The healthy cornea is remarkably resistant to infection, quickly clearing deliberately inoculated bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. Contrasting with the adjacent conjunctiva and other body surfaces, it also lacks a resident viable bacterial microbiome. Corneal resistance to microbes depends on intrinsic defenses involving tear fluid and the corneal epithelium. Dry eye, an ocular surface disease associated with discomfort and inflammation, can alter tear fluid composition and volume, and impact epithelial integrity. We previously showed that experimentally-induced dry eye (EDE) in mice does not increase corneal susceptibility to P. aeruginosa infection. Here, we explored if EDE alters corneal resistance to bacterial colonization. EDE was established in mice using scopolamine injections and dehumidified air-flow, and verified by phenol-red thread testing after 5 and 10 days. As expected, EDE corneas showed increased fluorescein staining versus controls consistent with compromised epithelial barrier function. Confocal imaging using mT/mG knock-in mice with red-fluorescent membranes revealed no other obvious morphological differences between EDE corneas and controls for epithelium, stroma, and endothelium. EDE corneas were imaged ex vivo and compared to controls after alkyne-functionalized D-alanine labeling of metabolically-active colonizing bacteria, or by FISH using a universal 16S rRNA gene probe. Both methods revealed very few viable bacteria on EDE corneas after 5 or 10 days (median of 0, upper quartile of ≤ 1 bacteria per field of view for each group [9-12 eyes per group]) similar to control corneas. Furthermore, there was no obvious difference in abundance of conjunctival bacteria, which included previously reported filamentous forms. Thus, despite reduced tear flow and apparent compromise to corneal barrier function (fluorescein staining), EDE murine corneas continue to resist bacterial colonization and maintain the absence of a resident viable bacterial microbiome.

Purpose

Female mice have been found to be resistant to streptozotocin (STZ)-induced diabetes, and pre-clinical research related to diabetic complications commonly omits females. The purpose of this study was to develop a method to induce diabetes in female mice, and to determine if retinas of diabetic female mice develop molecular changes and histopathological abnormalities comparable to those which develop in male diabetic mice.

Methods

To induce diabetes, animals of both sexes received daily intraperitoneal (i.p.) injection of STZ for 5 consecutive days at 55 mg/kg BW (a dose that is known to induce diabetes in male mice) or for females, 75 mg/kg BW of STZ. Retinal abnormalities that have been implicated in the development of the retinopathy (superoxide generation and expression of inflammatory proteins, iNOS and ICAM-1) were evaluated at 2 months of diabetes, and retinal capillary degeneration was evaluated at 8 months of diabetes.

Results

Daily i.p. injection of STZ for 5 consecutive days at a concentration of 55 mg/kg BW was sufficient to induce diabetes in 100% of male mice, but only 33% of female mice. However, females did become hyperglycemic when the dose of STZ administered was increased to 75 mg/kg BW. The resulting STZ-induced hyperglycemia in female and male mice was sustained for at least 8 months. After induction of the diabetes, both sexes responded similarly with respect to the oxidative stress, expression of iNOS, and degeneration of retinal capillaries, but differed in the limited population evaluated with respect to expression of ICAM-1.

Conclusions

The resistance of female mice to STZ-induced diabetes can be overcome by increasing the dose of STZ used. Female mice can, and should, be included in pre-clinical studies of diabetes and its complications.

Mammalian microRNAs (miRNAs) play a critical role in modulating the response of immune cells to stimuli. Cannabinoids are known to exert beneficial actions such as neuroprotection and immunosuppressive activities. However, the underlying mechanisms which contribute to these effects are not fully understood. We previously reported that the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) and the non-psychoactive cannabidiol (CBD) differ in their anti-inflammatory signaling pathways. Using lipopolysaccharide (LPS) to stimulate BV-2 microglial cells, we examined the role of cannabinoids on the expression of miRNAs. Expression was analyzed by performing deep sequencing, followed by Ingenuity Pathway Analysis to describe networks and intracellular pathways. miRNA sequencing analysis revealed that 31 miRNAs were differentially modulated by LPS and by cannabinoids treatments. In addition, we found that at the concentration tested, CBD has a greater effect than THC on the expression of most of the studied miRNAs. The results clearly link the effects of both LPS and cannabinoids to inflammatory signaling pathways. LPS upregulated the expression of pro-inflammatory miRNAs associated to Toll-like receptor (TLR) and NF-κB signaling, including miR-21, miR-146a and miR-155, whereas CBD inhibited LPS-stimulated expression of miR-146a and miR-155. In addition, CBD upregulated miR-34a, known to be involved in several pathways including Rb/E2f cell cycle and Notch-Dll1 signaling. Our results show that both CBD and THC reduced the LPS-upregulated Notch ligand Dll1 expression. MiR-155 and miR-34a are considered to be redox sensitive miRNAs, which regulate Nrf2-driven gene expression. Accordingly, we found that Nrf2-mediated expression of redox-dependent genes defines a Mox-like phenotype in CBD treated BV-2 cells. In summary, we have identified a specific repertoire of miRNAs that are regulated by cannabinoids, in resting (surveillant) and in LPS-activated microglia. The modulated miRNAs and their target genes are controlled by TLR, Nrf2 and Notch cross-talk signaling and are involved in immune response, cell cycle regulation as well as cellular stress and redox homeostasis.

Retinal OFF bipolar cells show distinct connectivity patterns with photoreceptors in the wild-type mouse retina. Some types are cone-specific while others penetrate further through the outer plexiform layer (OPL) to contact rods in addition to cones. To explore dendritic stratification of OFF bipolar cells in the absence of rods, we made use of the 'cone-full' Nrl-/- mouse retina in which all photoreceptor precursor cells commit to a cone fate including those which would have become rods in wild-type retinas. The dendritic distribution of OFF bipolar cell types was investigated by confocal and electron microscopic imaging of immunolabeled tissue sections. The cells' dendrites formed basal contacts with cone terminals and expressed the corresponding glutamate receptor subunits at those sites, indicating putative synapses. All of the four analyzed cell populations showed distinctive patterns of vertical dendritic invasion through the OPL. This disparate behavior of dendritic extension in an environment containing only cone terminals demonstrates type-dependent specificity for dendritic outgrowth in OFF bipolar cells: rod terminals are not required for inducing dendritic extension into distal areas of the OPL.

In support of the RNA world hypothesis, previous studies identified trimetaphosphate (Tmp) as a plausible energy source for RNA world organisms. In one of these studies, catalytic RNAs (ribozymes) that catalyze the triphosphorylation of RNA 5'-hydroxyl groups using Tmp were obtained by in vitro selection. One ribozyme (TPR1) was analyzed in more detail. TPR1 catalyzes the triphosphorylation reaction to a rate of 0.013 min-1 under selection conditions (50 mM Tmp, 100 mM MgCl2, 22°C). To identify a triphosphorylation ribozyme that catalyzes faster triphosphorylation, and possibly learn about its secondary structure TPR1 was subjected to a doped selection. The resulting ribozyme, TPR1e, contains seven mutations relative to TPR1, displays a previously unidentified duplex that constrains the ribozyme's structure, and reacts at a 24-fold faster rate than the parent ribozyme. Under optimal conditions (150 mM Tmp, 650 mM MgCl2, 40°C), the triphosphorylation rate of TRP1e reaches 6.8 min-1.

Mutations in the elongation of very long chain fatty acid 4 (ELOVL4) gene cause Stargardt macular dystrophy 3 (STGD3), a rare, juvenile-onset, autosomal dominant form of macular degeneration. Although several mouse models have already been generated to investigate the link between the three identified disease-causing mutations in the ELOVL4 gene, none of these models recapitulates the early-onset cone photoreceptor cell death observed in the macula of STGD3 patients. To address this specifically, we investigated the effect of mutant ELOVL4 in a mouse model with an all-cone retina. Hence, we bred mice carrying the heterozygously mutated Elovl4 gene on the R91W;Nrl-/- all-cone background and analyzed the retinal lipid composition, morphology, and function over the course of 1 year. We observed a reduction of total phosphatidylcholine-containing very long chain-polyunsaturated fatty acids (PC-VLC-PUFAs) by 39% in the R91W;Nrl-/-;Elovl4 mice already at 6 weeks of age with a pronounced decline of the longest forms of PC-VLC-PUFAs. Total levels of shorter-chain fatty acids (< C26) remained unaffected. However, this reduction in PC-VLC-PUFA content in the all-cone retina had no impact on morphology or function and did not accelerate retinal degeneration in the R91W;Nrl-/-;Elovl4 mice. Taken together, mutations in the ELOVL4 gene lead to cone degeneration in humans, whereas mouse models expressing the mutant Elovl4 show predominant rod degeneration. The lack of a phenotype in the all-cone retina expressing the mutant form of the protein supports the view that aberrant function of ELOVL4 is especially detrimental for rods in mice and suggests a more subtle role of VLC-PUFAs for cone maintenance and survival.

Ocular albinism type 1 (OA), caused by mutations in the OA1 gene, encodes a G-protein coupled receptor, OA1, localized in melanosomal membranes of the retinal pigment epithelium (RPE). This disorder is characterized by both RPE macro-melanosomes and abnormal decussation of ganglion cell axons at the brain's optic chiasm. We demonstrated previously that Oa1 specifically activates Gαi3, which also signals in the Oa1 transduction pathway that regulates melanosomal biogenesis. In this study, we screened the human Gαi3 gene, GNAI3, in DNA samples from 26 patients who had all clinical characteristics of OA but in whom a specific mutation in the OA1 gene had not been found, and in 6 normal control individuals. Using the Agilent HaloPlex Target Enrichment System and next-generation sequencing (NGS) on the Illumina MiSeq platform, we identified 518 variants after rigorous filtering. Many of these variants were corroborated by Sanger sequencing. Overall, 98.8% coverage of the GNAI3 gene was obtained by the HaloPlex amplicons. Of all variants, 6 non-synonymous and 3 synonymous were in exons, 41 in a non-coding exon embedded in the 3' untranslated region (UTR), 6 in the 5' UTR, and 462 in introns. These variants included novel SNVs, insertions, deletions, and a frameshift mutation. All were found in at least one patient but none in control samples. Using computational methods, we modeled the GNAI3 protein and its non-synonymous exonic mutations and determined that several of these may be the cause of disease in the patients studied. Thus, we have identified GNAI3 as a second gene possibly responsible for X-linked ocular albinism.

Purpose

Mitochondrial (mt) DNA damage is associated with age-related macular degeneration (AMD) and other human aging diseases. This study was designed to quantify and characterize mtDNA low-frequency heteroplasmy single nucleotide polymorphisms (SNPs) of three different tissues isolated from AMD subjects using Next Generation Sequencing (NGS) technology.

Methods

DNA was extracted from neural retina, [RPE+choroid] and blood from three deceased age-related macular degeneration (AMD) subjects. Entire mitochondrial genomes were analyzed for low-frequency heteroplasmy SNPs using NGS technology that independently sequenced both mtDNA strands. This deep sequencing method (average sequencing depth of 30,000; range 1,000-100,000) can accurately differentiate low-frequency heteroplasmy SNPs from DNA modification artifacts. Twenty-three 'hot-spot' heteroplasmy mtDNA SNPs were analyzed in 222 additional blood samples.

Results

Germline homoplasmy SNPs that defined mtDNA haplogroups were consistent in the three tissues of each subject. Analyses of SNPs with <40% heteroplasmy revealed the blood had significantly greater numbers of heteroplasmy SNPs than retina alone (p≤0.05) or retina+choroid combined (p = 0.008). Twenty-three 'hot-spot' mtDNA heteroplasmy SNPs were present, with three being non-synonymous (amino acid change). Four 'hot-spot' heteroplasmy SNPs (m.1120C>T, m.1284T>C, m.1556C>T, m.7256C>T) were found in additional samples (n = 222). Five heteroplasmy SNPs (m.4104A>G, m.5320C>T, m.5471G>A, m.5474A>G, m.5498A>G) declined with age. Two heteroplasmy SNPs (m.13095T>C, m.13105A>G) increased in AMD compared to Normal samples. In the heteroplasmy SNPs, very few transversion mutations (purine to pyrimidine or vice versa, associated with oxidative damage) were found and the majority were transition changes (purine to purine or pyrimidine to pyrimidine, associated with replication errors).

Conclusion

Within an individual, the blood, retina and [RPE+choroid] contained identical homoplasmy SNPs representing inherited germline mtDNA haplogroup. NGS methodology showed significantly more mtDNA heteroplasmy SNPs in blood compared to retina and [RPE+choroid], suggesting the latter tissues have substantial protection. Significantly higher heteroplasmy levels of m.13095T>C and m.13105A>G may represent potential AMD biomarkers. Finally, high levels of transition mutations suggest that accumulation of heteroplasmic SNPs may occur through replication errors rather than oxidative damage.

Purpose

Cytomegalovirus (CMV) retinitis remains a leading cause of blindness in countries with a high burden of AIDS. Although dilated fundus examinations are recommended for those with CD4 counts below 100 cells/μL, in practice only those with poor vision and/or symptoms are routinely referred for screening. Therefore, the predictive value of this common practice should be assessed.

Methods

This is a prospective cross-sectional study. Patients with known HIV and a CD4 count of less than 100 cells/μL attending an HIV clinic in Chiang Mai, Thailand completed a standardized questionnaire about visual symptoms and underwent visual acuity testing and dilated fundus examination. Participants without CMV retinitis were invited for repeated examinations every 3 months until their CD4 count exceeded 100 cells/μL. Patient-level statistical analyses were conducted to calculate diagnostic test characteristics, with bootstrapping to account for correlated data.

Results

Of 103 study participants, 16 had CMV retinitis diagnosed at some point during the study. Participants with CMV retinitis were more likely to complain of visual symptoms compared to those without CMV retinitis (p = 0.01), including scotoma (p = 0.0002), itchy or watery eyes (p < 0.0001), and eye pain (p = 0.003); they were also more likely to have visual acuity worse than Counting Fingers (p = 0.0003). However, the absence of eye symptoms and the absence of poor vision did not strongly affect the probability that a patient did not have disease (negative likelihood ratio 0.56 and 0.76, respectively).

Conclusions

Ocular symptoms and poor visual acuity were poor diagnostic indicators for the presence of CMV retinitis. Systematic screening of HIV patients with CD4 counts below 100 cells/μl should be carried out to detect disease at an early stage, when blindness can still be prevented.

Purpose

To determine the presence of structural changes in HIV retinae (i.e., photoreceptor density and retinal thickness in the macula) compared with age-matched HIV-negative controls.

Methods

Cohort of patients with known HIV under CART (combination Antiretroviral Therapy) treatment were examined with a flood-illuminated retinal AO camera to assess the cone photoreceptor mosaic and spectral-domain optical coherence tomography (SD-OCT) to assess retinal layers and retinal thickness.

Results

Twenty-four eyes of 12 patients (n = 6 HIV-positive and 6 HIV-negative) were imaged with the adaptive optics camera. In each of the regions of interest studied (nasal, temporal, superior, inferior), the HIV group had significantly less mean cone photoreceptor density compared with age-matched controls (difference range, 4,308-6,872 cones/mm2). A different subset of forty eyes of 20 patients (n = 10 HIV-positive and 10 HIV-negative) was included in the retinal thickness measurements and retinal layer segmentation with the SD-OCT. We observed significant thickening in HIV positive eyes in the total retinal thickness at the foveal center, and in each of the three horizontal B-scans (through the macular center, superior, and inferior to the fovea). We also noted that the inner retina (combined thickness from ILM through RNFL to GCL layer) was also significantly thickened in all the different locations scanned compared with HIV-negative controls.

Conclusion

Our present study shows that the cone photoreceptor density is significantly reduced in HIV retinae compared with age-matched controls. HIV retinae also have increased macular retinal thickness that may be caused by inner retinal edema secondary to retinovascular disease in HIV. The interaction of photoreceptors with the aging RPE, as well as possible low-grade ocular inflammation causing diffuse inner retinal edema, may be the key to the progressive vision changes in HIV-positive patients without overt retinitis.