Overview of Corneal Transplantation for the Nonophthalmologist.

Corneal transplant is a procedure that aims to replace dysfunctional corneal tissue with a transparent graft and is one of the most widely performed transplant surgeries, but its public and professional awareness is low outside of ophthalmology. Corneal tissue consists of 5 major layers that serve to maintain its structural integrity and refractive shape: the epithelium, Bowman's layer, the stroma, Descemet's membrane, and the endothelium. Failure or irreversible damage to any layer of the cornea may be an indication for corneal transplant, and variants of this procedure may be full thickness or selectively lamellar. Complications related to corneal transplantation may occur anywhere from during surgery to years afterward, including rejection, dehiscence, cataract, and glaucoma. Complications should be managed by an ophthalmologist, but other physicians should be aware of prophylactic medications. Topical immunosuppressants and steroids are effective for preventing and treating rejection episodes, whereas there is little evidence to support the use of systemic immunosuppression. Eye protection is recommended for any corneal transplant recipient. Physicians should counsel patients on corneal donation, especially if outside the United States, where donor tissue is in short supply.

Therapy for contact lens-related ulcers.

PURPOSE OF REVIEW: The current review covers the current literature and practice patterns of antimicrobial therapy for contact lens-related microbial keratitis (CLMK). Although the majority of corneal ulcers are bacterial, fungus and acanthamoeba are substantial contributors in CLMK and are harder to treat due to the lack of commercially available topical medications and low efficacy of available topical therapy. RECENT FINDINGS: Topical antimicrobials remain the mainstay of therapy for corneal ulcers. Fluoroquinolones may be used as monotherapy for small, peripheral bacterial ulcers. Antibiotic resistance is a persistent problem. Fungal ulcers are less responsive to topical medications and adjunct oral or intrastromal antifungal medications may be helpful. Acanthamoeba keratitis continues to remain a therapeutic challenge but newer antifungal and antiparasitic agents may be helpful adjuncts. Other novel and innovative therapies are being studied currently and show promise. SUMMARY: Contact lens-associated microbial keratitis is a significant health issue that can cause vision loss. Treatment remains a challenge but many promising diagnostics and procedures are in the pipeline and offer hope.

Reticular epithelial edema after penetrating keratoplasty in a patient taking netarsudil.

Netarsudil is a relatively new medication for the treatment of primary open-angle glaucoma and ocular hypertension. It has been associated with red eyes and burning after instillation. Reticular epitheliopathy is a relatively rare complication of netarsudil that has been described in patients with preexisting corneal edema. We report the case of a healthy 76-year-old woman who developed reticular epitheliopathy after full-thickness penetrating keratoplasty that completely resolved following discontinuation of the medication. In cases where netarsudil is initiated for treatment of glaucoma or, off-label, endothelial dysfunction, reticular epithelial edema should be considered in patients complaining of a decline in vision and severe pain.

In Vivo Knockdown of the Herpes Simplex Virus 1 Latency-Associated Transcript Reduces Reactivation from Latency.

During herpes simplex virus (HSV) latency, most viral genes are silenced, with the exception of one region of the genome encoding the latency-associated transcript (LAT). This long noncoding RNA was originally described as having a role in enhancing HSV-1 reactivation. However, subsequent evidence showing that the LAT blocked apoptosis and promoted efficient establishment of latency suggested that its effects on reactivation were secondary to establishment. Here, we utilized an adeno-associated virus (AAV) vector to deliver a LAT-targeting hammerhead ribozyme to HSV-1-infected neurons of rabbits after the establishment of HSV-1 latency. The rabbits were then induced to reactivate latent HSV-1. Using this model, we show that decreasing LAT levels in neurons following the establishment of latency reduced the ability of the virus to reactivate. This demonstrates that the HSV-1 LAT RNA has a role in reactivation that is independent of its function in establishment of latency. In addition, these results suggest the potential of AAV vectors expressing LAT-targeting ribozymes as a potential therapy for recurrent HSV disease such as herpes stromal keratitis, a leading cause of infectious blindness.IMPORTANCE Herpes simplex virus (HSV) establishes a lifelong infection and remains dormant (latent) in our nerve cells. Occasionally HSV reactivates to cause disease, with HSV-1 typically causing cold sores whereas HSV-2 is the most common cause of genital herpes. The details of how HSV reactivates are largely unknown. Most of HSV's genes are silent during latency, with the exception of RNAs made from the latency-associated transcript (LAT) region. While viruses that make less LAT do not reactivate efficiently, these viruses also do not establish latency as efficiently. Here we deliver a ribozyme that can degrade the LAT to the nerve cells of latently infected rabbits using a gene therapy vector. We show that this treatment blocks reactivation in the majority of the rabbits. This work shows that the LAT RNA is important for reactivation and suggests the potential of this treatment as a therapy for treating HSV infections.

Dual-Phase Iontophoresis for the Delivery of Antisense Oligonucleotides.

In support of ongoing research in the study of corneal and skin wound healing, we sought to improve on previously published results by using iontophoresis to deliver RNA interference-based oligonucleotides. By using a electromechanics-based approach, we were able to devise a two-phase solution that separated the buffering solution from the antisense oligonucleotide (ASO) solution. The separation was obtained by making the drug solution a higher density than the buffer, leading it to sink directly onto the tissue surface. This change immediately decreased the distance that the ASO would have to travel before delivery. The changes enabled delivery into ex vivo skin and corneas in 10 or fewer minutes and into in vivo corneas in 5 min. In vivo studies demonstrated short-term bioavailability of at least 24 h, a lack of chemical or thermal injury, a lack of interference in the healing of a corneal injury, and an antisense effect till at least day 7, but not day 14. The only side-effect observed was postdelivery edema that was not present when the vehicle alone was iontophoresed. This suggests that electro-osmotic flow from the delivery chamber was not the mechanism, but that the delivered solute likely increased the tissue's osmolarity. These results support the continued development and utilization of this ASO delivery approach in research-grade oligonucleotides to probe molecular biological pathways and in support of testing therapeutic ASOs in the skin and cornea.

Adeno-associated Virus Vectors Efficiently Transduce Mouse and Rabbit Sensory Neurons Coinfected with Herpes Simplex Virus 1 following Peripheral Inoculation.

UNLABELLED: Following infection of epithelial tissues, herpes simplex virus 1 (HSV-1) virions travel via axonal transport to sensory ganglia and establish a lifelong latent infection within neurons. Recent studies have revealed that, following intraganglionic or intrathecal injection, recombinant adeno-associated virus (rAAV) vectors can also infect sensory neurons and are capable of stable, long-term transgene expression. We sought to determine if application of rAAV to peripheral nerve termini at the epithelial surface would allow rAAV to traffic to sensory ganglia in a manner similar to that seen with HSV. We hypothesized that footpad or ocular inoculation with rAAV8 would result in transduction of dorsal root ganglia (DRG) or trigeminal ganglia (TG), respectively. To test this, we inoculated the footpads of mice with various amounts of rAAV as well as rAAV capsid mutants. We demonstrated that this method of inoculation can achieve a transduction rate of >90% of the sensory neurons in the DRG that innervate the footpad. Similarly, we showed that corneal inoculation with rAAV vectors in the rabbit efficiently transduced >70% of the TG neurons in the optic tract. Finally, we demonstrated that coinfection of mouse footpads or rabbit eyes with rAAV vectors and HSV-1 resulted in colocalization in nearly all of the HSV-1-positive neurons. These results suggest that rAAV is a useful tool for the study of HSV-1 infection and may provide a means to deliver therapeutic cargos for the treatment of HSV infections or of dysfunctions of sensory ganglia. IMPORTANCE: Adeno-associated virus (AAV) has been shown to transduce dorsal root ganglion sensory neurons following direct intraganglionic sciatic nerve injection and intraperitoneal and intravenous injection as well as intrathecal injection. We sought to determine if rAAV vectors would be delivered to the same sensory neurons that herpes simplex virus (HSV-1) infects when applied peripherally at an epithelial surface that had been treated to expose the underlying sensory nerve termini. For this study, we chose two well-established HSV-1 infection models: mouse footpad infection and rabbit ocular infection. The results presented here provide the first description of AAV vectors transducing neurons following delivery at the skin/epithelium/eye. The ability of AAV to cotransduce HSV-1-infected neurons in both the mouse and the rabbit provides an opportunity to experimentally explore and disrupt host and viral proteins that are integral to the establishment of HSV-1 latency, to the maintenance of latency, and to reactivation from latency in vivo.

Brothers with Smith-Lemli-Opitz syndrome.

Abnormal cholesterol metabolism is the cause of SLOS, with low cholesterol levels and elevated levels of cholesterol precursors thought to contribute to the clinical findings in this syndrome. Management of SLOS involves early intervention with appropriate therapies for identified disabilities, genetic counseling for families, nutritional consultations, educational interventions, and behavioral management. Although no randomized dietary studies have been conducted, cholesterol supplementation continues to be a common recommendation for persons with SLOS, because it may result in clinical improvement and has few adverse effects (Nowaczyk, 2013). Even with early detection and treatment (e.g., sibling B in this case report), persons with SLOS often have significant behavioral issues and cognitive and developmental delays that require a team approach by parents, educators, specialists, and primary care providers.

The progression of haze formation in rabbit corneas following phototherapeutic keratectomy.

PURPOSE: To determine the topographical location and time course of development of corneal haze in a phototherapeutic keratectomy model using slit lamp examination, macrophotography, quantitative image analysis, and immunofluorescence staining of corneal sections. METHODS: Rabbit corneas were ablated with an excimer laser and were observed and graded for haze via slit lamp, imaged, and graded by macrophotography. Corneal sections were stained for α-smooth muscle actin (α-SMA) and tenascin-C (TNC). The distribution of haze imaged in the macrophotographs and density of α-SMA and TNC staining were compared. A daily image time course of haze formation was generated using macrophotography. RESULTS: The first signs of corneal haze were apparent shortly after reepithelialization. The haze was distributed as a ring at the wound margin in all cases, while nearly all corneas also had some central islands of haze initiation. With time, the haze spread within the ablated zone and intensified. The pattern of immunofluorescent staining for α-SMA and TNC at the wound margin mirrored the haze distribution, spread, and intensification with time. CONCLUSIONS: The initiation and spread of subepithelial haze begins shortly after reepithelialization. The haze then spreads from the loci of initiation and becomes more dense with time, maturing as early as 14 days after wounding. The improved temporal and spatial resolution provided by these data improve the current model of light-scattering haze formation in wounded corneas, which will improve the design of studies aimed at maintaining corneal clarity following acute injury or surgery.

Reactivation of herpes zoster keratitis in an adult after varicella zoster vaccination.

PURPOSE: In this study, the case of a patient who presented with reactivation of herpes zoster (HZ) keratitis and worsening of neurotrophic keratopathy, keratouveitis, and keratoconjunctivitis sicca after vaccination with live attenuated HZ vaccine (Zostavax) is described. METHOD: This is a retrospective case review. RESULTS: A 63-year-old man, with a history of HZ keratouveitis and neurotrophic keratopathy that had been quiescent for 3.5 years off medication, presented with keratouveitis 2 weeks after Zostavax administration. Oral acyclovir and topical prednisolone acetate and cyclopentolate were started, with subsequent improvement in inflammation and visual acuity. However, the patient was unable to be tapered completely off the steroids. CONCLUSIONS: HZ keratouveitis is the result of cell-mediated immunity (CMI) directed toward viral antigens within the eye. The live attenuated HZ vaccine, Zostavax, boosts the recipient's CMI to prevent reactivation of HZ. However, patients with a history of HZ keratitis may have persistent viral antigens in their corneas and can develop recurrence of keratouveitis because of the vaccine-induced increase in CMI. Vaccination should be undertaken with caution in patients with a history of HZ ophthalmicus.

Topical Corticosteroids in the Management of Bacterial Keratitis.

Bacterial keratitis can cause significant morbidity from ulceration of the cornea and the resultant scarring. The use of steroids to decrease these complications is controversial with arguments for and against their use. The SCUT (Steroids for Corneal Ulcers Trial) was initiated in 2006 to definitively determine whether steroids in bacterial keratitis were beneficial or harmful. While the SCUT showed no benefit or harm overall, subgroup analyses showed that larger, more central ulcers with very poor initial visual acuity may benefit. On the other hand, Nocardia ulcers that were treated with steroids had worse outcomes. The study did have some limitations as the patient population was not typical for bacterial keratitis in the United States, and there were some criticisms of the therapeutic approach so the question is still not definitively answered.

Proteolytic processing of connective tissue growth factor in normal ocular tissues and during corneal wound healing.

PURPOSE: Connective tissue growth factor (CTGF) is a fibrogenic cytokine that is up-regulated by TGF-ß and mediates most key fibrotic actions of TGF-ß, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. This study addresses the role of proteolytic processing of CTGF in human corneal fibroblasts (HCF) stimulated with TGF-ß, normal ocular tissues and wounded corneas. METHODS: Proteolytic processing of CTGF in HCF cultures, normal animal eyes, and excimer laser wounded rat corneas were examined by Western blot. The identity of a 21-kDa band was determined by tandem mass spectrometry, and possible alternative splice variants of CTGF were assessed by 5' Rapid Amplification of cDNA Ends (RACE). RESULTS: HCF stimulated by TGF-ß contained full length 38-kDa CTGF and fragments of 25, 21, 18, and 13 kDa, while conditioned medium contained full length 38- and a 21-kDa fragment of CTGF that contained the middle "hinge" region of CTGF. Fragmentation of recombinant CTGF incubated in HCF extracts was blocked by the aspartate protease inhibitor, pepstatin. Normal mouse, rat, and rabbit whole eyes and rabbit ocular tissues contained abundant amounts of C-terminal 25- and 21-kDa fragments and trace amounts of 38-kDa CTGF, although no alternative transcripts were detected. All forms of CTGF (38, 25, and 21 kDa) were detected during healing of excimer ablated rat corneas, peaking on day 11. CONCLUSIONS: Proteolytic processing of 38-kDa CTGF occurs during corneal wound healing, which may have important implications in regulation of corneal scar formation.

A connective tissue growth factor signaling receptor in corneal fibroblasts.

PURPOSE: To biochemically characterize the receptor for connective tissue growth factor (CTGF) of human corneal fibroblasts (HCF). METHODS: Radiolabeled recombinant human CTGF was used to determine the specificity and time course of binding to low-passage cultures of HCF. The affinity and number of receptors present were calculated by Scatchard and best-fit analyses. In vitro immunoprecipitation assays with radiolabeled CTGF and soluble mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF-2-R) alone, or with CTGF-related growth factors were conducted. Additionally, (125)I-CTGF-binding and CTGF-stimulated proliferation were measured in cultures of M6P/IGF-2-R knockout fibroblasts. RESULTS: Binding of (125)I-CTGF to fibroblast cultures was significantly displaced by CTGF, but not by related growth factors. Scatchard plot analysis indicated the presence of both a high-affinity, low-abundance binding site, and a low-affinity, high-abundance binding site; whereas, the best-fit analysis suggests a single high-affinity, low-abundance binding site. A 280 kDa complex containing cross-linked (125)I-CTGF was immunoprecipitated by antibodies to CTGF or M6P/IGF-2-R. M6P/IGF-2-R knockout cells have a reduced proliferative response to TGF-ß, and don't proliferate at all in response to CTGF. CONCLUSIONS: CTGF binds to the M6P/IGF-2-R with high affinity, and the M6P/IGF-2-R is required for CTGF-stimulated proliferation in fibroblasts. These observations suggest that the M6P/IGF-2-R may be a new antifibrotic target.