Brian C. Gilger

Ophthalmic Drug Delivery Systems for the Treatment of Retinal Diseases: Basic Research to Clinical Applications

The basic science part of this article focuses on the anatomic barriers to the five major modes of ocular drug delivery: intraocular, periocular, hybrid, topical, and systemic. The second half is a review of the clinical and regulatory components of translational science.

CHARACTERIZATION OF T-LYMPHOCYTES IN THE ANTERIOR UVEA OF EYES WITH CHRONIC EQUINE RECURRENT UVEITIS

Equine recurrent uveitis (ERU), a chronic, recurrent inflammation primarily of the anterior uveal tract, is the most common cause of blindness in horses. Recently, T-lymphocytes have been found to be the most numerous cell type to infiltrate the anterior uveal of horses with ERU. In the present study, we characterized the T-lymphocyte population in the anterior uveal tract of eyes of horses with chronic ERU by evaluating the microscopic appearance (histopathologic features), the T-lymphocyte subsets, and the relative levels and amounts of T-lymphocyte cytokine mRNA in the anterior uvea. Seven inflamed eyes (from six horses with chronic ERU) and 5 normal eyes (from five horses with nonocular problems) were studied. After clinical examination, the eyes were removed, ocular fluids were aspirated, and anterior uveal tissues (iris and ciliary body) were processed for histologic and molecular (RNA isolation) analyses. Histologic examination by hematoxylin and eosin (H and E) staining and immunohistochemistry evaluating T-lymphocyte subsets (anti-CD4, CD8, CD5) were performed for each sample. RNA samples were analyzed for levels of messenger (m) RNA specific for interleukin (IL)-2, 4, and interferon-gamma (IFNgamma) by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). Eyes with ERU exhibited characteristic clinical signs, including corneal edema, aqueous flare, posterior synechia, corpora nigra degeneration, and cataract formation. Histologically, infiltration of the uveal tract with lymphocytes, plasma cells, and macrophages was most evident in the ciliary body and base of the iris. Loss of tissue structure (destruction) was most evident in the ciliary processes. Infiltrating lymphocytes were predominantly CD4+ T-cells (e.g. 48% CD4+ and 18% CD8+ in the ciliary body stroma), as determined by immunohistochemistry. Few inflammatory cells were observed in the normal eyes. The QRT-PCR results revealed increased transcription of IL-2 and IFNgamma and low IL-4 mRNA expression in eyes with chronic ERU compared to normal eyes, demonstrating a Thelper (Th) 1-like inflammatory response in eyes with ERU.

Treatment of Acute Posterior Uveitis in a Porcine Model by Injection of Triamcinolone Acetonide Into the Suprachoroidal Space Using Microneedles

PURPOSE To evaluate the effect of triamcinolone acetonide (TA) administered into the suprachoroidal space (SCS) using a microneedle and compare it with intravitreal (IVT) TA injections in a porcine model of acute posterior segment inflammation. MATERIALS An IVT injection of balanced salt solution (BSS) or lipopolysaccharide (LPS) was followed 24 hours later with an injection of 0.2 mg or 2.0 mg of TA into the SCS or IVT. The SCS was accessed using microneedles in a minimally invasive procedure. Ocular inflammatory scores and IOP measurements were collected daily, whereas electroretinography, optical coherence tomography, and wide-field ocular fundus photography was performed on -1, 0, and 3 days after treatment. Aqueous and vitreous humor cell counts and protein levels and histopathology were also compared. RESULTS Delivery of TA to the SCS using microneedles was simple, effective, and not associated with adverse effects or toxicity. SCS injection of low (0.2 mg) and high doses (2.0 mg) of TA was as effective in reducing acute inflammation in the ocular posterior segment as high-dose IVT injection. Low-dose SCS TA was also effective in reducing inflammation; however, low-dose IVT TA was not. CONCLUSIONS Results from this study suggest that 0.2 mg and 2.0 mg of SCS TA was as effective in reducing inflammation as 2.0 mg IVT TA injection in a model of acute posterior segment inflammation. There were no adverse effects, increased IOP, or evidence of procedural or drug toxicity following injection of TA into the SCS in porcine eyes.

Efficacy of Doxycycline, Azithromycin, or Trovafloxacin for Treatment of Experimental Rocky Mountain Spotted Fever in Dogs

ABSTRACT Dogs were experimentally inoculated with Rickettsia rickettsii (canine origin) in order to compare the efficacies of azithromycin and trovafloxacin to that of the current antibiotic standard, doxycycline, for the treatment of Rocky Mountain spotted fever. Clinicopathologic parameters, isolation of rickettsiae in tissue culture, and PCR amplification of rickettsial DNA were used to evaluate the response to therapy or duration of illness (untreated infection control group) in the four groups. Concentrations of the three antibiotics in plasma and blood cells were measured by high-performance liquid chromatography. Doxycycline and trovafloxacin treatments resulted in more-rapid defervescence, whereas all three antibiotics caused rapid improvement in attitudinal scores, blood platelet numbers, and the albumin/total-protein ratio. Based upon detection of retinal vascular lesions by fluorescein angiography, trovafloxacin and doxycycline substantially decreased rickettsia-induced vascular injury to the eye, whereas the number of ocular lesions in the azithromycin group did not differ from that in the infection control group. As assessed by tissue culture isolation, doxycycline resulted in the earliest apparent clearance of viable circulating rickettsiae; however, rickettsial DNA could still be detected in the blood of some dogs from all four groups on day 21 postinfection, despite our inability to isolate viable rickettsiae at that point. As administered in this study, trovafloxacin was as efficacious as doxycycline but azithromycin proved less efficacious, possibly due to the short duration of administration.

Long-term outcome after implantation of a suprachoroidal cyclosporine drug delivery device in horses with recurrent uveitis.

OBJECTIVE To determine the long-term efficacy, complications, and duration of effect of a cyclosporine (CsA) suprachoroidal implant (CSI) in horses with equine recurrent uveitis (ERU). METHODS Horses with ERU were treated with a 6-mm diameter, 25 mg, reservoir matrix CsA implant in the deep sclera adjacent to the suprachoroidal space. Horses with follow-up >1 year were examined for frequency of uveitis episodes, complications, and vision at last recheck. RESULTS Data from 151 eyes of 133 horses from the USA and Europe that had CsA devices implanted for ERU were reviewed. Follow-up time ranged from 13 to 85 months after surgery, with a mean and median follow-up time of 28.9 and 26.3 months, respectively. Overall, at last follow-up 78.8% of eyes were considered visual and the overall mean frequency of uveitis episodes after CSI was 0.09 ± SD 0.08 episodes per month. The most common complications leading to vision loss at last follow-up were persistent uveitis episodes (54%), glaucoma (22%), mature cataracts (16%), and retinal detachment (6%). Persistent uveitis episodes tended to be the highest cause of vision loss in horses with <24 months and >48 months of follow-up. CONCLUSIONS This study demonstrated the long-term maintenance of vision of horses with ERU implanted with a CSI. The increased vision loss related to uveitis episode of inflammation in eyes after the likely depletion of CsA from the CSI suggests that a repeat CSI may be required at or before 48 months after surgery.

Production of ELOVL4 transgenic pigs: a large animal model for Stargardt-like macular degeneration

Background Truncation mutations in the elongation of very long chain fatty acids-4 (AF277094, MIM #605512) (ELOVL4) gene cause Stargardt-like macular dystrophy type 3 (STGD3). Mice expressing truncated ELOVL4 develop rapid retinal degeneration, but are poor STGD3 models since mice lack a macula. Photoreceptor topography in the pig retina is more similar to that in humans as it includes the cone rich, macula-like area centralis. The authors generated transgenic pigs expressing human disease-causing ELOVL4 mutations to better model the pathobiology of this macular disease. Methods Pronuclear DNA microinjection and somatic cell nuclear transfer were used to produce transgenic pigs for two different ELOVL4 mutations: the 5 base pair deletion (5 bpdel) and the 270 stop mutation (Y270terEYFP). Retinal transgene expression, morphology and electrophysiology were examined. Results The authors obtained four lines of Y270terEYFP and one line of 5 bpdel transgenic animals. Direct fluorescence microscopy indicated that the Y270terEYFP protein is expressed in photoreceptors and mislocalised within the cell. Immunohistochemical examination of transgenic pigs showed photoreceptor loss and disorganised inner and outer segments. Electroretinography demonstrated diminished responses in both transgenic models. Conclusions These transgenic pigs provide unique animal models for examining macular degeneration and STGD3 pathogenesis.

Advancements in ocular drug delivery

This review covers both noninvasive and invasive ophthalmic drug delivery systems that can have application to therapy of veterinary ophthalmic diseases. Noninvasive approaches include gel technologies, permeation enhancement via pro-drug development, solubilization agents and nanoparticle technologies, iontophoresis, microneedles, drug-eluting contact lenses and eye misters, and microdroplets. More invasive systems include both eroding implants and noneroding technologies that encompass diffusion based systems, active pumps, intraocular lenses, suprachoroidal drug delivery, and episcleral reservoirs. In addition to addressing the physiologic challenges of achieving the necessary duration of delivery, tissue targeting and patient compliance, the commercial development factors of biocompatibility, sterilization, manufacturability and long-term stability will be discussed.

Role of bacteria in the pathogenesis of recurrent uveitis in horses from the southeastern United States

OBJECTIVE To determine the role of intraocular bacteria in the pathogenesis of equine recurrent uveitis (ERU) in horses from the southeastern United States by evaluating affected eyes of horses with ERU for bacterial DNA and intraocular production of antibodies against Leptospira spp. SAMPLE POPULATION Aqueous humor, vitreous humor, and serum samples of 24 clinically normal horses, 52 horses with ERU, and 17 horses with ocular inflammation not associated with ERU (ie, non-ERU inflammation). PROCEDURES Ribosomal RNA quantitative PCR (real-time PCR) assay was used to detect bacterial DNA in aqueous humor and vitreous humor from clinically normal horses (n = 12) and horses with chronic (> 3-month) ERU (28). Aqueous humor and serum were also evaluated for anti-Leptospira antibody titers from clinically normal horses (n = 12), horses with non-ERU inflammation (17), and horses with confirmed chronic ERU (24). RESULTS Bacterial DNA was not detected in aqueous humor or vitreous humor of horses with ERU or clinically normal horses. No significant difference was found in titers of anti-Leptospira antibodies in serum or aqueous humor among these 3 groups. Only 2 horses, 1 horse with ERU and 1 horse with non-ERU inflammation, had definitive intraocular production of antibodies against Leptospira organisms. CONCLUSIONS AND CLINICAL RELEVANCE In horses from the southeastern United States, Leptospira organisms may have helped initiate ERU in some, but the continued presence of the organisms did not play a direct role in the pathogenesis of this recurrent disease.

Porcine global flash multifocal electroretinogram: possible mechanisms for the glaucomatous changes in contrast response function.

PURPOSE The aim of this study was to obtain a better understanding of the cellular contributions to the porcine global flash mfERG by using a pharmacologic dissection method, together with the method using variation of stimulus contrast which has been used to demonstrate mfERG changes in human glaucoma. METHODS Global flash mfERGs with different stimulus-contrast settings (99%, 65%, 49% or 29%) were recorded from 14 eyes of ten 6-week-old Yorkshire pigs in control conditions and after suppression of inner retinal responses with inhalation of isoflurance (ISO), and injections of tetrodotoxin (TTX) and N-methyl-d-aspartic acid (NMDA). ON- and OFF-pathway responses were isolated by injection of 2-amino-4-phosphonobutyric acid (APB) and cis-2,3-piperidinedicarboylic acid (PDA). RESULTS The porcine global flash mfERG consisted of an early direct component (DC) and a late induced component (IC). ISO and TTX removed inner retinal contributions to the IC; NMDA application further abolished the oscillatory wavelets in the DC and removed the residual IC waveform. The inner retina contributed regular oscillation-like wavelets (W1, W2 and W3) to the DC and shaped the IC. After removing the inner retinal contributions, the porcine global flash mfERG waveform becomes comparable to that obtained with conventional mfERG stimulation. The remaining waveform (smoothed DC) was mainly contributed by the ON- and OFF-bipolar cells as revealed after APB or PDA injection. Photoreceptors contributed a small signal to the leading edge of N1. The characteristic of contrast response function of DC was demonstrated to be contributed by the inner retinal oscillation-like wavelets. CONCLUSION We believe that the DC of the porcine global flash mfERG is mainly composed of contributions from photoreceptors, and ON- and OFF-bipolar cells, where inner retinal activity partially shaped the DC with superimposed regular wavelets. However, the IC is dominated by inner retinal activity. The contrast response functions of DC consisted of both outer retinal response and oscillation-like wavelets of the inner retinal response. Both contain different characteristics during contrast modulation of the stimulus, where the changes of W2 of the inner retinal response seem independent of contrast modulation. The DC contrast response feature depends mainly on the relative contribution of inner retinal activities; the loss of inner retinal cells may alter the DC contrast response function, making it tend toward linearity.

Pharmacologically defined components of the normal porcine multifocal ERG

Multifocal electroretinograms (mfERG) from isoflurane anesthetized pigs were recorded and sequential application of TTX, NMDA, APB and PDA were used to identify contributions to the mfERG from inner retinal neurons, ON-pathway, OFF-pathway and photoreceptors. The cellular origins of the first-order kernel (K1) and the first slice of the second-order kernel (K2.1) porcine mfERG are contributed from both inner and outer retina. For the K1 waveform, the n1 involved responses of cone photoreceptors and OFF-bipolar cells. The leading edge of p1 is dominated by ON-bipolar cell depolarization. The rear edge of p1, n2 and p2 are dominated by ON-bipolar activities and shaped by the activities of OFF-bipolar cells and retinal cells with NMDAr and voltage-gated sodium channels other than ganglion cells. The p3 is mainly inner retinal activities. For the K2.1 waveform, the p1 and n1 are the summation of activities of ON-, OFF-bipolar cells and retinal cells rich in NMDAr and voltage-gated sodium channels other than ganglion cells. The p2 seems to be related to the ganglion cells. Better understanding of the cellular origins of the normal porcine mfERG will be useful for comparing and defining the functional changes that may occur in diseased retinas.