Genistein transfersome-embedded topical delivery system for skin melanoma treatment: in vitro and ex vivo evaluations.

Skin melanoma is considered the most dangerous form of skin cancer due to its association with high risk of metastasis, high mortality rate and high resistance to different treatment options. Genistein is a natural isoflavonoid with known chemotherapeutic activity. Unfortunately, it has low bioavailability due to its poor aqueous solubility and excessive metabolism. In the current study, genistein was incorporated into transferosomal hydrogel to improve its bioavailability. The prepared transferosomal formulations were characterized regarding: particle size; polydispersity index; zeta potential; encapsulation efficiency; TEM; FTIR; DSC; XRD; in vitro drug release; viscosity; pH; ex vivo anti-tumor activity on 3D skin melanoma spheroids and 1-year stability study at different storage temperatures. The optimized formulation has high encapsulation efficiency with an excellent particle size that will facilitate its penetration through the skin. The transfersomes have a spherical shape with sustained drug release profile. The anti-tumor activity evaluation of genistein transfersome revealed that genistein is a potent chemotherapeutic agent with enhanced penetration ability through the melanoma spheroids when incorporated into transfersomes. Stability study results demonstrate the high physical and chemical stability of our formulations. All these outcomes provide evidence that our genistein transferosomal hydrogel is a promising treatment option for skin melanoma.

Evaluation of Pregabalin bioadhesive multilayered microemulsion IOP-lowering eye drops.

In spite of available treatment options, glaucoma continues to be a leading cause of irreversible blindness in the world. Current glaucoma medications have multiple limitations including: lack of sustained action; requirement for multiple dosing per day, ocular irritation and limited options for drugs with different mechanisms of action. Previously, we demonstrated that pregabalin, a drug with high affinity and selectivity for CACNA2D1, lowered IOP in a dose-dependent manner. The current study was designed to evaluate pregabalin microemulsion eye drops and to estimate its efficacy in humans using in silico methods. Molecular docking studies of pregabalin against CACNA2D1 of mouse, rabbit, and human were performed. Pregabalin microemulsion eye drops were characterized using multiple in vivo studies and its stability was evaluated over one year at different storage conditions. Molecular docking analyses and QSPR of pregabalin confirmed its suitability as a new IOP-lowering medication that functions using a new mechanism of action by binding to CACNA2D1 in all species evaluated. Because of its prolonged corneal residence time and corneal penetration enhancement, a single topical application of pregabalin ME can provide an extended IOP reduction of more than day in different animal models. Repeated daily dosing for 2 months confirms the lack of any tachyphylactic effect, which is a common drawback among marketed IOP-lowering medications. In addition, pregabalin microemulsion demonstrated good physical stability for one year, and chemical stability for 3-6 months if stored below 25 °C. Collectively, these outcomes greatly support the use of pregabalin eye drops as once daily IOP-lowering therapy for glaucoma management.

Influence of the calcium voltage-gated channel auxiliary subunit (CACNA2D1) absence on intraocular pressure in mice.

The etiology of elevated intraocular pressure (IOP), a major risk factor for glaucoma (optic nerve atrophy), is poorly understood despite continued efforts. Although the gene variant of CACNA2D1 (encoding α2δ1), a calcium voltage-gated channel auxiliary subunit, has been reported to be associated with primary open-angle glaucoma, and the pharmacological mitigation of α2δ1 activity by pregabalin lowers IOP, the cellular basis for α2δ1 role in the modulation of IOP remains unclear. Our recent findings reveled readily detectable levels of α2δ1 and its ligand thrombospondin in the cytoskeletome fraction of human trabecular meshwork (TM) cells. To understand the direct role of α2δ1 in the modulation of IOP, we evaluated α2δ1 null mice for changes in IOP and found a moderate (∼10%) but significant decrease in IOP compared to littermate wild type control mice. Additionally, to gain cellular insights into α2δ1 antagonist (pregabalin) induced IOP changes, we assessed pregabalin's effects on human TM cell actin cytoskeletal organization and cell adhesive interactions in comparison with a Rho kinase inhibitor (Y27632), a known ocular hypotensive agent. Unlike Y27632, pregabalin did not have overt effects on cell morphology, actin cytoskeletal organization, or cell adhesion in human TM cells. These results reveal a modest but significant decrease in IOP in α2δ1 deficient mice, and this response appears to be not associated with the contractile and cell adhesive characteristics of TM cells based on the findings of pregabalin effects on isolated TM cells. Therefore, the mechanism by which pregabalin lowers IOP remains elusive.

Evaluation of an online modular eating disorders training (PreparED) to prepare healthcare trainees: a survey study.

BACKGROUND: Eating disorders (EDs) are serious, complex disorders for which broad-based clinical training is lacking. This study aimed to evaluate the efficacy of a free, brief, web-based curriculum, PreparED, in increasing comfort and confidence with, and knowledge about EDs in healthcare trainees, and to obtain program feedback from key stakeholders (i.e., learners). METHODS: This programmatic evaluation study was designed as a quantitative, repeated measures (i.e., pre- and post-test intervention) investigation. A convenience sample of two groups of healthcare trainees across geographically diverse training sites completed an anonymous survey pre- and post- engagement with PreparED. The survey included items to assess prior exposure to EDs, as well as program feasibility. The main educational outcomes included (1) Confidence and Comfort with EDs and (2) Knowledge of EDs. User experience variables of interest were likeability, usability, and engagement with the training modules. Mixed effects linear regression was used to assess the association between PreparED and educational outcome variables. RESULTS: Participants (N = 67) included 41 nutrition graduate students and 26 nurse practitioner students recruited from Teacher's College/Columbia University in New York, NY, USA, Columbia University School of Nursing in New York, NY, USA and North Dakota State University School of Nursing in Fargo, ND, USA. Confidence/Comfort scores and Knowledge scores significantly improved following engagement with PreparED (ß = for effect of intervention = 1.23, p < 0.001, and 1.69, p < 0.001, respectively). Neither training group nor prior exposure to EDs moderated the effect on outcomes. All learners agreed the program was easy to follow; the overwhelming majority (89.4%) felt the length of the modules was "just right." All participants perceived that PreparED had increased their knowledge of EDs, and the majority (94.0%) reported greater confidence in and comfort with caring for people with these disorders, including assessment of symptoms, awareness of associated medical complications, and likelihood of future screening. CONCLUSIONS: Findings suggest that brief, user-friendly, online courses can improve knowledge and attitudes about EDs, filling a critical gap in healthcare training.

Computational methods in glaucoma research: Current status and future outlook.

Advancements in computational techniques have transformed glaucoma research, providing a deeper understanding of genetics, disease mechanisms, and potential therapeutic targets. Systems genetics integrates genomic and clinical data, aiding in identifying drug targets, comprehending disease mechanisms, and personalizing treatment strategies for glaucoma. Molecular dynamics simulations offer valuable molecular-level insights into glaucoma-related biomolecule behavior and drug interactions, guiding experimental studies and drug discovery efforts. Artificial intelligence (AI) technologies hold promise in revolutionizing glaucoma research, enhancing disease diagnosis, target identification, and drug candidate selection. The generalized protocols for systems genetics, MD simulations, and AI model development are included as a guide for glaucoma researchers. These computational methods, however, are not separate and work harmoniously together to discover novel ways to combat glaucoma. Ongoing research and progresses in genomics technologies, MD simulations, and AI methodologies project computational methods to become an integral part of glaucoma research in the future.

Exploring Early-Stage Retinal Neurodegeneration in Murine Pigmentary Glaucoma: Insights From Gene Networks and miRNA Regulation Analyses.

Purpose: Glaucoma is a group of heterogeneous optic neuropathies characterized by the progressive degeneration of retinal ganglion cells. However, the underlying mechanisms have not been understood completely. We aimed to elucidate the genetic network associated with the development of pigmentary glaucoma with DBA/2J (D2) mouse model of glaucoma and corresponding genetic control D2-Gpnmb (D2G) mice carrying the wild type (WT) Gpnmb allele. Methods: Retinas isolated from 13 D2 and 12 D2G mice were subdivided into 2 age groups: pre-onset (1-6 months: samples were collected at approximately 1-2, 2-4, and 5-6 months) and post-onset (7-15 months: samples were collected at approximately 7-9, 10-12, and 13-15 months) glaucoma were compared. Differential gene expression (DEG) analysis and gene-set enrichment analyses were performed. To identify micro-RNAs (miRNAs) that target Gpnmb, miRNA expression levels were correlated with time point matched mRNA expression levels. A weighted gene co-expression network analysis (WGCNA) was performed using the reference BXD mouse population. Quantitative real-time PCR (qRT-PCR) was used to validate Gpnmb and miRNA expression levels. Results: A total of 314 and 86 DEGs were identified in the pre-onset and post-onset glaucoma groups, respectively. DEGs in the pre-onset glaucoma group were associated with the crystallin gene family, whereas those in the post-onset group were related to innate immune system response. Of 1329 miRNAs predicted to target Gpnmb, 3 miRNAs (miR-125a-3p, miR-3076-5p, and miR-214-5p) were selected. A total of 47 genes demonstrated overlapping with the identified DEGs between D2 and D2G, segregated into their time-relevant stages. Gpnmb was significantly downregulated, whereas 2 out of 3 miRNAs were significantly upregulated (P < 0.05) in D2 mice at both 3-and 10-month time points. Conclusions: These findings suggest distinct gene-sets involved in pre-and post-glaucoma in the D2 mouse. We identified three miRNAs regulating Gpnmb in the development of murine pigmentary glaucoma.

A Novel Mouse Model for Late-Onset Retinal Degeneration (L-ORD) Develops RPE Abnormalities Due to the Loss of C1qtnf5/Ctrp5.

Late-onset retinal degeneration (L-ORD) is an autosomal dominant macular dystrophy resulting from mutations in the gene CTRP5/C1QTNF5. A mouse model (Ctrp5+/-) for the most common S163R developed many features of human clinical disease. We generated a novel homozygous Ctrp5 gene knock-out (Ctrp5-/-) mouse model to further study the mechanism of L-ORD. The retinal morphology of these mice was evaluated by retinal imaging, light microscopy, and transmission electron microscopy (TEM) at 6, 11, and 18.5 mo. Expression of Ctrp5 was analyzed using immunostaining and qRT-PCR. The Ctrp5-/- mice showed lack of both Ctrp5 transcript and protein. Presence of a significantly larger number of autofluorescent spots was observed in Ctrp5-/- mice compared to the WT (P < 0.0001) at 19 mo. Increased RPE stress with vacuolization and thinning was observed as early as 6 mo in Ctrp5-/- mice. Further, ultrastructural analyses revealed a progressive accumulation of basal laminar sub-RPE deposits in Ctrp5-/- mice from 11 mo. The Ctrp5-/- mice shared retinal and RPE pathology that matches with that previously described for Ctrp5+/- mice suggesting that pathology in these mice results from the loss of functional CTRP5 and that the presence of CTRP5 is critical for normal RPE and retinal function.

Current Advancements in Mouse Models of Retinal Disease.

The field of retinal degenerative (RDs) disease study has been in a state of exponential growth from discovering the underlying genetic components of such diseases as age-related macular degeneration (AMD) and retinitis pigmentosa (RP) to the first gene therapy developed and approved for human Leber congenital amaurosis. However, a source for high-fidelity animal models of these complex, multifactorial, and/or polygenic diseases is a need that has yet to be fulfilled. While models for AMD and RP do exist, they often require aging the animals for a year or more, feeding special diets, or introduction of external modulators such as exposure to cigarette smoke. Currently, work is being done to uncover high-fidelity naturally occurring models of these retinal diseases with the hope and intent of providing the vision community the tools it needs to better understand, treat, and, one day, cure the patients suffering from these devastating afflictions.

Cellular and Molecular Mechanisms of Pathogenesis Underlying Inherited Retinal Dystrophies.

Inherited retinal dystrophies (IRDs) are congenital retinal degenerative diseases that have various inheritance patterns, including dominant, recessive, X-linked, and mitochondrial. These diseases are most often the result of defects in rod and/or cone photoreceptor and retinal pigment epithelium function, development, or both. The genes associated with these diseases, when mutated, produce altered protein products that have downstream effects in pathways critical to vision, including phototransduction, the visual cycle, photoreceptor development, cellular respiration, and retinal homeostasis. The aim of this manuscript is to provide a comprehensive review of the underlying molecular mechanisms of pathogenesis of IRDs by delving into many of the genes associated with IRD development, their protein products, and the pathways interrupted by genetic mutation.

Eating behavior in atypical anorexia nervosa.

OBJECTIVE: Atypical anorexia nervosa (AN) has been increasingly identified in the community and in clinical settings. Initial studies indicate that psychological symptoms are similar or more severe among patients with atypical AN, as compared with AN. This study examined whether eating behavior differed among patients with AN (n = 98), patients with atypical AN (n = 18), and healthy controls (HC, n = 75). METHOD: Adults and adolescents chose what to eat from a standardized, laboratory-based multi-item meal. Total intake, macronutrient composition, diet variety, and energy density were compared between groups. RESULTS: Both AN and atypical AN severely restricted caloric intake as compared with HC (431 ± 396 kcal and 340 ± 338 kcal vs. 879 ± 350 kcal, F2,188  = 35.4, p < .001). Individuals with AN and atypical AN did not differ in the mean intake of total calories or percentage of calories from fat (15.2 ± 25.2% vs. 11.5 ± 16.9%). DISCUSSION: This study demonstrates that individuals with atypical AN are at least as restrictive in their food intake as individuals with AN, and the restriction of dietary fat is particularly notable. Examination of eating behavior in a larger sample would be useful to replicate these findings. The current study highlights the need to understand maladaptive eating behavior in atypical AN in order to develop appropriate treatment recommendations. PUBLIC SIGNIFICANCE: Atypical anorexia nervosa is emerging as a prevalent eating disorder in community and clinical populations. The findings that patients with atypical anorexia nervosa limit calorie and fat intake in a pattern similar to that of patients with anorexia nervosa highlights the need for research to identify appropriate treatment strategies for normalization of eating patterns.

Early Course of Symptom Development in Anorexia Nervosa.

PURPOSE: Anorexia nervosa (AN) commonly begins in adolescence; however, detailed knowledge of symptom trajectories, including their temporal sequence, is less well elucidated. The purpose of the present study is to describe the onset and duration of disordered eating behaviors prior to a diagnosis of AN, examine concordance between child and parent report, and examine the relationships between timing of symptom onset and illness severity. METHODS: Seventy-one adolescents (ages 12-18 years) and their parents were interviewed about dieting, restriction, loss of control/binge eating, purging, excessive/compulsive exercise, weight history, and amenorrhea. Body mass index percentiles were calculated, and adolescents completed the Eating Disorder Examination-Questionnaire. RESULTS: Restriction, being underweight, dieting, and excessive exercise were reported by most of the sample; purging, loss-of-control eating, and having been overweight were reported by less than a third. Dieting typically emerged first, on average around age 14; the remainder of behaviors tended to emerge between ages 14 and 14½; and average age of formal diagnosis was slightly over 15 years. Dyads had good agreement regarding presence and timing of all behaviors except for dieting, for which children reported about 6 months earlier onset/longer duration, compared to parents. Although older age at interview was associated with lower body mass index percentile and higher EDE-Q score, neither age of onset nor duration was associated with severity when controlling for current age. DISCUSSION: Teens and parents describe a similar sequence of behavior changes leading up to a diagnosis of AN that typically begins with dieting and occurs over an approximate 1- to 1½-year period. Querying teens and parents about eating behavior changes may aid in identification and early intervention in AN; adolescents with normal weight who engage in persistent dieting or restrictive eating may warrant more frequent weight monitoring.

Potential Similarities in Sex Difference in Key Genes and Their Expression, Network, EQTL and Pathways between COVID-19 and Chronic Kidney Disease Based on Mouse Model.

COVID-19 and chronic kidney disease (CKD) share similarity in sex bias and key genes in the disease pathway of sex difference. We investigated the sex difference of molecular pathways of four key players of these two diseases using an existing large set of whole genome expression profiles from the kidneys of female and male mouse models. Our data show that there is little to no correlation at the whole genome expression level between female and male mice among these four genes. There are considerable sex differences among genes in upstream regulation, Ace2 complex interaction, and downstream pathways. Snap25 and Plcb4 may play important roles in the regulation of the expression level of Adam17, Tmprss2, and Cd146 in females. In males, Adh4 is a candidate gene for the regulation of Adam17, while Asl, Auts2, and Rabger1 are candidates for Tmprss2. Within the Ace2 complex, Cd146 directly influences the expression level of Adam17 and Ace2 in the female, while in the male Adam potentially has a stronger influence on Ace2 than that of Tmprss2. Among the top 100 most related genes, only one or two genes from four key genes and 11 from the control B-Actin were found to be the same between sexes. Among the top 10 sets of genes in the downstream pathway of Ace2, only two sets are the same between the sexes. We concluded that these known key genes and novel genes in CKD may play significant roles in the sex difference in the CKD and COVID-19 disease pathways.

Chronic Proinflammatory Signaling Accelerates the Rate of Degeneration in a Spontaneous Polygenic Model of Inherited Retinal Dystrophy.

Collectively, retinal neurodegenerative diseases are comprised of numerous subtypes of disorders which result in loss of a varying cell types in the retina. These diseases can range from glaucoma, which results in retinal ganglion cell death, to age-related macular degeneration and retinitis pigmentosa, which result in cell death of the retinal pigment epithelium, photoreceptors, or both. Regardless of the disease, it's been recently found that increased release of proinflammatory cytokines and proliferation of active microglia result in a remarkably proinflammatory microenvironment that assists in the pathogenesis of the disease; however, many of the details of these inflammatory events have yet to be elucidated. In an ongoing study, we have used systems genetics to identify possible models of spontaneous polygenic age-related macular degeneration by mining the BXD family of mice using single nucleotide polymorphism analyses of known genes associated with the human retinal disease. One BXD strain (BXD32) was removed from the study as the rate of degeneration observed in these animals was markedly increased with a resultant loss of most all photoreceptors by 6 months of age. Using functional and anatomical exams including optokinetic nystamography, funduscopy, fluorescein angiography, and optical coherence tomography, along with immunohistochemical analyses, we show that the BXD32 mouse strain exhibits a severe neurodegenerative phenotype accompanied by adverse effects on the retinal vasculature. We also expose the concurrent establishment of a chronic proinflammatory microenvironment including the TNFα secretion and activation of the NF-κB and JAK/STAT pathways with an associated increase in activated macrophages and phagoptosis. We conclude that the induced neuronal death and proinflammatory pathways work synergistically in the disease pathogenesis to enhance the rate of degeneration in this spontaneous polygenic model of inherited retinal dystrophy.

Genome-wide association study finds multiple loci associated with intraocular pressure in HS rats.

Elevated intraocular pressure (IOP) is influenced by environmental and genetic factors. Increased IOP is a major risk factor for most types of glaucoma, including primary open angle glaucoma (POAG). Investigating the genetic basis of IOP may lead to a better understanding of the molecular mechanisms of POAG. The goal of this study was to identify genetic loci involved in regulating IOP using outbred heterogeneous stock (HS) rats. HS rats are a multigenerational outbred population derived from eight inbred strains that have been fully sequenced. This population is ideal for a genome-wide association study (GWAS) owing to the accumulated recombinations among well-defined haplotypes, the relatively high allele frequencies, the accessibility to a large collection of tissue samples, and the large allelic effect size compared to human studies. Both male and female HS rats (N = 1,812) were used in the study. Genotyping-by-sequencing was used to obtain ∼3.5 million single nucleotide polymorphisms (SNP) from each individual. SNP heritability for IOP in HS rats was 0.32, which agrees with other studies. We performed a GWAS for the IOP phenotype using a linear mixed model and used permutation to determine a genome-wide significance threshold. We identified three genome-wide significant loci for IOP on chromosomes 1, 5, and 16. Next, we sequenced the mRNA of 51 whole eye samples to find cis-eQTLs to aid in identification of candidate genes. We report 5 candidate genes within those loci: Tyr, Ctsc, Plekhf2, Ndufaf6 and Angpt2. Tyr, Ndufaf6 and Angpt2 genes have been previously implicated by human GWAS of IOP-related conditions. Ctsc and Plekhf2 genes represent novel findings that may provide new insight into the molecular basis of IOP. This study highlights the efficacy of HS rats for investigating the genetics of elevated IOP and identifying potential candidate genes for future functional testing.

In Silico Screening and In Vivo Evaluation of Potential CACNA2D1 Antagonists as Intraocular Pressure-Reducing Agents in Glaucoma Therapy.

Glaucoma is a leading cause of permanent vision loss and current drugs do not halt disease progression. Thus, new therapies targeting different drug targets with novel mechanisms of action are urgently needed. Previously, we identified CACNA2D1 as a novel modulator of intraocular pressure (IOP) and demonstrated that a topically applied CACNA2D1 antagonist-pregabalin (PRG)-lowered IOP in a dose-dependent manner. To further validate this novel IOP modulator as a drug target for IOP-lowering pharmaceutics, a homology model of CACNA2D1 was built and docked against the NCI library, which is one of the world's largest and most diverse compound libraries of natural products. Acivicin and zoledronic acid were identified using this method and together with PRG were tested for their plausible IOP-lowering effect on Dutch belted rabbits. Although they have inferior potency to PRG, both of the other compounds lower IOP, which in turn validates CACNA2D1 as a valuable drug target in treating glaucoma.

Proinflammatory Pathways Are Activated in the Human Q344X Rhodopsin Knock-In Mouse Model of Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a hereditary disease of the retina that results in complete blindness. Currently, there are very few treatments for the disease and those that exist work only for the recessively inherited forms. To better understand the pathogenesis of RP, multiple mouse models have been generated bearing mutations found in human patients including the human Q344X rhodopsin knock-in mouse. In recent years, the immune system was shown to play an increasingly important role in RP degeneration. By way of electroretinography, optical coherence tomography, funduscopy, fluorescein angiography, and fluorescent immunohistochemistry, we show degenerative and vascular phenotypes, microglial activation, photoreceptor phagocytosis, and upregulation of proinflammatory pathway proteins in the retinas of the human Q344X rhodopsin knock-in mouse. We also show that an FDA-approved pharmacological agent indicated for the treatment of rheumatoid arthritis is able to halt activation of pro-inflammatory signaling in cultured retinal cells, setting the stage for pre-clinical trials using these mice to inhibit proinflammatory signaling in an attempt to preserve vision. We conclude from this work that pro- and autoinflammatory upregulation likely act to enhance the progression of the degenerative phenotype of rhodopsin Q344X-mediated RP and that inhibition of these pathways may lead to longer-lasting vision in not only the Q344X rhodopsin knock-in mice, but humans as well.

COVID-19 vaccine: Call for employees in international transportation industries and international travelers as the first priority in global distribution.

While countries are in a hurry to obtain SARS-CoV-2 vaccine, we are concerned with the availability of vaccine and whether a vaccine will be available to all in need. We predicted three possible scenarios for vaccine distributions and urge an international united action on the worldwide equitable access. In case the international community does not reach a consensus on how to distribute the vaccine to achieve worldwide equitable access, we call for a distribution plan that includes the employees in international transportation industries and international travelers to halt the disease transmission and promote the recovery of the global economy.

Enhanced Corneal Penetration of a Poorly Permeable Drug Using Bioadhesive Multiple Microemulsion Technology.

Corneal penetration is a key rate limiting step in the bioavailability of topical ophthalmic formulations that incorporate poorly permeable drugs. Recent advances have greatly aided the ocular delivery of such drugs using colloidal drug delivery systems. Ribavirin, a poorly permeable BCS class-III drug, was incorporated in bioadhesive multiple W/O/W microemulsion (ME) to improve its corneal permeability. The drug-loaded ME was evaluated regarding its physical stability, droplet size, PDI, zeta potential, ultrastructure, viscosity, bioadhesion, in vitro release, transcorneal permeability, cytotoxicity, safety and ocular tolerance. Our ME possessed excellent physical stability, as it successfully passed several cycles of centrifugation and freeze-thaw tests. The formulation has a transparent appearance due to its tiny droplet size (10 nm). TEM confirmed ME droplet size and revealed its multilayered structure. In spite of the high aqueous solubility and the low permeability of ribavirin, this unique formulation was capable of sustaining its release for up to 24 h and improving its corneal permeability by 3-fold. The in vitro safety of our ME was proved by its high percentage cell viability, while its in vivo safety was confirmed by the absence of any sign of toxicity or irritation after either a single dose or 14 days of daily dosing. Our ME could serve as a vehicle for enhanced ocular delivery of drugs with different physicochemical properties, including those with low permeability.

Systems Genetics of Optic Nerve Axon Necrosis During Glaucoma.

In this study, we identify genomic regions that modulate the number of necrotic axons in optic nerves of a family of mice, some of which have severe glaucoma, and define a set of high priority positional candidate genes that modulate retinal ganglion cell (RGC) axonal degeneration. A large cohort of the BXD family were aged to greater than 13 months of age. Optic nerves from 74 strains and the DBA/2J (D2) parent were harvested, sectioned, and stained with p-phenylenediamine. Numbers of necrotic axons per optic nerve cross-section were counted from 1 to 10 replicates per genotype. Strain means and standard errors were uploaded into GeneNetwork 2 for mapping and systems genetics analyses (Trait 18614). The number of necrotic axons per nerve ranged from only a few hundred to more than 4,000. Using conventional interval mapping as well as linear mixed model mapping, we identified a single locus on chromosome 12 between 109 and 112.5 Mb with a likelihood ratio statistic (LRS) of ~18.5 (p genome-wide ~0.1). Axon necrosis is not linked to locations of major known glaucoma genes in this family, including Gpnmb, Tyrp1, Cdh11, Pou6f2, and Cacna2d1. This indicates that although these genes contribute to pigmentary dispersion or elevated IOP, none directly modulates axon necrosis. Of 156 positional candidates, eight genes-CDC42 binding protein kinase beta (Cdc42bpb); eukaryotic translation initiation factor 5 (Eif5); BCL2-associated athanogene 5 (Bag5); apoptogenic 1, mitochondrial (Apopt1); kinesin light chain 1 (Klc1); X-ray repair cross complementing 3 (Xrcc3); protein phosphatase 1, regulatory subunit 13B (Ppp1r13b); and transmembrane protein 179 (Tmem179)-passed stringent criteria and are high priority candidates. Several candidates are linked to mitochondria and/or axons, strengthening their plausible role as modulators of ON necrosis. Additional studies are required to validate and/or eliminate plausible candidates. Surprisingly, IOP and ON necrosis are inversely correlated across the BXD family in mice >13 months of age and these two traits share few genes among their top ocular and retinal correlates. These data suggest that the two traits are independently modulated or that a more complex and multifaceted approach is required to reveal their association.

Once Daily Pregabalin Eye Drops for Management of Glaucoma.

Elevated intraocular pressure (IOP) is the most significant risk factor contributing to visual field loss in glaucoma. Unfortunately, the deficiencies associated with current therapies have resulted in reduced efficacy, several daily dosings, and poor patient compliance. Previously, we identified the calcium voltage-gated channel auxiliary subunit alpha2delta 1 gene (Cacna2d1) as a modulator of IOP and demonstrated that pregabalin, a drug with high affinity and selectivity for CACNA2D1, lowered IOP in a dose-dependent manner. Unfortunately, IOP returned to baseline at 6 h after dosing. In the current study, we develop a once daily topical pregabalin-loaded multiple water-in-oil-in-water microemulsion formulation to improve drug efficacy. We characterize our formulations using multiple in vitro and in vivo evaluations. Our lead formulation provides continuous release of pregabalin for up to 24 h. Because of its miniscule droplet size (<20 nm), our microemulsion has a transparent appearance and should not blur vision. It is also stable at one month of storage at temperatures ranging from 5 to 40 °C. Our formulation is nontoxic, as illustrated by a cell toxicity study and slit-lamp biomicroscopic exams. CACNA2D1 is highly expressed in both the ciliary body and the trabecular meshwork, where it functions to modulate IOP. A single drop of our lead pregabalin formulation reduces IOP by greater than 40%, which does not return to baseline until >30 h post-application. Although there were no significant differences in the amplitude of IOP reduction between the formulations we tested, a significant difference was clearly observed in their duration of action. Our multilayered microemulsion is a promising carrier that sustains the release and prolongs the duration of action of pregabalin, a proposed glaucoma therapeutic.