Ocular Delivery of Predatory Bacteria with Cryomicroneedles Against Eye Infection.

The development of potent antibiotic alternatives with rapid bactericidal properties is of great importance in addressing the current antibiotic crisis. One representative example is the topical delivery of predatory bacteria to treat ocular bacterial infections. However, there is a lack of suitable methods for the delivery of predatory bacteria into ocular tissue. This work introduces cryomicroneedles (cryoMN) for the ocular delivery of predatory Bdellovibrio bacteriovorus (B. bacteriovorus) bacteria. The cryoMN patches are prepared by freezing B. bacteriovorus containing a cryoprotectant medium in a microneedle template. The viability of B. bacteriovorus in cryoMNs remains above 80% as found in long-term storage studies, and they successfully impede the growth of gram-negative bacteria in vitro or in a rodent eye infection model. The infection is significantly relieved by nearly six times through 2.5 days of treatment without substantial effects on the cornea thickness and morphology. This approach represents the safe and efficient delivery of new class of antimicrobial armamentarium to otherwise impermeable ocular surface and opens up new avenues for the treatment of ocular surface disorders.

Ocular drug delivery targeted by iontophoresis in the suprachoroidal space using a microneedle.

Treatment of many posterior-segment ocular indications would benefit from improved targeting of drug delivery to the back of the eye. Here, we propose the use of iontophoresis to direct delivery of negatively charged nanoparticles through the suprachoroidal space (SCS) toward the posterior pole of the eye. Injection of nanoparticles into the SCS of the rabbit eye ex vivo without iontophoresis led to a nanoparticle distribution mostly localized at the site of injection near the limbus and <15% of nanoparticles delivered to the most posterior region of SCS (>9 mm from the limbus). Iontophoresis using a novel microneedle-based device increased posterior targeting with >30% of nanoparticles in the most posterior region of SCS. Posterior targeting increased with increasing iontophoresis current and increasing application time up to 3 min, but further increasing to 5 min was not better, probably due to the observed collapse of the SCS within 5 min after injection ex vivo. Reversing the direction of iontophoretic flow inhibited posterior targeting, with just ~5% of nanoparticles reaching the most posterior region of SCS. In the rabbit eye in vivo, iontophoresis at 0.14 mA for 3 min after injection of a 100 µL suspension of nanoparticles resulted in ~30% of nanoparticles delivered to the most posterior region of the SCS, which was consistent with ex vivo findings. The procedure was well tolerated, with only mild, transient tissue effects at the site of injection. We conclude that iontophoresis in the SCS using a microneedle has promise as a method to target ocular drug delivery within the eye, especially toward the posterior pole.

Hypertonic Saline Injection Model of Experimental Glaucoma in Rats.

A reliable method of creating chronic elevation of intraocular pressure (IOP) in rodents is an important tool in reproducing and studying the mechanisms of optic nerve injury that occur in glaucoma. In addition, such a model could provide a valuable method for testing potential neuroprotective treatments. This paper outlines the basic methods for producing obstruction of aqueous humor outflow and IOP elevation by injecting hypertonic saline (a sclerosant) into the aqueous outflow pathway. This is one of several rodent glaucoma models in use today. In this method, a plastic ring is placed around the equator of the eye to restrict injected saline to the limbus. By inserting a small glass microneedle in an aqueous outflow vein in the episclera and injecting hypertonic saline toward the limbus, the saline is forced into Schlemm's canal and across the trabecular meshwork. The resultant inflammation and scarring of the anterior chamber angle occurs gradually, resulting in a rise in IOP after approximately 1 week. This article will describe the equipment necessary for producing this model and the steps of the technique itself.

The Microbead Occlusion Model of Ocular Hypertension in Mice.

Glaucoma is a common optic neuropathy that leads to vision loss through the degeneration of retinal ganglion cells (RGCs) and their axons. RGC degeneration in glaucoma is associated with sensitivity to intraocular pressure (IOP) and elevated IOP (also known as ocular hypertension) is the primary modifiable risk factor. Ocular hypertension is the primary characteristic of rodent models for glaucoma research. Intracameral injection of microbeads has evolved as a preferred method of IOP elevation in rodents, particularly in mice. Here, we outline the protocol and method for the Microbead Occlusion Model in mice. We highlight the importance of anesthesia choice and the utilization of glass micropipettes in combination with a micromanipulator and microsyringe pump for the successful execution of the model.

Preliminary study of a controllable device for subtenon drug infusion in a rabbit model.

BACKGROUND AND OBJECTIVE: Conventional methods to treat intraocular diseases are invasive or associated with adverse effects. A minimally invasive means of sustained-release drug delivery to the vitreous is required. This study evaluated a novel device for subtenon drug delivery to the vitreous, relative to a single subconjunctival injection. METHODS: Sixty adult New Zealand White rabbits were randomly assigned to receive demethylvancomycin (DMV) by continuous subtenon delivery with the flow rate of 0.1 ml/hr for 24 hr, or as a single 0.3 ml subconjunctival injection in the right eyes. Rabbits were killed in subgroups of six at 1, 3, 6, 12 and 24 hr. The DMV concentration of the vitreous humour of the right eye was analysed by high-performance liquid chromatography. RESULTS: Overall, the vitreous DMV concentration of the subtenon group was significantly higher than that of the subconjunctival group (F = 25.928, p = 0.001). The DMV concentration of the subtenon group was also significantly higher than that of the subconjunctival group at 3, 6, 12 and 24 hr (t = 2.457, 5.064, 3.085, 4.207; p = 0.04, 0.01, 0.018, 0.004, respectively). In the subtenon group, the DMV concentration reached maximum (2.41 ± 0.67 µg/ml) at 6 hr, and at 24 hr was 2.37 ± 1.23 µg/ml. In the subconjunctival group, the DMV concentration reached maximum (0.48 ± 0.27 µg/ml) at 1 hr and declined to 0.09 ± 0.05 µg/ml at 24 hr. CONCLUSION: Subtenon application with this novel minimally invasive design is an effective method for delivering an appropriate drug to the vitreous in a sustained and controllable amount.

A minimally invasive adjustable-depth blunt injector for delivery of pharmaceuticals into the posterior pole.

PURPOSE: To investigate the feasibility and safety of a novel minimally invasive adjustable-depth blunt injector for pharmaceuticals delivery into the posterior segment. METHODS: Indocyanine green (ICG), sodium fluorescein and iron oxide nanoparticles (IONPs) were injected using the new injector into the extravascular spaces of the choroid (EVSC) compartment of rabbits and cadaver pig eyes. Spectral domain optical coherence tomography (SD-OCT), fundus imaging and histology analysis were performed for assessment of injection safety and efficacy. RESULTS: Indocyanine green, fluorescein and IONPs were detected across the EVSC in rabbit eyes, covering over 80 per cent of the posterior eye surface. Injected IONPs were retained in the EVSC for at least 2 weeks following injection. No retinal detachment, choroidal haemorrhage or inflammation was detected in any of the injected eyes. In cadaver pig eyes, ICG was detected across the EVSC. CONCLUSIONS: This novel minimally invasive delivery system may be used to safely deliver large volumes of pharmaceuticals into a new treatment reservoir compartment - the EVSC which can serve as a depot, in close proximity to the retina, covering most of the surface of the back of the eye without insertion of surgical instruments under the central retina. This system is predicted to enhance the therapeutic effect of treatments for posterior eye disorders.

Circumferential flow of particles in the suprachoroidal space is impeded by the posterior ciliary arteries.

Microneedle injection into the suprachoroidal space (SCS) enables targeted drug delivery for treatment of posterior segment diseases. This study sought to identify and characterize anatomical barriers to circumferential spread of particles in the SCS of rabbit and human cadaver eyes. These barriers could make targeting specific regions within the SCS challenging. A hollow microneedle (33-gauge, 750 µm long) was used to inject fluorescent particles into albino New Zealand White rabbit eyes ex vivo at six different positions around the limbus and a limited number of conditions in vivo. SCS injections were also performed in human cadaver eyes 8 mm and 2 mm from the optic nerve (ON). Eyes were dissected and particle distribution was quantified. In rabbit eyes, injections made in the superior or inferior hemispheres (even when injected temporally immediately adjacent to the long posterior ciliary artery (LPCA)) did not significantly cross into the other hemisphere, apparently due to a barrier formed by the LPCA. The vortex veins had a minor effect on particle deposition, limited to only around the vortex vein root. In human eyes, the short posterior ciliary arteries (SPCAs) prevented circumferential spread towards the macula and ON. In conclusion, the rabbit LPCA and the human SPCA were anatomical barriers to particle spread within the SCS. Therefore, design of drug delivery protocols targeting the SCS need to account for barriers formed by anatomical structures in order for injected drug to reach target tissues.

Tomografía de coherencia óptica de dominio espectral en la toxicidad retiniana por Celestone® / Spectral domain optical coherence tomography in Celestone® retinal toxicity

CASO CLÍNICO: Tras la administración intravítrea inadvertida de Celestone® (betametasona acetato y fosfato sódico, cloruro de benzalconio) en una paciente, la tomografía de coherencia óptica de dominio espectral (OCT-SD) mostraba hiperreflectividad, adelgazamiento e irregularidad en espículas en las capas internas de la mácula. Estos hallazgos tempranos serían el resultado del efecto tóxico directo del fármaco o sus excipientes. La alteración en la línea de los elipsoides y la aparición de espacios quísticos tabicados fueron hallazgos tardíos. DISCUSIÓN: La OCT-SD puede ayudar a conocer mejor la fisiopatología del daño retiniano y a diagnosticar las complicaciones asociadas, aportando información con valor pronóstico

CASE REPORT: After inadvertent intravitreal injection of Celestone® (betamethasone sodium phosphate and acetate, benzalkonium chloride) in a patient, the macular spectral domain optical coherence tomography (SD-OCT) images showed hyper-reflectivity, thinning, and irregular spikes in the inner retinal layers. These early findings could explain the toxic secondary anatomical changes due to the drug itself and/or to its excipients. Late and permanent SD-OCT findings included changes in the ellipsoid zone and cystic-like spaces. DISCUSSION: In this case, SD-OCT images can help to better understand the pathophysiology of the retinal damage and to diagnose the associated complications, providing information with prognostic value

Trans-Corneal Subretinal Injection in Mice and Its Effect on the Function and Morphology of the Retina.

PURPOSE: To introduce a practical method of subretinal injection in mice and evaluate injection-induced retinal detachment (RD) and damage using a dynamic imaging system, electrophysiology, and histology. METHODS: After full dilation of a 2-month-old C57BL/6J mouse pupil, the cornea near the limbus was punctured with a 30 ½-gague disposable beveled needle. A 33 ½-gauge blunt needle was inserted through the corneal perforation into the anterior chamber, avoiding the lens before going deeper into the vitreous cavity, and penetrating the inner retina to reach the subretinal space. The mice were divided into four groups: in group 1, about 80-100% of the retina was filled with subretinally injected solution; in group 2, approximately 50-70% of the retina was filled with injected solution; in group 3, the procedures were stopped before solution injection; and non-injected eyes were used as the negative control in group 4. An optical coherence tomography (OCT) imaging system was used to monitor retinal reattachment during the first three days following the injections. Histological and functional changes were examined by light microscopy and electroretinography (ERG) at five weeks post-injection. RESULTS: After a short-term training, a 70% success rate with 50% or more coverage (i.e., retinal blebs occupied 50% or more retinal area and filled with the injected solution) with minimal injection-related damages can be achieved. Bleb formation was associated with retinal detachment (RD) between the neuroretina and the retinal pigment epithelium (RPE) layer. Partial RD could be observed at post-injection day 1, and by day 2 most of the retina had reattached. At 5 weeks post-injection, compared to uninjected control group 4, the b-wave amplitudes of ERG decreased 22% in group 1, 16% in group 2, and 7% in group 3; the b-wave amplitudes were statistically different between the uninjected group and the groups with either 50-70% or 80-100% coverage. The subretinal injection-induced RD reattached and became stable at five weeks post-injection, although some photoreceptor damage could still be observed in and around the injection sites, especially in 80-100% coverage group. CONCLUSIONS: Trans-corneal subretinal injection is effective and practical, although subretinal injection-related damages can cause some morphological and functional loss.

Optimizing gene transfer to conventional outflow cells in living mouse eyes.

The mouse eye has physiological and genetic advantages to study conventional outflow function. However, its small size and shallow anterior chamber presents technical challenges to efficient intracameral delivery of genetic material to conventional outflow cells. The goal of this study was to optimize methods to overcome this technical hurdle, without damaging ocular structures or compromising outflow function. Gene targeting was monitored by immunofluorescence microscopy after transduction of adenovirus encoding green fluorescent protein driven by a CMV promoter. Guided by a micromanipulator and stereomicroscope, virus was delivered intracamerally to anesthetized mice by bolus injection using a 33 gauge needle attached to Hamilton syringe or infusion with glass micropipette connected to syringe pump. The total number of particles introduced remained constant, while volume of injected virus solution (3-10 µl) was varied for each method and time of infusion (3-40 min) tested. Outflow facility and intraocular pressure were monitored invasively using established techniques. Unlike bolus injections or slow infusions, introduction of virus intracamerally during rapid infusions (3 min) at any volume tested preferentially targeted trabecular meshwork and Schlemm's canal cells, with minimal transduction of neighboring cells. While infusions resulted in transient intraocular pressure spikes (commensurate with volume infused, Δ40-70 mmHg), eyes typically recovered within 60 min. Transduced eyes displayed normal outflow facility and tissue morphology 3-6 days after infusions. Taken together, fast infusion of virus solution in small volumes intracamerally is a novel and effective method to selectively deliver agents to conventional outflow cells in living mice.

PDMS embedded microneedles as a controlled release system for the eye.

PURPOSE: To demonstrate intraocular drug delivery using a novel device fabricated by embedding hollow glass microneedles within a soft and flexible poly (dimethylsiloxane) (PDMS) substrate for ease of device insertion into the eye. METHODS: Hollow glass microneedles (5 µm ID tips), fabricated using standard glass drawing techniques, were assembled into a photolithographically micropatterned PDMS substrate. The microneedles were fluidically coupled to a drug reservoir through a 300 µm microchannel to test for in vitro release of 6-aminoquinolone (144 Da) and Rose Bengal (1044 Da). Intravitreal delivery in ex vivo bovine eyes was also studied. RESULTS: The microneedles penetrated UV-crosslinked collagen and excised bovine sclera without breaking or delaminating from the PDMS matrix. A total of 45 ng of 6-aminoquinolone and 16 µg of Rose Bengal was released into buffered saline over a 20-min infusion at a delivery rate of 50 µL/min. Microinjection of Rose Bengal for 8 h into ex vivo bovine vitreous resulted in a total mass accumulation of 0.0202 mg into both phases of the vitreous humor and to the uveal face of the sclera without clogging of the internal needle microchannel. CONCLUSIONS: PDMS-embedded microneedles offer an integrated method of drug targeting to the intraocular tissues using a less invasive and less painful approach when compared with macroscale hypodermic needles. The release rates from the microneedles were controllable on demand using a syringe pump and were independent of the properties of the drugs tested. The device demonstrated a new hybrid approach of coupling rigid microneedles strong enough to penetrate the tough, fibrous sclera with a soft and pliable PDMS substrate that could conform to the contours of the eye.

Optimized technique for subretinal injections in mice.

Subretinal injections in mice become increasingly important. Currently, the most prominent application is in gene therapy of inherited eye diseases by means of viral vector delivery to photoreceptors or the retinal pigment epithelium (RPE). Since there are no large animal models for most of these diseases, genetically modified mouse models are commonly used in preclinical proof-of-concept studies. However, because of the relatively small mouse eye, adverse effects of the subretinal delivery procedure itself may interfere with the therapeutic outcome. The protocol described here concerns a transscleral pars plana subretinal injection in small eyes, and may be used for but not limited to virus-mediated gene transfer.

Development of a Lorentz-force actuated intravitreal jet injector.

Intravitreal injection is a common treatment in ophthalmology, but it can lead to numerous complications. Needle-free jet injection has been shown to successfully deliver fluid to various layers of skin, and, by its nature, may reduce intravitreal injection complications. From injection trials into ex vivo rabbit eyes, we find that needle-free jet injection can be used for intravitreal drug delivery. A custom-designed control scheme, characterized in this study, is crucial to this delivery. The system is capable of delivering 40 µL of fluid to the posterior vitreous humor, with an injection duration less than 100 ms and scleral entry site less than 350 µm in diameter.

Use of triamcinolone to judge needle depth in big-bubble deep anterior lamellar keratoplasty.

PURPOSE: To report a technique for judging needle depth in the corneal stroma during big-bubble deep anterior lamellar keratoplasty (DALK). METHODS: With the anterior chamber full of air, a dilute suspension of washed triamcinolone crystals is injected to "wash" over the air bubble. Some crystals are trapped between air and endothelium, making the posterior limit of the cornea easier to identify. The needle is then inserted into deep stroma, and big-bubble DALK is performed in the usual manner. RESULTS: This technique was used in 10 keratoconic eyes undergoing big-bubble DALK. No adverse effects of triamcinolone use were found in any eyes. CONCLUSIONS: This technique provides another option for surgeons who wish to accurately judge needle depth during big-bubble DALK.

Severe aseptic orbital cellulitis with subtenon carboplatin for intraocular retinoblastoma.

Retinoblastoma is a rare intraocular tumor of childhood. Chemoreduction followed by laser or cryotherapy is the treatment of choice. Subtenon carboplatin injection is also an accepted treatment modality for vitreous seeds, along with systemic chemotherapy. Transient periocular edema, optic neuropathy and fibrosis of orbital tissues are the known side effects of subteneon carboplatin injection. We report a case of severe aseptic orbital cellulitis with necrosis and prolapse of the conjunctiva 48 h after the injection, which resolved well on only conservative management.

Injectability of silicone oil-based tamponade agents.

BACKGROUND/AIMS: High viscosity silicone oils are used as tamponade agents to increase the resistance to emulsification; however, this makes the oils more difficult to inject. Increasing the extensional viscosity is one way to reduce emulsification. This study aimed to evaluate how silicone oils with increased extensional viscosity behave in terms of their ease of injection. METHODS: The shear viscosity and the length of time taken to inject 9 ml of Siluron 1000, Siluron 2000, Siluron 5000, SiliconMate, a 56/44 w/w blend of Siluron 1000/Siluron 5000 (Blend A) and a 90/10 w/w blend of Siluron 1000/PDMS 423kDa molecular weight (Blend B) were examined. RESULTS: The shear viscosity of Siluron 1000, Siluron 2000 and Siluron 5000 were within the expected ranges. The shear viscosity of Blend A was 2283 mPa s, Blend B was 4710 mPa s and SiliconMate was 995.3 mPa s. Siluron 1000 and SiliconMate had the shortest injection times as expected due to their lower shear viscosities. Comparison of Siluron 2000 and Blend A demonstrated that Siluron 2000 was easier to inject. Similarly, Blend B was easier to inject than Siluron 5000. CONCLUSION: Silicone oil blends containing small percentages of a high molecular weight additive are easier to inject than single grade oils of the equivalent shear viscosity.