglia. The degree of microglia activation may influence the extent of retina injury following an inflammatory stimulus. Cytokines, released by activated microglia, regulate the influx of inflammatory cells to the damaged area. Thus, therapeutic strategy to reduce cytokine expression in microglia would be neuroprotective. Sinomenine, an alkaloid isolated from the stem and root of Sinomenium acutum, has long been recognized as an anti-inflammatory drug for rheumatoid arthritis and also inhibits macrophage activation. In this study, we activated retinal microglia in culture with advanced glycation end products (AGEs) treatment and attempted to determine whether sinomenine could reduce the production of cytokines from the activated microglia at both gene and protein levels. Changes in inflammatory cytokines, TNF alpha, IL-1 beta and IL-6, were measured by semi-quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) both in the presence and absence of AGEs. The effect of sinomenine on levels of reactive oxygen species (ROS) and the nuclear translocation of NF-kB p65 were studied with a laser confocal scanning microscope. AGEs treatment induced a significant release of TNF alpha, IL-1 beta, and IL-6 from retinal microglia. Sinomenine could inhibit release of these cytokines. Sinomenine attenuated ROS production in a dose-dependent fashion and reduced the nuclear translocation of NF-kB p65 in AGEs-activated retinal microglia in culture.

Role of NF-kappaB and MAPKs in light-induced photoreceptor apoptosis.

PURPOSE: To elucidate the role of nuclear factor kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPKs) in light-induced apoptosis of photoreceptors in culture and to explore the potential inhibitory effect of minocycline and sulforaphane on apoptosis. METHODS: Apoptosis of 661W cells was induced by exposure to light and was detected by terminal dUTP transferase nick end labeling (TUNEL). The mRNA expression and protein production of 10 chemokines and noxious factors were examined by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The protein expression of the p65 subunit of NF-kappaB, and the MAPKs p-p38, p-p44/42, and p-JNK were examined by Western blot and immunofluorescence analyses. RESULTS: After exposure to light for 4 hours, 60% to 70% of the 661W cells underwent apoptosis. The expression of five selected chemokines and noxious factors was upregulated. The protein expression of the p65 subunit of NF-kappaB was downregulated, and the expression of the MAPKs p-p38, p-p44/42, and p-JNK was upregulated. Pretreatment with SB203580 for 1 hour inhibited light-induced upregulation of p-p38 and inhibited photoreceptor apoptosis. Pretreatment with minocycline or sulforaphane for 1 hour inhibited light-induced downregulation of the NF-kappaB p65 subunit and inhibited photoreceptor apoptosis. CONCLUSIONS: Apoptotic photoreceptors secrete chemokines and noxious factors to induce an immunologic response. The NF-kappaB and MAPK pathways both are involved in light-induced 661W photoreceptor apoptosis. Minocycline and sulforaphane inhibit light-induced photoreceptor apoptosis, partly through an NF-kappaB-dependent mechanism, but not through the MAPK pathway.

Minocycline delayed photoreceptor death in rds mice through iNOS-dependent mechanism.

PURPOSE: To elucidate the role of activated microglia and nitric oxide (NO) in photoreceptor apoptosis in rds mice, and to investigate the effect of minocycline treatment on rds mice. METHODS: Photoreceptor apoptosis in rds mice was detected by terminal dUTP transferase nick end labeling (TUNEL). Retinal microglial cells were identified by CD11b antibody. The mRNA expression of inducible nitric oxide synthase (iNOS) and chemokines were examined by reverse transcription polymerase chain reaction (RT-PCR) assay. The protein expression of iNOS was examined by immunohistochemistry and Western blotting analysis. The rds mice were treated intra-peritoneally from the second postnatal day (P2) with minocycline. RESULTS: Accompanying photoreceptor degeneration in rds mice, microglia were activated and immigrated from inner retinal layer (IRL) to outer nuclear layer (ONL), and the expression of iNOS was up-regulated. Minocycline treatment reduced the iNOS expression and decreased the initial photoreceptor apoptosis, but did not provide long term ameliorative effect on the photoreceptor cell loss of rds mice. CONCLUSIONS: NO played a major role in the initial photoreceptor apoptosis in rds mice. The migration of activated microglia to the ONL contributed to the subsequent photoreceptor cell death; minocycline treatment ameliorated the photoreceptor apoptosis in rds mice, and this protective effect was partly through iNOS-suppressive mechanism.