The Association Between Craniofacial Fracture Patterns and Traumatic Optic Neuropathy.

Traumatic optic neuropathy (TON) is a rare but potentially devastating complication of craniofacial trauma. Approximately half of patients with TON sustain permanent vision loss. In this study, we sought to identify the most common fracture patterns associated with TON. We performed a retrospective review of craniomaxillofacial CT scans of trauma patients who presented to the R Adams Cowley Shock Trauma Center from 2015 to 2017. Included were adult patients who had orbital fractures with or without other facial fractures. Patients diagnosed with TON by a formal ophthalmologic examination were analyzed. Craniofacial fracture patterns were identified. Bivariate analysis and multivariate logistic regression were performed to identify craniofacial fracture patterns most commonly associated with TON. A total of 574 patients with orbital fractures who met inclusion criteria [15 (2.6%)] were diagnosed with TON. The median [interquartile range (IQR)] age was 44 (28-59) years. Patients with optic canal fractures and sphenoid sinus fractures had greater odds of TON compared with patients who did not have these fracture types [adjusted odds ratio (aOR) 95% confidence interval (CI) 31.8 (2.6->100), 8.1 (2.7-24.4), respectively]. Patients who sustain optic canal and sphenoid sinus fractures in the setting of blunt facial trauma are at increased odds of having a TON. Surgeons and other physicians involved in the care of these patients should be aware of this association.

Effectiveness of Trachomatous Trichiasis Case-identification Approaches in Ethiopia.

BACKGROUND: Trachoma control programs use multiple approaches to identify individuals with trachomatous trichiasis (TT). Evidence is limited regarding which approaches are most effective and cost-efficient. METHODS: We evaluated the effectiveness of two TT case-identification approaches in Ethiopia: community mobilization to encourage self-referral for centralized screening and house-to-house screenings conducted by case finders. We compared the number of true cases found per 1000 population and costs associated with case identification under each approach, stratified by villages that received one or multiple screening visits. RESULTS: We conducted screenings in 396 villages. In villages receiving one house-to-house visit, case finders identified 14,229 suspected cases, of whom 10,513 (73.9%) presented for TT confirmation. A median of 17.2% (interquartile range [IQR]: 9.1%-27.8%) of those presenting truly had TT (positive predictive value). In single-visit villages, the community mobilization approach yielded higher rates of confirmed cases than the house-to-house approach (1.5 [IQR: 1.1, 2.6] vs. 1.1 [IQR: 0.5, 1.9] cases per 1000 population), and the median cost of identifying a TT case was less ($5.59 vs. $31.18) using community mobilization than house-to-house. In multiple-visit villages, additional screening visits increased the median rate of confirmed cases to 2.5 per 1000 population in community mobilization villages, but the rate remained unchanged in house-to-house villages. CONCLUSIONS: Community mobilization-based TT case finding had a higher yield than house-to-house, at a substantially lower cost. Future research should examine whether additional tools to aid case finders in their diagnosis increases case-finding efficiency and accuracy and whether TT prevalence and surgical program duration impact case-finding success.

Regulation of Opsin Gene Expression by DNA Methylation and Histone Acetylation.

One important role of epigenetic regulation is controlling gene expression in development and homeostasis. However, little is known about epigenetics' role in regulating opsin expression. Cell cultures (HEK 293, Y79, and WERI) producing different levels of opsins were treated with 5-aza-2'-deoxycytidine (5-Aza-dc) and/or sodium butyrate (SB) or suberoylanilide hydroxamic acid (SAHA) for 72 h. Global DNA methylation, site-specific methylation, and expressions of opsins were measured by LUMA assay, bisulfite pyrosequencing, and qPCR, respectively. Mouse retinal explants from wild-type P0/P1 pups were ex vivo cultured with/without 5-Aza-dc or SAHA for 6 days. The morphology of explants, DNA methylation, and expressions of opsins was examined. The drugs induced global DNA hypomethylation or increased histone acetylation in cells, including DNA hypomethylation of rhodopsin (RHO) and L-opsin (OPN1LW) and a concomitant increase in their expression. Further upregulation of RHO and/or OPN1LW in HEK 293 or WERI cells was observed with 5-Aza-dc and either SB or SAHA combination treatment. Mouse retinal explants developed normally but had drug-dependent differential DNA methylation and expression patterns of opsins. DNA methylation and histone acetylation directly regulate opsin expression both in vitro and ex vivo. The ability to manipulate opsin expression using epigenetic modifiers enables further study into the role of epigenetics in eye development and disease.

A New Surgical Technique for Postoperative Trachomatous Trichiasis.

INTRODUCTION: The World Health Organization has identified management of postoperative trichiasis (PTT) as one of the key remaining areas of focus needed to eliminate blinding trachoma as a public health problem. We developed the Bevel-Rotation Advancement Procedure (B-RAP) to treat individuals who need repeat trichiasis surgery. METHODS: Scarring caused by trichiasis surgery can cause the eyelid to become thick and distorted, making repeat surgery more difficult. To minimize eyelid thickness following B-RAP, a beveled incision of the tarsus is made allowing a marginal rotation of the eyelash fragment. Dissection between the anterior and posterior lamellae above the beveled incision and removal of scar tissue allows the marginal rotation to be combined with a posterior lamellar advancement to treat severely scarred eyelids with PTT and eyelid contour abnormalities (ECAs). RESULTS: Two surgeons performed B-RAP on 44 eyelids of 30 patients with PTT. The number of prior trachomatous trichiasis (TT) surgeries ranged from 2 to more than 4. At the 3-6 months postoperative visit, 37 eyelids (84%) had no recurrence of PTT. Three eyelids had central lashes touching; the remaining eyelids with recurrent PTT had nasal and temporal lashes touching. Fifteen eyelids (34%) had ECAs, but only 1 was severe. CONCLUSIONS: B-RAP was developed considering the altered eyelid anatomy found in the postsurgical eyelid with TT. Thinning of the eyelash fragment and removal of postoperative scar tissue improves the ability to advance and stabilize the eyelash fragment after external rotation. B-RAP shows promise as a procedure for improving outcomes of repeat trichiasis surgery.

Evaluation and implementation of a mannequin-based surgical simulator for margin-involving eyelid laceration repair - a pilot study.

BACKGROUND: Repair of margin-involving eyelid lacerations is a challenge for beginning ophthalmology residents, yet no commercially-available simulation models exist for learning this skill. The objective of the study was to modify a mannequin-based surgical simulator originally developed for trachomatous trichiasis surgery training to teach margin-involving eyelid laceration repair and to evaluate its success within a residency wet-lab environment. METHODS: We modified a previously developed mannequin-based training system for trachomatous trichiasis surgery into a simulator for margin-involving eyelid laceration repair. Six ophthalmology residents from a tertiary care academic institution performed at least one simulated margin-involving eyelid laceration repair using the surgical simulator between September 2019 and March 2020. Each session was video recorded. Two oculoplastic surgeons reviewed the videos in a blinded fashion to assess surgical proficiency using a standardized grading system. Participants were surveyed on their comfort level with eyelid laceration repair pre- and post-completion of simulation. They were also queried on their perceived usefulness of the surgical simulator compared to past methods and experiences. RESULTS: Six residents completed 11 simulation surgeries. For three residents who completed more than one session, a slight increase in their skills assessment score and a decrease in operative time over two to three simulation sessions were found. Self-reported comfort level with margin-involving eyelid laceration repairs was significantly higher post-simulation compared to pre-simulation (p = 0.02). Residents ranked the usefulness of our surgical simulator higher than past methods such as fruit peels, surgical skill boards, gloves, and pig feet (p = 0.03) but lower than operating room experience (p = 0.02). Residents perceived the surgical simulator to be as useful as cadaver head and emergency department/consult experience. CONCLUSIONS: We developed a surgical simulator for teaching eyelid laceration repair and showed its utility in developing trainees' surgical skills. Our surgical simulator was rated to be as useful as a cadaver head but is more readily available and cost effective.

A multiplex immunoassay of serum biomarkers for the detection of uveal melanoma.

BACKGROUND: Approximately 50% of uveal melanoma (UM) patients develop metastases preferentially in the liver leading to death within 15 months. Currently, there is no effective treatment for metastatic UM, in part because the tumor burden is typically high when liver metastases are detected through abnormal liver function tests (LFTs) or imaging studies. The use of LFTs results followed by diagnostic tests has high specificity and predictive values but low sensitivity, and better tests are needed for early diagnosis of the primary tumor as well as its metastatic spread. To evaluate serum biomarkers for the early detection of UM, multiplex immunoassays were developed. METHODS: Magnetic bead-based multiplex immunoassays were developed for the selected serum biomarkers using a Bio-Plex 200 system. The dynamic ranges, lower limits of detection and quantification, cross-reactivity, and intra- and inter-assay precision were assessed. All proteins were analyzed in sera of 48 patients diagnosed with UM (14 metastatic, 9 disease-free (DF) ≥ 5 years, 25 unknown) and 36 healthy controls. The performance of the biomarkers was evaluated individually and in combination for their ability to detect UM. RESULTS: A 7-plex immunoassay of OPN, MIA, CEACAM-1, MIC-1, SPON1, POSTN and HSP27 was developed with negligible cross-reactivity, recovery of 84-105%, and intra-assay and inter-assay precision of 2.3-7.5% or 2.8-20.8%, respectively. Logistic regression identified a two-marker panel of HSP27 and OPN that significantly improved the individual biomarker performance in discriminating UM from healthy controls. The improved discrimination of a two-marker panel of MIA and MIC-1 was also observed between metastatic UM and DF, however not statistically significant due to the small sample size. CONCLUSIONS: The multiplex immunoassay provides sufficient analytical performance to evaluate serum biomarkers that complement each other in detection of UM, and warrants further validation with a larger number of patient samples.

Virtual Fitting, Selection, and Cutting of Preformed Anatomic Orbital Implants.

PURPOSE: To evaluate the usage of preoperative digital anatomic implant evaluation in internal orbital fractures. METHODS: An IRB-approved review of cases of orbital fracture repair was conducted and cases where digital implant modeling was performed were selected for comprehensive review. The surgical time of these cases was also compared with surgeon and implant matched controls. RESULTS: A total of 25 patients and 26 orbits underwent preoperative virtual fitting and were reviewed. There were no complications or revision surgeries needed. Postoperative assessment demonstrated accuracy to the preoperative target with an average maximum deviation of 1.9 mm. CONCLUSIONS: Preoperative digital fracture assessment, implant manufacturer and size selection, and virtual cutting guide creation provides additional tools for orbital surgeons to achieve anatomic restoration without significant differences in operating time.

Conditional knockdown of DNA methyltransferase 1 reveals a key role of retinal pigment epithelium integrity in photoreceptor outer segment morphogenesis.

Dysfunction or death of photoreceptors is the primary cause of vision loss in retinal and macular degenerative diseases. As photoreceptors have an intimate relationship with the retinal pigment epithelium (RPE) for exchange of macromolecules, removal of shed membrane discs and retinoid recycling, an improved understanding of the development of the photoreceptor-RPE complex will allow better design of gene- and cell-based therapies. To explore the epigenetic contribution to retinal development we generated conditional knockout alleles of DNA methyltransferase 1 (Dnmt1) in mice. Conditional Dnmt1 knockdown in early eye development mediated by Rx-Cre did not produce lamination or cell fate defects, except in cones; however, the photoreceptors completely lacked outer segments despite near normal expression of phototransduction and cilia genes. We also identified disruption of RPE morphology and polarization as early as E15.5. Defects in outer segment biogenesis were evident with Dnmt1 exon excision only in RPE, but not when excision was directed exclusively to photoreceptors. We detected a reduction in DNA methylation of LINE1 elements (a measure of global DNA methylation) in developing mutant RPE as compared with neural retina, and of Tuba3a, which exhibited dramatically increased expression in mutant retina. These results demonstrate a unique function of DNMT1-mediated DNA methylation in controlling RPE apicobasal polarity and neural retina differentiation. We also establish a model to study the epigenetic mechanisms and signaling pathways that guide the modulation of photoreceptor outer segment morphogenesis by RPE during retinal development and disease.

Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells.

Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adeno-associated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 10(9) vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs.

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death.

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.

Prediction of promoters and enhancers using multiple DNA methylation-associated features.

BACKGROUND: Regulatory regions (e.g. promoters and enhancers) play an essential role in human development and disease. Many computational approaches have been developed to predict the regulatory regions using various genomic features such as sequence motifs and evolutionary conservation. However, these DNA sequence-based approaches do not reflect the tissue-specific nature of the regulatory regions. In this work, we propose to predict regulatory regions using multiple features derived from DNA methylation profile. RESULTS: We discovered several interesting features of the methylated CpG (mCpG) sites within regulatory regions. First, a hypomethylation status of CpGs within regulatory regions, compared to the genomic background methylation level, extended out >1000 bp from the center of the regulatory regions, demonstrating a high degree of correlation between the methylation statuses of neighboring mCpG sites. Second, when a regulatory region was inactive, as determined by histone mark differences between cell lines, methylation level of the mCpG site increased from a hypomethylated state to a hypermethylated state, the level of which was even higher than the genomic background. Third, a distinct set of sequence motifs was overrepresented surrounding mCpG sites within regulatory regions. Using 5 types of features derived from DNA methylation profiles, we were able to predict promoters and enhancers using machine-learning approach (support vector machine). The performances for prediction of promoters and enhancers are quite well, showing an area under the ROC curve (AUC) of 0.992 and 0.817, respectively, which is better than that simply based on methylation level, especially for prediction of enhancers. CONCLUSIONS: Our study suggests that DNA methylation features of mCpG sites can be used to predict regulatory regions.

Characterization of tissue-specific differential DNA methylation suggests distinct modes of positive and negative gene expression regulation.

BACKGROUND: DNA methylation plays an important role in regulating gene expression during many biological processes. However, the mechanism of DNA-methylation-dependent gene regulation is not fully understood. Here, we explore two possible DNA methylation regulatory mechanisms with opposite modes of gene expression regulation. RESULTS: By comparing the genome-wide methylation and expression patterns in different tissues, we find that majority of tissue-specific differentially methylated regions (T-DMRs) are negatively correlated with expression of their associated genes (negative T-DMRs), consistent with the classical dogma that DNA methylation suppresses gene expression; however, a significant portion of T-DMRs are positively correlated with gene expression (positive T-DMRs). We observe that the positive T-DMRs have similar genomic location as negative T-DMRs, except that the positive T-DMRs are more enriched in the promoter regions. Both positive and negative T-DMRs are enriched in DNase I hypersensitivity sites (DHSs), suggesting that both are likely to be functional. The CpG sites of both positive and negative T-DMRs are also more evolutionarily conserved than the genomic background. Interestingly, the putative target genes of the positive T-DMR are enriched for negative regulators such as transcriptional repressors, suggesting a novel mode of indirect DNA methylation inhibition of expression through transcriptional repressors. Likewise, two distinct sets of DNA sequence motifs exist for positive and negative T-DMRs, suggesting that two distinct sets of transcription factors (TFs) are involved in positive and negative regulation mediated by DNA methylation. CONCLUSIONS: We find both negative and positive association between T-DMRs and gene expression, which implies the existence of two different mechanisms of DNA methylation-dependent gene regulation.

EMT-associated factors promote invasive properties of uveal melanoma cells.

PURPOSE: Transcription factors regulating the epithelial-to-mesenchymal transition (EMT) program contribute to carcinogenesis and metastasis in many tumors, including cutaneous melanoma. However, little is known about the role of EMT factors in the growth and metastatic dissemination of uveal melanoma cells. Here, we analyzed the expression and functions of the EMT factors ZEB1, Twist1, and Snail1 in uveal melanoma cell lines and primary tumors. METHODS: ZEB1, Twist1, and Snail1 mRNA levels were measured using qPCR in five uveal melanoma cell lines and in 30 primary tumors. Gene expression was used to determine class 1 and class 2 signatures in the primary tumors. Short hairpin RNA was used to downregulate the expressions of the EMT factors; then, growth and transwell invasion assays were performed. RESULTS: ZEB1, Twist1, and Snail1 were expressed in all five uveal melanoma lines, with ZEB1 having the highest protein levels. ZEB1 mRNA was significantly elevated in highly metastatic class 2 primary tumors for which survival data were not available, whereas a high gene expression of Twist1 was associated with a worse prognosis in a separate tumor cohort analyzed by expression profiling. The genetic downregulation of ZEB1 in OCM1, OMM1, and 92.1 resulted in a more than 50% reduction in invasion, but only suppressed growth in OMM1 cells. Suppression of Twist1 in Mel290 and OMM1 reduced growth and invasion by more than 50%. The downregulation of Snail1 in the 92.1 cell line reduced invasion by 50%, but did not interfere with growth. CONCLUSIONS: The downregulation of ZEB1, Twist1, and Snail1 reduces the invasive properties of uveal melanoma cells, and the elevated mRNA levels of ZEB1 and Twist1 are associated with a more aggressive clinical phenotype in uveal melanoma samples. Therefore, these factors could represent new therapeutic targets in patients with ocular melanoma.

Integrative analysis of tissue-specific methylation and alternative splicing identifies conserved transcription factor binding motifs.

The exact role of intragenic DNA methylation in regulating tissue-specific gene regulation is unclear. Recently, the DNA-binding protein CTCF has been shown to participate in the regulation of alternative splicing in a DNA methylation-dependent manner. To globally evaluate the relationship between DNA methylation and tissue-specific alternative splicing, we performed genome-wide DNA methylation profiling of mouse retina and brain. In protein-coding genes, tissue-specific differentially methylated regions (T-DMRs) were preferentially located in exons and introns. Gene ontology and evolutionary conservation analysis suggest that these T-DMRs are likely to be biologically relevant. More than 14% of alternatively spliced genes were associated with a T-DMR. T-DMR-associated genes were enriched for developmental genes, suggesting that a specific set of alternatively spliced genes may be regulated through DNA methylation. Novel DNA sequences motifs overrepresented in T-DMRs were identified as being associated with positive and/or negative regulation of alternative splicing in a position-dependent context. The majority of these evolutionarily conserved motifs contain a CpG dinucleotide. Some transcription factors, which recognize these motifs, are known to be involved in splicing. Our results suggest that DNA methylation-dependent alternative splicing is widespread and lay the foundation for further mechanistic studies of the role of DNA methylation in tissue-specific splicing regulation.

Exposure rate of smooth surface tunnel porous polyethylene implants after enucleation.

PURPOSE: To determine the exposure rate after enucleation using the smooth surface tunnel (SST) and SST-EZ porous polyethylene (PP) orbital implants (Stryker Kalamazoo, MI, U.S.A.). METHODS: The authors performed an institutional review board-approved retrospective review of 150 consecutively placed SST and SST-EZ PP implants after enucleation. Patient records were reviewed for comorbidities, surgical details, follow-up length, and presence of implant exposure. All prior published studies on porous orbital implant insertion were reviewed, and the exposure rate for patients undergoing enucleation with primary implant insertion was studied for comparison. RESULTS: Of the 150 implants, 30 were excluded due to follow up of less than 8 weeks. Of the remaining 120 implants, 4 (3.3%) became exposed during the follow-up period (mean, 2.1 years), 2 of which were in children who underwent chemotherapy for retinoblastoma. The authors calculated a 7.1% exposure rate for all porous implant materials placed primarily after enucleation from 58 previously published studies. CONCLUSIONS: The SST and SST-EZ PP implants are well tolerated when placed at the time of enucleation. The theoretical advantage of a smooth anterior surface may indeed lead to a lower rate of exposure compared with standard unwrapped porous materials as demonstrated by the relatively low exposure rate using this implant.

Reconstruction of periorbital soft tissue defects.

Because of the complex anatomy and fine mechanics of the periorbital soft tissues, the reconstruction of this region can be particularly daunting. Through a structured assessment of the defect, based on subunit analysis and thorough understanding of the surgical layers, we believe to allow the reconstructive surgeon to develop an algorithmic approach to these complex problems. The sequela of a suboptimal reconstruction do not only result in an inferior aesthetic result, but also have the potential for long-term functional problems such as epiphora, dry eye, ptosis, eyelid retraction, and thus requiring secondary surgery. There is no better time to aim for a perfect reconstruction than at the time of the initial surgery. In this chapter, we hope to encourage the reader to strengthen and recapitulate these analytical skills and present the most commonly used and studied techniques to help achieve a reproducible functional and aesthetically appealing result.

Impact of hospital volume and specialization on the cost of orbital trauma care.

PURPOSE: To examine the impact of hospital volume and specialization on the cost of orbital trauma care. DESIGN: Comparative case series and database study. PARTICIPANTS: Four hundred ninety-nine patients who underwent orbital reconstruction at either a high-volume regional eye trauma center, its academic parent institution, or all other hospitals in Maryland between 2004 and 2009. METHODS: We used a publicly available database of hospital discharge data to identify the study population's clinical and cost characteristics. Multivariate models were developed to determine the impact of care setting on hospital costs while controlling for patient demographic and clinical variables. MAIN OUTCOME MEASURES: Mean hospital costs accrued during hospital admission for orbital reconstruction in 3 separate care settings. RESULTS: Almost half (n = 248) of all patients received surgical care at the regional eye trauma center and had significantly lower adjusted mean hospital costs ($6194; 95% confidence interval [CI], $5709-$6719) compared with its parent institution ($8642; 95% CI, $7850-$9514) and all other hospitals ($12,692; 95% CI, $11,467-$14,047). A subpopulation analysis selecting patients with low comorbidity scores also was performed. The eye trauma center continued to have lower adjusted costs ($4277; 95% CI, $4112-$4449) relative to its parent institution ($6595; 95% CI, $5838-$7451) and other hospitals ($7150; 95% CI, $5969-$8565). CONCLUSIONS: Higher volume and specialization seen at a regional eye trauma center are associated with lower costs in the surgical management of orbital trauma.

Dynamic usage of alternative splicing exons during mouse retina development.

Alternative processing of pre-mRNA plays an important role in protein diversity and biological function. Previous studies on alternative splicing (AS) often focused on the spatial patterns of protein isoforms across different tissues. Here we studied dynamic usage of AS across time, during murine retina development. Over 7000 exons showed dynamical changes in splicing, with differential splicing events occurring more frequently in early development. The overall splicing patterns for exclusive and inclusive exons show symmetric trends and genes with symmetric splicing patterns that tend to have similar biological functions. Furthermore, we observed that within the retina, retina-enriched genes that are preferentially expressed at the adult stage tend to have more dynamically spliced exons compared to other genes, suggesting that genes maintaining retina homeostasis also play an important role in development via a series of AS events. Interestingly, the transcriptomes of retina-enriched genes largely reflect the retinal developmental process. Finally, we identified a number of candidate cis-regulatory elements for retinal AS by analyzing the relative occurrence of sequence motifs in exons or flanking introns. The occurrence of predicted regulatory elements showed strong correlation with the expression level of known RNA binding proteins, suggesting the high quality of the identified cis-regulatory elements.

Intraglandular injection of botulinum toxin a reduces tear production in rabbits.

PURPOSE: To develop an animal model and investigate the dose-dependent effect of an intraglandular injection of botulinum toxin A (BTX-A) on tear production. METHODS: In a volume of 0.1-ml, 0.625-, 1.25-, or 2.5-U BTX-A was injected transconjunctivally in the superolateral lobe of the lacrimal gland of adult New Zealand white female rabbits. In the contralateral lacrimal gland, 0.1 ml of 0.9% sodium chloride was injected. Prior to injection and at 1-week postinjection, photographs were taken to evaluate pre- and postoperative eyelid position. Fluorescein and Rose Bengal stain were used to evaluate the corneal surface, and Schirmer test was used to assess tear production. RESULTS: Glands injected with the intermediate (1.25 U) and the highest (2.5 U) doses of BTX-A displayed a statistically significant decrease in tear production (p = 0.002 and 0.007, respectively) compared with the contralateral saline-injected glands at 1 week. No corneal pathologic factors from excessive dryness were observed following the injection. While postinjection ptosis was observed (p = 0.025), no difference was seen between BTX-A and saline-injected eyes. CONCLUSIONS: In rabbits, intraglandular injection of BTX-A resulted in decreased tear production at 1 week. No additional reduction in tear production was seen with a BTX-A dose greater than 1.25 U, suggesting glandular receptor saturation at this dose. Despite suppression of tear production, no corneal pathologic factors were observed. Further studies are needed to refine this animal model with the ultimate goal of determining optimum delivery route and concentration to reduction in tear production while minimizing side effects in patients.