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.

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.

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.

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.

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.

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.

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.

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.

Minocycline and sulforaphane inhibited lipopolysaccharide-mediated retinal microglial activation.

PURPOSE: To elucidate the inhibitory effect of minocycline and sulforaphane on lipopolysaccharide (LPS)-induced retinal microglial activation and the mechanisms through which they exerted their inhibitory effects. METHODS: Primary retinal microglial cultures were exposed to LPS with or without minocycline and sulforaphane. The mRNA expression of monocyte chemotactic protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, eotaxin, regulated upon activation normal T-cell expressed and secreted (RANTES) protein, and interleukin (IL)-10 were examined by reverse transcription polymerase chain reaction (RT-PCR) assay. The mRNA expression of inducible nitric oxide synthase (iNOS) and subsequent nitric oxide (NO) production were examined by RT-PCR assay and Griess reagent assay. Protein expression of the p65 subunit of nuclear factor-kappaB (NF-kappaB) and p-p38, p-p44/42 and p-JNK mitogen-activated protein kinases (MAPKs) were examined by Western blot and immunofluorescent analysis. RESULTS: Cultured retinal microglial cells were activated following exposure to LPS. The mRNA expression and protein production of eotaxin, RANTES, and IL-10 and the mRNA expression of iNOS and subsequent NO production were upregulated. The protein expression of p-p38, p-JNK, and the p65 subunit of NF-kappaB were also upregulated. However, the protein expression of p-p44/42 was not significantly changed. Pretreatment with minocycline or sulforaphane for 1 h before LPS administration inhibited LPS-induced microglial morphological change and inhibited LPS-induced upregulation of p-p38, but had no effect on the expression of p-p44/42, p-JNK, and the p65 subunit of NF-kappaB. CONCLUSIONS: Minocycline and sulforaphane inhibited LPS-induced retinal microglial activation, Western blot and immunofluorescent studies showed decreased p-p38 MAPK expression. We suggested that the inhibitory effect of minocycline and sulforaphane was partly through a p38 MAPK-dependent mechanism.

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.

An international strategic plan to preserve and restore vision: four curricula of ophthalmic education.

PURPOSE: To highlight the four International Curricula of Ophthalmic Education developed by the Task Forces of the International Council of Ophthalmology, published in Klinische Monatsblätter für Augenheilkunde in November 2006. DESIGN: A global perspective of developing educational curricula as tools to improve eye care. METHODS: Review of the experience and conclusions of the four international panels. RESULTS: The Task Force on Resident and Specialist Education developed a curriculum consisting of 15 topics in basic, standard, and advanced levels to provide flexibility of educational programs of the ophthalmic specialist in different locations across the world. The curricula were designed to be an educational tool to stimulate multiple levels of training of the ophthalmic specialist. The Task Force on Ophthalmic Education of Medical Students designed a curriculum covering 11 topics and provides illustrative materials for teachers and students. The Task Force strongly advocates the ophthalmology curriculum to be part of the core program of general medical schools education. The Task Force on Para-ophthalmic Vision Specialist Education developed a curriculum to highlight the importance of a team approach to eye care, consisting of ophthalmic specialists and paraophthalmic personnel to produce maximum efficiency. The Task Force on Continuing Medical Education (CME) designed a curriculum exploring the principles, elements, categories, and administration of CME activities in a variety of topics. CONCLUSIONS: These curricula shifted the traditional apprentice system of education to a curriculum-based training program in which goals, expectations, competencies, and technical training are defined to improve eye care worldwide.

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.

[Pathological study of retinal excitotoxicity following subretinal injection of NMDA].

OBJECTIVE: To study neuronal excitotoxicity induced by subretinal injection of glutamate analogues. METHODS: We injected 30 mmol/L N-methyl-D-aspartate (NMDA) in 10 microl of DMEM/F12 into subretinal blebs in 24 one-month-old rabbits that were killed 0.5, 1, 2 or 7 days after injection. The retinas were studied histopathologically by light and electronic microscope. The horizontal cells, rod bipolar cells and amacrine cells were labeled by antiserum against calbindin, protein kinase Calpha, and calretinin, respectively. TUNEL labeling was performed to identify apoptotic cells. RESULTS: Compared with vehicle injected control retina, treated retina showed an early and severe morphological changes in both amacrine and ganglion cells and scattered densification in photoreceptor nuclei at acute stage (<1 d), all retinal neurons damage at the intermediate phase (2 d), and a significant loss of cells in all layers of the retina at the chronic phase (7 d), respectively. Horizontal, amacrine and ganglion cells were reduced after subretinal injection of NMDA. The number of rod bipolar cells had little change. The morphological changes of neuronal death included apoptosis, necrosis, hydropic degeneration and karyorrhectic cell death. TUNEL positive nuclei were significantly (P < 0.01) increased in all retinal layers at the acute phases after NMDA administration. CONCLUSIONS: NMDA administration into sub-retina induces excitotoxicity in photoreceptor, horizontal, rod bipolar, amacrine, and ganglion cells of retina, which is different from the other models of retinal cultures and intravitreal injection that only cause neuronal death in the inner retina.

Identification of sequential events and factors associated with microglial activation, migration, and cytotoxicity in retinal degeneration in rd mice.

PURPOSE: To elucidate the role of activated microglia in the photoreceptor apoptosis of rd mice by identifying sequential events and factors associated with microglial activation, migration, and cytotoxicity during retinal degeneration. METHODS: Photoreceptor apoptosis in rd mice at postnatal days (P)8, 10, 12, 14, 16, and 18 was detected by terminal dUTP transferase nick end labeling (TUNEL). Retinal microglia were identified by CD11b antibody. Expression of chemokine mRNA, including monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, regulated on activation normal T-cell expressed and secreted (RANTES), interferon-gamma-inducible 10-kDa protein (IP-10), and fractalkine in the retina were examined by reverse transcription-polymerase chain reaction (RT-PCR) assay. Production of tumor necrosis factor (TNF)-alpha in the dystrophic retina was studied by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry analysis. Microglial expression of TNF-alpha was determined by double immunolabeling. RESULTS: Whereas photoreceptor apoptosis in the rd mice started at P10 and reached a peak at P16, activation and migration of microglial cells were observed at P10 and peaked at P14. The expression of MCP-1, MCP-3, MIP-1alpha, MIP-1beta, and RANTES transcripts were noted at P8 and reached a peak at P12. Production of TNF-alpha was noted in the outer nuclear layer (ONL) of the rd mice at P8 and reached a peak at P12. At the peak of microglial activity, TNF-alpha was predominantly expressed in the activated microglial cells in the ONL. CONCLUSIONS: Activation of microglia, as well as expression of their signaling molecules (chemokines) and microglia-derived toxic factor (TNF-alpha), coincides with or precedes the occurrence of photoreceptor apoptosis, suggesting activated microglia play a major role in retinal degeneration in rd mice. The chemokines MCP-1, MCP-3, MIP-1alpha, MIP-1beta, and RANTES are involved in activation and recruitment of the microglia to the degenerating photoreceptor cell layer. TNF-alpha, produced by the activated microglia, may accentuate the photoreceptor cell death.

Activation of microglia and chemokines in light-induced retinal degeneration.

PURPOSE: Microglial cells, which are activated and recruited by chemokines, have been shown to play crucial roles in neuronal degenerations of the central nervous system (CNS). This study investigated the activation and migration of retinal microglial cells and expression of chemokines in retinas in light-induced photoreceptor degeneration in mice. METHODS: Ninety-five Balb/cJ mice were kept in cyclic light for 1 week followed by dark adaptation for 48 h prior to light exposure of 3 h at 3.5 Klux. Animals were enthuanized at various times after light exposure. Terminal deoxynucleotidyl transferase-mediated dUTP nick end label (TUNEL) assay, rat-anti-mouse CD11b and 5D4 antibodies, isolectin-B4, and a chemokine-specific gene array were used to detect DNA fragmentation during retinal degeneration, to label retinal microglial cells, and to determine the expression of retinal chemokines and chemokine receptors, respectively. Reverse-transcriptase coupled polymerase chain reactions (RT-PCRs) were conducted on selected chemokine mRNAs to confirm the gene array findings. RESULTS: After intense light exposure, TUNEL-positive cells were noted in the outer nuclear layer (ONL) of the retina at 3 h, and their presence were noticeably increased at 1 day but declined at 3 days and 7 days after light exposure. In contrast, CD11b- or isolectin-B4-positive cells were seen in the ONL as early as 6 h and their presence increased significantly at 1 day and 3 days after light exposure. These cells displayed a round or ovoid morphology at 6 h and 1 day but assumed a more ameboid configuration at 3 days. By 7 day, the number of the microglial cells declined in the ONL and they became ramified, and were present mostly in the subretinal space. 5D4-positive cells with large cell bodies were only noted at 3 day and 7 day but not earlier. With chemokine-specific gene array analysis, we identified four chemokines and two chemokine receptors showing significant increases in their gene expressions. Among them, monocyte chemoattractant protein-3 (MCP-3), showed a remarkable 4.4 fold increase in its gene expression. RT-PCR confirmed a marked increase of MCP-3 expression in retinas at 3 h to 1 day, and a return to normal at 3 days following light injury. CONCLUSIONS: Retinal chemokines such as MCP-3 and their receptors are involved in the activation and migration of retinal microglia in light-induced retinal degeneration, which in turn modulate the apoptotic loss of photoreceptor cells in the outer retina.

Minocycline inhibits LPS-induced retinal microglia activation.

Retinal neurodegenerative disease 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 retinal injury following an inflammatory stimulus. Cytokines released by activated microglia regulate the influx of inflammatory cells to the damaged area. Thus, a therapeutic strategy to reduce cytokine expression in microglia would be neuroprotective. Minocycline, a semisynthetic tetracycline derivative, is known to protect rodent brain from ischemia and to inhibit microglial activation. In this study, we activated retinal microglia in culture with lipopolysaccharide (LPS) and attempted to determine whether minocycline could reduce the production of cytokines from activated microglia at both gene and protein levels. Changes in inflammatory cytokines, TNF-alpha and IL-1beta, were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) in the presence or absence of LPS. We also measured the levels of nitric oxide (NO) by the nitrate reductase method under similar conditions. LPS treatment induced a significant upregulation of the mRNA and release of TNF-alpha, IL-1beta, and NO from retinal microglia. Minocycline inhibited these releases. Thus, minocycline might exert its antiinflammatory effect on microglia by inhibiting the expression and release of TNF-alpha, IL-1beta, and NO.

Frosted branch angiitis secondary to macular choroidal neovascularization in a chinese woman.

BACKGROUND: This is perhaps the first report of frosted branch angiitis secondary to choroidal neovascularization (CNV) from China. We describe a case of frosted branch angiitis associated with macular CNV. CASE: A 19-year-old Chinese woman complained of metamorphopsia and blurred vision in the right eye on August 27, 2002. OBSERVATIONS: Fluorescein angiography demonstrated CNV in the macula. Five months later, focal and atrophic choroidal scars were observed in the macular area. She complained of blurred vision again on April 14, 2003. Iridocyclitis and frosted branch angiitis were found in both fundi. Following treatment with systemic acycloguanosine and corticosteroids for 4 months, the fundi recovered almost completely. CONCLUSIONS: A case of frosted branch angiitis secondary to macular choroidal neovascularization is reported in a Chinese woman. The relation between frosted branch angiitis and CNV remains to be further explored.

Early glial responses after acute elevated intraocular pressure in rats.

PURPOSE: To study the responses of glial cells to a short-term elevation in intraocular pressure (IOP) in rats. METHODS: Adult Sprague-Dawley albino rats, 45 to 55 days old, were given India ink intracamerally. After 7 days, 200 spots of laser burn over 360 degrees were delivered by an Argon laser (620-637 nm; 200 mW; 200 mm; 0.2 seconds) aimed at the ink deposits in the trabecular meshwork. IOP was recorded and eye tissues at 12 hours and 1, 3, 5, 7, or 14 days after laser were examined by immunohistochemistry with antibodies against glial fibrillary acidic protein (GFAP), vimentin, S-100, ED1, and OX42. To evaluate neuronal loss, the number of cells in the retinal ganglion cell layer (RGCL) was counted on flat preparations of retinas at various times after elevation of IOP. RESULTS: Significant elevation of IOP from 1 to 7 days and loss of cells in the RGCL from 3 days onward were noted after trabecular laser photocoagulation. In the inner retina, there was a gradual and sustained increase in GFAP and S-100 immunoreactivity, but only a transient increase in vimentin immunoreactivity. No remarkable changes in GFAP, vimentin, and S-100 immunoreactivity were noted at the optic nerve head (ONH). ED1- and OX42-labeled cells were noted in the choroidal plexus, the parapapillary region of the optic nerve, and the ONH from 3 days onward, whereas expression in the retina was unremarkable. CONCLUSIONS: There is differential expression of glial cell markers in the retina and the ONH, with early loss of cells in the RGCL in response to the elevation of IOP. Macroglia such as astrocytes and Müller cells may be involved in the pathophysiology of retinal ganglion cell death or retinal repair, and activated microglial/phagocytic cells may play an important role in modulating the changes in the ONH that occur with the elevation of IOP.