Mouse Ocular Tissues Research Articles

Development of a Temperature and pH Dual-Sensitive In-Situ Gel for Treating Allergic Conjunctivitis.

The purpose of this study was to improve the efficacy of olopatadine hydrochloride (OT) in treating allergic conjunctivitis (AC). To achieve this goal, we developed an eye formulation without antimicrobial agents using a temperature-pH dual-sensitive in situ gel technology combined with heat sterilization. Various types of carbomers were evaluated and their optimal doses determined. The prescription containing poloxamer 407 (P407) and poloxamer 188 (P188) was optimized using central composite design for response surface methodology (CCD-RSM). The final optimized dual-sensitive in situ gel (TP-gel) consisted of 0.1% olopatadine hydrochloride, 18.80% P407, 0.40% P188, 0.30% Pemulen™TR-1(TR-1), 4.0% mannitol, and 0.08% Tri(hydroxymethyl)aminomethane(Tris).Sterilization was performed at a temperature of 121℃ for a duration of 20 min. Experimental results showed that TP-gel had good safety profile and remained on the ocular surface for approximately (65.83 ± 8.79) minutes, which is four times longer than eye drops. The expression levels of IL-13, IL-17, and OVA-IgE in mouse ocular tissues with allergic conjunctivitis treated with TP-gel were significantly reduced. This suggests that TP-gel has the potential to be an effective treatment method for allergic conjunctivitis.

Differential expression of PIEZO1 and PIEZO2 mechanosensitive channels in ocular tissues implicates diverse functional roles

PIEZO1 and PIEZO2 are mechanosensitive ion channels that regulate many important physiological processes including vascular blood flow, touch, and proprioception. As the eye is subject to mechanical stress and is highly perfused, these channels may play important roles in ocular function and intraocular pressure regulation. PIEZO channel expression in the eye has not been well defined, in part due to difficulties in validating available antibodies against PIEZO1 and PIEZO2 in ocular tissues. It is also unclear if PIEZO1 and PIEZO2 are differentially expressed. To address these questions, we used single-molecule fluorescence in situ hybridization (smFISH) together with transgenic reporter mice expressing PIEZO fusion proteins under the control of their endogenous promoters to compare the expression and localization of PIEZO1 and PIEZO2 in mouse ocular tissues relevant to glaucoma. We detected both PIEZO1 and PIEZO2 expression in the trabecular meshwork, ciliary body, and in the ganglion cell layer (GCL) of the retina. Piezo1 mRNA was more abundantly expressed than Piezo2 mRNA in these ocular tissues. Piezo1 but not Piezo2 mRNA was detected in the inner nuclear layer and outer nuclear layer of the retina. Our results suggest that PIEZO1 and PIEZO2 are differentially expressed and may have distinct roles as mechanosensors in glaucoma-relevant ocular tissues.

Pathogenesis and Virulence of Chlamydia Trachomatis

The pathogenesis of C. trachomatis disease is a multi-step process that includes: (1) infectivity and exposure to the organism (2) Susceptibility to infection and sickness related to the host's genetic makeup. Recurrence and chronic infections are also common in at-risk teenage and young adult groups. Antibiotic resistance to the primary medications used to treat C. trachomatis is becoming increasingly widespread, even with the correct diagnosis. Chlamydial infection can prevent tumor necrosis factor (TNF)-an-induced physiological apoptosis. Failure to adequately prevent, identify, treat, and remove infection increases the risk of pathogenicity and illness. The plasmid glycoproteins 1–8 (pGP1–8) encode eight open reading frames and most Chlamydia species. In the United Kingdom, there has been a recent increase in the prevalence of such illnesses, whereas, in the Scandinavian nations, there has been a drop, albeit there has been a minor increase in recent years (owing to the development of nucleic acid testing technologies, to some extent). However, it should be noted that reliable monitoring systems and population-based data are Chlamydia trachomatis 4 gitis. Chlamydia trachomatis is made plasma or accessible; moreover, it’s weakened in the vaginal canal of the mouse and nonhuman primate ocular tissue. The plasmid-free organisms ‘in vivo but not in vitro traits were completely mimeographs when pGP3 was inadequate, demonstrating that plasmid-encoded pGP3 is a critical virulence factor in vivo. Moreover, leading to a shortage of cost-effective moment in time tests, including methodologies consistent with strain typing during therapeutic, and the overall degree underlying therapeutic failure is foreign. Those disadvantages were exacerbated because the rest of the genders’ infestations were undiagnosed, allowing continuous silence propagation and developmental defects. The popular medications C. tachometers are becoming more prevalent with proper identification.

Establishment of an Acanthamoeba keratitis mouse model confirmed by amoebic DNA amplification

Acanthamoeba castellanii, the causative agent of Acanthamoeba keratitis (AK), occurs mainly in contact lens users with poor eye hygiene. The findings of many in vitro studies of AK, as well as the testing of therapeutic drugs, need validation in in vivo experiments. BALB/c mice were used in this study to establish in vivo AK model. A. castellanii cell suspensions (equal mixtures of trophozoites and cysts) were loaded onto 2-mm contact lens pieces and inserted into mouse eyes that were scratched using an ophthalmic surgical blade under anesthesia and the eyelids of the mice were sutured. The AK signs were grossly observed and PCR was performed using P-FLA primers to amplify the Acanthamoeba 18S-rRNA gene from mouse ocular tissue. The experimental AK mouse model was characterized by typical hazy blurring and melting of the mouse cornea established on day 1 post-inoculation. AK was induced with at least 0.3 × 105A. castellanii cells (optimal number, 5 × 104), and the infection persisted for two months. The PCR products amplified from the extracted mouse eye DNA confirmed the development of Acanthamoeba-induced keratitis during the infection periods. In conclusion, the present AK mouse model may serve as an important in vivo model for the development of various therapeutic drugs against AK.

The role of aquaporin-4 in optic nerve head astrocytes in experimental glaucoma.

To study aquaporin channel expression in astrocytes of the mouse optic nerve (ON) and the response to IOP elevation in mice lacking aquaporin 4 (AQP4 null). C57BL/6 (B6) and AQP4 null mice were exposed to bead-induced IOP elevation for 3 days (3D-IOP), 1 and 6 weeks. Mouse ocular tissue sections were immunolabeled against aquaporins 1(AQP1), 4(AQP4), and 9(AQP9). Ocular tissue was imaged to identify normal AQP distribution, ON changes, and axon loss after IOP elevation. Ultrastructure examination, cell proliferation, gene expression, and transport block were also analyzed. B6 mice had abundant AQP4 expression in Müller cells, astrocytes of retina and myelinated ON (MON), but minimal AQP4in prelaminar and unmyelinated ON (UON). MON of AQP4 nulls had smaller ON area, smaller axon diameter, higher axon density, and larger proportionate axon area than B6 (all p≤0.05). Bead-injection led to comparable 3D-IOP elevation (p = 0.42) and axonal transport blockade in both strains. In B6, AQP4 distribution was unchanged after 3D-IOP. At baseline, AQP1 and AQP9 were present in retina, but not in UON and this was unaffected after IOP elevation in both strains. In 3D-IOP mice, ON astrocytes and microglia proliferated, more in B6 than AQP4 null. After 6 week IOP elevation, axon loss occurred equally in the two mouse types (24.6%, AQP4 null vs. 23.3%, B6). Lack of AQP4 was neither protective nor detrimental to the effects of IOP elevation. The minimal presence of AQP4 in UON may be a vital aspect of the regionally specific phenotype of astrocytes in the mouse optic nerve head.

Expression of SARS-CoV-2 receptor ACE2 and TMPRSS2 in human primary conjunctival and pterygium cell lines and in mouse cornea.

PurposeTo determine the expressions of SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) and type II transmembrane serine protease (TMPRSS2) genes in human and mouse ocular cells and comparison to other tissue cells.MethodsHuman conjunctiva and primary pterygium tissues were collected from pterygium patients who underwent surgery. The expression of ACE2 and TMPRSS2 genes was determined in human primary conjunctival and pterygium cells, human ocular and other tissue cell lines, mesenchymal stem cells as well as mouse ocular and other tissues by reverse transcription-polymerase chain reaction (RT-PCR) and SYBR green PCR.ResultsRT-PCR analysis showed consistent expression by 2 ACE2 gene primers in 2 out of 3 human conjunctival cells and pterygium cell lines. Expression by 2 TMPRSS2 gene primers could only be found in 1 out of 3 pterygium cell lines, but not in any conjunctival cells. Compared with the lung A549 cells, similar expression was noted in conjunctival and pterygium cells. In addition, mouse cornea had comparable expression of Tmprss2 gene and lower but prominent Ace2 gene expression compared with the lung tissue.ConclusionConsidering the necessity of both ACE2 and TMPRSS2 for SARS-CoV-2 infection, our results suggest that conjunctiva would be less likely to be infected by SARS-CoV-2, whereas pterygium possesses some possibility of SARS-CoV-2 infection. With high and consistent expression of Ace2 and Tmprss2 in cornea, cornea rather than conjunctiva has higher potential to be infected by SARS-CoV-2. Precaution is necessary to prevent possible SARS-CoV-2 infection through ocular surface in clinical practice.

Ectopically Expressed Human Olfactory Receptor OR51E1: a Search for Novel Molecular Probes

Olfactory receptors comprise the largest family of G protein‐couples receptors (GPCRs) ‐ ~1400 functional genes in the mouse (Olfrs) and ~400 in the human (ORs). Most genes were assigned to it based solely on the sequence similarity. Recent studies demonstrated that many Olfrs are expressed outside of the nasal cavity and possibly involved in physiological processes such as muscle regeneration and controlling blood pressure. These ectopically expressed olfactory receptors might recognize cues other than odorants and be involved in processes independent of the sense of smell. Ligands were identified for fewer than 100 of ORs and Olfrs.Using transcriptomics analysis followed by RT‐PCR and in situ RNA hybridization we previously showed expression of several Olfrs and other olfactory signaling genes expressed in the mouse ocular tissues. We focused our attention on Olfr558 and demonstrated its expression in blood vessels of the eye. The human ortholog of Olfr558 (OR51E1 or PSGR2) was shown to be expressed in the prostate and dramatically up‐regulated in prostate cancer and is an established biomarker of malignancy. The human and mouse genes encoding this receptor are almost identical (94%), which is unique for GPCRs in general, and particularly for sensory chemoreceptors. This unusual evolutionary conservation points to the importance of its physiological function. While the function of this receptor or its native ligand is unknown, in vitro it responds to some short‐ and medium‐chain fatty acids (e.g., butyrate and valerate) by activating adenylyl cyclase. We found that growth of human prostate cancer cells that express OR51E1 is inhibited by valerate. The knockdown of OR51E1 gene with shRNA eliminated this effect of valerate.While butyrate and valerate can be used in such experiments, their low affinity and selectivity complicates interpretation of the data. Additionally, they are quickly metabolized in the body. Thus, there is a clear need for novel probes that can be used to dissect OR51E1 function. To address this need, we established a stable cell line overexpressing OR51E1 and demonstrated that in response to known OR51E1 agonists this cell line increases intracellular cAMP level. We then used this cell line for screening of a diverse chemical library of 9600 compounds, showing its suitability for high throughput screening. To demonstrate that our expression system can be used for expression of other ORs, we generated stable cell lines overexpressing two other receptors – OR51E2 and OR2AT4. We showed that both receptors are expressed as proteins of the expected molecular weight and localize on the cell surface. Since these ORs represents a large class of previously unexplored yet druggable targets, this project facilitates investigation of the entire family of ectopic olfactory receptors.

The master transcription factor SOX2, mutated in anophthalmia/microphthalmia, is post-transcriptionally regulated by the conserved RNA-binding protein RBM24 in vertebrate eye development.

Mutations in the key transcription factor, SOX2, alone account for 20% of anophthalmia (no eye) and microphthalmia (small eye) birth defects in humans-yet its regulation is not well understood, especially on the post-transcription level. We report the unprecedented finding that the conserved RNA-binding motif protein, RBM24, positively controls Sox2 mRNA stability and is necessary for optimal SOX2 mRNA and protein levels in development, perturbation of which causes ocular defects, including microphthalmia and anophthalmia. RNA immunoprecipitation assay indicates that RBM24 protein interacts with Sox2 mRNA in mouse embryonic eye tissue. and electrophoretic mobility shift assay shows that RBM24 directly binds to the Sox2 mRNA 3'UTR, which is dependent on AU-rich elements (ARE) present in the Sox2 mRNA 3'UTR. Further, we demonstrate that Sox2 3'UTR AREs are necessary for RBM24-based elevation of Sox2 mRNA half-life. We find that this novel RBM24-Sox2 regulatory module is essential for early eye development in vertebrates. We show that Rbm24-targeted deletion using a constitutive CMV-driven Cre in mouse, and rbm24a-CRISPR/Cas9-targeted mutation or morpholino knockdown in zebrafish, results in Sox2 downregulation and causes the developmental defects anophthalmia or microphthalmia, similar to human SOX2-deficiency defects. We further show that Rbm24 deficiency leads to apoptotic defects in mouse ocular tissue and downregulation of eye development markers Lhx2, Pax6, Jag1, E-cadherin and gamma-crystallins. These data highlight the exquisite specificity that conserved RNA-binding proteins like RBM24 mediate in the post-transcriptional control of key transcription factors, namely, SOX2, associated with organogenesis and human developmental defects.

Characterization of mouse ocular response to a 35-day spaceflight mission: Evidence of blood-retinal barrier disruption and ocular adaptations

The health risks associated with spaceflight-induced ocular structural and functional damage has become a recent concern for NASA. The goal of the present study was to characterize the effects of spaceflight and reentry to 1 g on the structure and integrity of the retina and blood-retinal barrier (BRB) in the eye. To investigate possible mechanisms, changes in protein expression profiles were examined in mouse ocular tissue after spaceflight. Ten week old male C57BL/6 mice were launched to the International Space Station (ISS) on Space-X 12 at the Kennedy Space Center (KSC) on August, 2017. After a 35-day mission, mice were returned to Earth alive. Within 38 +/− 4 hours of splashdown, mice were euthanized and ocular tissues were collected for analysis. Ground control (GC) and vivarium control mice were maintained on Earth in flight hardware or normal vivarium cages respectively. Repeated intraocular pressure (IOP) measurements were performed before the flight launch and re-measured before the mice were euthanized after splashdown. IOP was significantly lower in post-flight measurements compared to that of pre-flight (14.4–19.3 mmHg vs 16.3–20.3 mmHg) (p < 0.05) for the left eye. Flight group had significant apoptosis in the retina and retinal vascular endothelial cells compared to control groups (p < 0.05). Immunohistochemical analysis of the retina revealed that an increased expression of aquaporin-4 (AQP-4) in the flight mice compared to controls gave strong indication of disturbance of BRB integrity. There were also a significant increase in the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and a decrease in the expression of the BRB-related tight junction protein, Zonula occludens-1 (ZO-1). Proteomic analysis showed that many key proteins and pathways responsible for cell death, cell cycle, immune response, mitochondrial function and metabolic stress were significantly altered in the flight mice compared to ground control animals. These data indicate a complex cellular response that may alter retina structure and BRB integrity following long-term spaceflight.

Impact of Spaceflight and Artificial Gravity on the Mouse Retina: Biochemical and Proteomic Analysis.

Astronauts are reported to have experienced some impairment in visual acuity during their mission on the International Space Station (ISS) and after they returned to Earth. There is emerging evidence that changes in vision may involve alterations in ocular structure and function. To investigate possible mechanisms, changes in protein expression profiles and oxidative stress-associated apoptosis were examined in mouse ocular tissue after spaceflight. Nine-week-old male C57BL/6 mice (n = 12) were launched from the Kennedy Space Center on a SpaceX rocket to the ISS for a 35-day mission. The animals were housed in the mouse Habitat Cage Unit (HCU) in the Japan Aerospace Exploration Agency (JAXA) “Kibo” facility on the ISS. The flight mice lived either under an ambient microgravity condition (µg) or in a centrifugal habitat unit that produced 1 g artificial gravity (µg + 1 g). Habitat control (HC) and vivarium control mice lived on Earth in HCUs or normal vivarium cages, respectively. Quantitative assessment of ocular tissue demonstrated that the µg group induced significant apoptosis in the retina vascular endothelial cells compared to all other groups (p < 0.05) that was 64% greater than that in the HC group. Proteomic analysis showed that many key pathways responsible for cell death, cell repair, inflammation, and metabolic stress were significantly altered in µg mice compared to HC animals. Additionally, there were more significant changes in regulated protein expression in the µg group relative to that in the µg + 1 g group. These data provide evidence that spaceflight induces retinal apoptosis of vascular endothelial cells and changes in retinal protein expression related to cellular structure, immune response and metabolic function, and that artificial gravity (AG) provides some protection against these changes. These retinal cellular responses may affect blood–retinal barrier (BRB) integrity, visual acuity, and impact the potential risk of developing late retinal degeneration.

Serotype survey of AAV gene delivery via subconjunctival injection in mice.

AAV gene therapy approaches in the posterior eye resulted in the first FDA-approved gene therapy-based drug. However, application of AAV vectorology to the anterior eye has yet to enter even a Phase I trial. Furthermore, the simple and safe subconjunctival injection has been relatively unexplored in regard to AAV vector transduction. To determine the utility of this route for the treatment of various ocular disorders, a survey of gene delivery via natural AAV serotypes was performed and correlated to reported cellular attachment factors. AAV serotypes packaged with a self-complementary reporter were administered via subconjunctival injection to WT mice. Subconjunctival injection of AAV vectors was without incidence; however, vector shedding in tears was noted weeks following administration. AAV transduction was serotype dependent in anterior segment tissues including the eye lid, conjunctiva, and cornea, as well as the periocular tissues including muscle. Transgene product in the cornea was highest for AAV6 and AAV8, however, their corneal restriction was remarkably different; AAV6 appeared restricted to the endothelium layer while AAV8 efficiently transduced the stromal layer. Reported AAV cellular receptors were not well correlated to vector transduction; although, in some cases they were conserved among mouse and human ocular tissues. Subconjunctival administration of particular AAV serotypes may be a simple and safe targeted gene delivery route for ocular surface, muscular, corneal, and optic nerve diseases.

Fingolimod Suppresses a Cascade of Core Vicious Cycle in Dry Eye NOD Mouse Model: Involvement of Sphingosine-1-Phosphate Receptors in Infiltrating Leukocytes.

The purpose of this study was to evaluate the inhibitory mechanism of fingolimod and the involvement of sphingosine-1-phosphate receptors (S1PRs) and cytokines-matrix metalloproteinases (MMPs)/MAP kinases (MAPKs) signaling in a dry eye disease (DED) mouse model. Sixty-four male NOD mice (DED model) and 16 age-matched BALB/c mice were used. In a preliminary experiment, 16 NOD mice were randomly divided into a positive control group and fingolimod-treated groups, with 8 BALB/c mice serving as wild-type control. In a subsequent, separate study, 48 NOD mice were randomly divided into 6 groups: fingolimod-treated groups at three different concentrations (0.05%, 0.005%, and 0.001%), normal saline group, untreated group, and fingolimod+W146 group. Animals received normal saline or fingolimod eyedrops three times daily until euthanasia 2 months later. Mice in the fingolimod+W146 group received daily intraperitoneal injections of W146 (0.1 mg/kg/day). Proinflammatory mediators were assessed by a protein array. Activities of MMP-2 and MMP-9 were evaluated by zymography. MAPKs and S1PRs were examined by Western blots and immunohistochemistry. Infiltrating cells and inhibitory mechanisms were assessed. In the positive control group, levels of inflammatory mediators and S1PRs were upregulated. By comparison, fingolimod treatment significantly suppressed such markers which were significantly reversed by W146 (P < 0.01). Importantly, by double immunofluorescence staining, leukocytes were confirmed involved in DED in the NOD mouse model. Leukocytes are involved in DED in the NOD mouse model. The therapeutic mechanisms of fingolimod may be associated with inhibitory roles of "cytokines-MMPs/MAPKs" cycle in NOD mouse ocular surface tissues by mediating S1PRs in infiltrating leukocytes.

Modified Hiraoka TEM grid staining apparatus and technique using 3D printed materials and gadolinium triacetate tetrahydrate, a nonradioactive uranyl acetate substitute

The Hiraoka transmission electron microscopy (TEM) grid staining apparatus, which allowed multiple grids to be batch stained together on a plate, is no longer commercially available. Because the need for such a device still exists, we developed a modified Hiraoka TEM grid staining apparatus by combining a Leica AC20 grid plate holder with a modified 3D printed grid loading holder and trays lined with ParafilmR. This apparatus was then used to test a post-section contrasting method that uses gadolinium triacetate tetrahydrate solution, a nonradioactive uranyl acetate substitute, which produces similar staining results to uranyl acetate when used with lead stain in mouse ocular tissue for TEM.

Lactoferrin Expression in Human and Murine Ocular Tissue

ABSTRACT Purpose: Lactoferrin (LF) is a multifunctional protein known to provide innate defense due to its antimicrobial and anti-inflammatory properties. In the eye, LF has been identified in the tears and vitreous humor. Its presence in other ocular tissues has not been determined. Our aim is to assess the presence of LF in the cornea, iris, retina and retinal pigment epithelium (RPE) of humans and mice. Methods: To test for the endogenous production of LF, reverse transcription polymerase chain reaction was performed in cultured human cells from the cornea and RPE and in murine tissues. To confirm LF localization in specific ocular tissue, immunohistochemistry was performed on flat mounts of cornea, retina and RPE in human donor eyes. The presence of LF was assessed by western blotting in human and mouse ocular tissue and human culture cells (cornea and RPE). To verify antibody specificity, purified human LF and transferrin (TF) were used on 1D and 2D western blots. Results: LF gene expression was confirmed in the cornea and RPE cell cultures from humans, suggesting that LF is an endogenously produced protein. PCR results from mouse ocular tissue showed LF expression in cornea, iris, RPE, but not in retina. These results were also consistent with immunohistochemical localization of LF in human donor tissue. Antibody reaction for human LF was specific and western blotting showed its presence in the cornea, iris and RPE tissues. A faint reaction for the retina was observed but was likely due to contamination from other ocular tissues. Multiple commercially available antibodies for murine LF cross-reacted with TF, so no reliable results were obtained for murine western blot. Conclusion: LF is expressed in multiple eye tissues of humans and mice. This widespread expression and multifunctional activity of LF suggests that it may play an important role in protecting eye tissues from inflammation-associated diseases.

Characterization of Human, Mouse, and Frog Rhodopsin Microdomains Within Native Disc Membranes

Rhodopsin is the light-activated receptor located in the disc membranes of rod photoreceptor cells in the retina and initiates vision via the phototransduction signaling cascade. There are divergent views on rhodopsin's quaternary organization within native disc membranes. The classical view posits that rhodopsin molecules function as freely diffusing monomers. However, recent evidence suggests that rhodopsin oligomerizes and forms higher order structures within the membrane. An accurate description of signaling events in phototransduction and of associated disease mechanisms is reliant on a comprehensive understanding of how rhodopsin is organized within native disc membranes. The aim of the current study was to determine and quantify the physiological arrangement of several vertebrate rhodopsins within their native disc membranes using atomic force microscopy (AFM). AFM is a microscopic method that allows for the imaging of membrane proteins in their native environment under physiological conditions. Disc membranes, with 90% of the total protein content comprised of rhodopsin, were isolated from human, mouse, and frog ocular tissue. AFM images of single-bilayer disc membranes revealed that these vertebrate disc membranes have similar topographies. Topographic features in these images indicate that rhodopsin is organized into microdomains and that the formation of these microdomains is not an effect of a low temperature environment. The microdomains formed by rhodopsin from each species were quantified and comparatively analyzed. By characterizing these microdomains, a baseline has been established for the organization of rhodopsin in human, mouse, and frog disc membranes. These characterizations of microdomain rhodopsin organization may serve as a guide in future investigations which will need to address the importance and role of such an organization on phototransduction and diseased states.

Spaceflight Environment Induces Mitochondrial Oxidative Damage in Ocular Tissue

A recent report shows that more than 30% of the astronauts returning from Space Shuttle missions or the International Space Station (ISS) were diagnosed with eye problems that can cause reduced visual acuity. We investigate here whether spaceflight environment-associated retinal damage might be related to oxidative stress-induced mitochondrial apoptosis. Female C57BL/6 mice were flown in the space shuttle Atlantis (STS-135), and within 3-5 h of landing, the spaceflight and ground-control mice, similarly housed in animal enclosure modules (AEMs) were euthanized and their eyes were removed for analysis. Changes in expression of genes involved in oxidative stress, mitochondrial and endothelial cell biology were examined. Apoptosis in the retina was analyzed by caspase-3 immunocytochemical analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Levels of 4-hydroxynonenal (4-HNE) protein, an oxidative specific marker for lipid peroxidation were also measured. Evaluation of spaceflight mice and AEM ground-control mice showed that expression of several genes playing central roles in regulating the mitochondria-associated apoptotic pathway were significantly altered in mouse ocular tissue after spaceflight compared to AEM ground-control mice. In addition, the mRNA levels of several genes, which are responsible for regulating the production of reactive oxygen species were also significantly up-regulated in spaceflight samples compared to AEM ground-control mice. Further more, the level of HNE protein was significantly elevated in the retina after spaceflight compared to controls. Our results also revealed that spaceflight conditions induced significant apoptosis in the retina especially inner nuclear layer (INL) and ganglion cell layer (GCL) compared to AEM ground controls. The data provided the first evidence that spaceflight conditions induce oxidative damage that results in mitochondrial apoptosis in the retina. This data suggest that astronauts may be at increased risk for late retinal degeneration.

A Mutation in SLC24A1 Implicated in Autosomal-Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder that can be associated with impaired night vision. The last decade has witnessed huge progress in ophthalmic genetics, including the identification of three genes implicated in the pathogenicity of autosomal-recessive CSNB. However, not all patients studied could be associated with mutations in these genes and thus other genes certainly underlie this disorder. Here, we report a large multigeneration family with five affected individuals manifesting symptoms of night blindness. A genome-wide scan localized the disease interval to chromosome 15q, and recombination events in affected individuals refined the critical interval to a 10.41 cM (6.53 Mb) region that harbors SLC24A1, a member of the solute carrier protein superfamily. Sequencing of all the coding exons identified a 2 bp deletion in exon 2: c.1613_1614del, which is predicted to result in a frame shift that leads to premature termination of SLC24A1 (p.F538CfsX23) and segregates with the disorder under an autosomal-recessive model. Expression analysis using mouse ocular tissues shows that Slc24a1 is expressed in the retina around postnatal day 7. In situ and immunohistological studies localized both SLC24A1 and Slc24a1 to the inner segment, outer and inner nuclear layers, and ganglion cells of the retina, respectively. Our data expand the genetic basis of CSNB and highlight the indispensible function of SLC24A1 in retinal function and/or maintenance in humans.

Complement factor H and factor B expression in RPE cells

Abstract Purpose Age‐related macular degeneration (AMD) is the leading cause of untreatable blindness in the developed world. The pathogenesis of AMD is not fully understood. Recent evidence suggests that local inflammation in particular complement activation plays an important role. We aim to understand how complement activation is regulated at retina/choroidal interface. Methods The expression and distribution of complement factor H (CFH) and factor B (CFB) in mouse ocular tissues were examined by immunohistochemistry. Regulation of CFH and CFB gene expression by various cytokines or photoreceptor outer segments (POS) was investigated in vitro in cultured RPE cells. Changes in CFH or CFB gene expression after treatment were evaluated by RT‐PCR. Results In normal mouse eyes, CFH was detected in corneal epithelial cells, ciliary body, RPE cells, Bruch’s membrane and choroidal vessels. There is no significant change in either the expression level or the distribution pattern of CFH in ocular tissues of different ages of mice. CFB was exclusively detected in RPE cells in normal mice. The expression of CFB in RPE cells increases with age. In vitro in RPE cultures, the expression of CFH was negatively regulated by cytokine TNF‐alpha and IL‐6, whereas the expression of CFB was positively regulated by TNF‐alpha and IFN‐gamma. Short‐term incubation of RPE cells with POS did not alter the expression of CFH or CFB, whereas long‐term incubation of RPE cells with POS significantly down‐regulated CFH expression but up‐regulated CFB expression. Conclusion Complement regulatory factors CFH and CFB are produced locally in the retina/choroidal interface by RPE cells. The production of CFH and CFB in RPE cells is regulated differently by various cytokines and oxidized POS.

Mapping ribosomal RNA transcription activity in the mouse eye

All cells must make ribosomes, in which rRNA transcription is the rate-limiting step; however, some cells may require more ribosomes than others. Cell-type specific regulation of rRNA synthesis has been largely ignored in the past, because of the inability to measure rRNA transcription rate in situ. Here we map rRNA transcription activity in individual cells in mouse ocular tissues detected by a novel in situ hybridization technique, which detects the full-length transcripts (47S pre-rRNA) as well as various rRNA processing intermediates. In the adult mouse eye, the corneal and lens epithelia and some retinal neurons contain a higher level of 47S pre-rRNA and rRNA processing intermediates, which are regulated developmentally in neonates prior to eye opening. In the cornea and lens epithelia, the higher rRNA level of 47S rRNA correlates with cell proliferation, which is consistent with the notion that dividing cells require more protein synthesis. Interestingly, in some retinal neurons, the high level of 47S pre-rRNA does not correlate with mature rRNA accumulation or protein synthesis, suggesting the existence of unappreciated biochemical needs of these cells.

668. Non-Viral Gene Delivery for Ocular Diseases with Compacted DNA Nanoparticles

Top of pageAbstract Our objective is to develop an effective and robust therapeutic vector system which can be utilized in the treatment of genetically-based blinding diseases. In the present study we have evaluated the ability of compacted DNA nanoparticles to transfect mouse ocular tissues in vivo. EGFP expression plasmids transcriptionally-controlled by the CMV promoter were compacted into neutral-charged DNA nanoparticles (having a diameter < 15 nm) using polyethylene glycol-substituted lysine peptides and injected subretinally or intravitreally into the eyes of adult BALB/c mice. Both delivery routes were examined in order to target photoreceptors, RPE, and optic nerve cells (subretinal) or inner retinal cells (intravitreal). In a set of control animals, naked plasmid DNA was injected at the same concentration as the nanoparticles or a mock injection was performed. After 2 days post-injection, mice were euthanized and the retinas were assayed for EGFP expression by quantitative real-time RT-PCR (qRT-PCR) and immunohistochemistry. Significant levels of EGFP expression were obtained by either subretinal or intravitreal delivery of the compacted DNA nanoparticles. Immunohistochemistry demonstrated the ability of these nanoparticles to transfect and express EGFP in vivo at extraordinarily high efficiencies. After subretinal injection, EGFP was detected in almost 100% of the photoreceptors and abundant expression was observed in the inner nuclear layer, RPE, and optic nerve. By qRT-PCR, EGFP mRNA levels were comparable in abundance to rhodopsin mRNA. Mice injected intravitreally showed very efficient EGFP expression in the cells of the inner retina, including the ganglion cell layer. After either subretinal or intravitreal delivery, minimal or no expression of EGFP was detected with naked plasmid or mock-injected controls. Ocular delivery of the DNA nanoparticles did not induce any apparent toxicity. Compacted DNA nanoparticles can efficiently target post-mitotic cells of the retina and yield high levels of transgene expression. Transfection of different retinal cell layers can be achieved by the route of delivery. This non-viral system is a safe and very effective tool for ocular gene therapy. Further studies are underway utilizing this technology to rescue several well characterized animal models of retinal disease.