A Degradable Sustained-Release Drug Delivery System for Bleb-Forming Glaucoma Surgery.

Fibrosis of the filtering bleb is one of the main causes of failure after bleb-forming glaucoma surgery. Intraoperative application of mitomycin C (MMC) is the current gold standard to reduce the fibrotic response. However, MMC is cytotoxic and one-time application is often insufficient. A sustained-release drug delivery system (DDS), loaded with MMC, may be less cytotoxic and equally or more effective. Two degradable (polycaprolactone (PCL) and polylactic-co-glycolic acid (PLGA)) MMC-loaded DDSs are developed. Release kinetics are first assessed in vitro followed by rabbit implants in conjunction with the PRESERFLO MicroShunt. As a control, the MicroShunt is implanted with adjunctive use of a MMC solution. Rabbits are euthanized at postoperative day (POD) 28 and 90. The PLGA and PCL DDSs release (on average) 99% and 75% of MMC, respectively. All groups show functioning blebs until POD 90. Rabbits implanted with a DDS show more inflammation with avascular thin-walled blebs when compared to the control. However, collagen is more loosely arranged. The PLGA DDS shows less inflammation, less foreign body response (FBR), and more complete degradation at POD 90 when compared to the PCL DDS. Further optimization with regard to dosage is required to reduce side effects to the conjunctiva.

The Influence of Design Modifications and Microstructured Surface Topographies on Bleb Survival after Glaucoma Tube Shunt Implantation.

Lowering intraocular pressure (IOP) by placement of a glaucoma shunt is an effective treatment for glaucoma. However, fibrosis of the outflow site can hamper surgical outcome. In this study, the antifibrotic effect of adding an endplate (with or without microstructured surface topographies) to a microshunt made of poly(styrene-block-isobutylene-block-styrene) is investigated. New Zealand white rabbits are implanted with a control implant (without endplate) and modified implants. Afterward, bleb morphology and IOP is recorded for 30 days. After killing of the animals, eyes are collected for histology, Addition of an endplate extended bleb survival, Topography-990 has the longest recorded bleb-survival time. Histology reveals that the addition of an endplate increases the presence of myofibroblasts, macrophages, polymorphonuclear cells, and foreign body giant cells compared to the control. However, an increased capsule thickness and inflammatory response are observed in the groups with surface topographies, The addition of an endplate results in prolonged bleb survival, demonstrating that engineering of the shape of glaucoma implants could prolong bleb functionality. Future research should further elaborate the effect of surface topographies on long-term bleb survival, since an increased presence of pro-fibrotic cells and increased capsule thickness are observed compared to the control.

Wound Healing Response After Bleb-Forming Glaucoma Surgery With a SIBS Microshunt in Rabbits.

Purpose: The PreserFlo MicroShunt is an innovative implant for the surgical treatment of glaucoma. Although usually effective, surgeries can still fail due to fibrosis. This study was conducted to gain insight into the histological aspects of the fibrotic response and find potential targets to reduce postoperative fibrosis. Methods: Fifteen New Zealand White rabbits were implanted with a microshunt and followed up for 40 days. Animals were euthanized at postoperative days (PODs) 1, 5, and 40 to collect eyes for histological evaluation. Bleb formation and ocular health were assessed by slit-lamp (SL) biomicroscopy and optical coherence tomography (OCT). Intraocular pressure (IOP) was measured using rebound tonometry. Results: Blebs failed after approximately 2 weeks based on bleb survival and IOP measurements. No severe complications were observed with OCT and SL. Histology revealed a wide variety of cells, in the bleb and around the microshunt, including polymorphonuclear leucocytes (PMNs), myofibroblasts, and foreign body giant cells, at different PODs. Conclusions: Implantation of a poly(styrene-b-isobutylene-b-styrene) microshunt in rabbits resulted in the occurrence of a wide variety of cells during the wound-healing response. Future research should further elucidate the potential of these (earlier often overlooked) cells to target the fibrotic response in vivo-for example, by developing novel antifibrotic drugs, methods for sustained delivery of medications, or augmenting material properties. Translational Relevance: Current antifibrotic therapies aim to inhibit myofibroblasts; however, a wide variety of cells are involved in the fibrotic response. Future research focusing on these cells could offer novel methods for reducing the fibrotic response after glaucoma surgery.

Animal models and drug candidates for use in glaucoma filtration surgery: A systematic review.

Glaucoma, a degenerative disease of the optic nerve, is the leading cause of irreversible blindness worldwide. Currently, there is no curative treatment. The only proven treatment is lowering intraocular pressure (IOP), the most important risk factor. Glaucoma filtration surgery (GFS) can effectively lower IOP. However, approximately 10% of all surgeries fail yearly due to excessive wound healing, leading to fibrosis. GFS animal models are commonly used for the development of novel treatment modalities. The aim of the present review was to provide an overview of available animal models and anti-fibrotic drug candidates. MEDLINE and Embase were systematically searched. Manuscripts until September 1st, 2021 were included. Studies that used animal models of GFS were included in this review. Additionally, the snowball method was used to identify other publications which had not been identified through the systematic search. Two hundred articles were included in this manuscript. Small rodents (e.g. mice and rats) are often used to study the fibrotic response after GFS and to test drug candidates. Due to their larger eyes, rabbits are better suited to develop medical devices. Novel drugs aim to inhibit specific pathways, e.g. through the use of modulators, monoclonal antibodies, aqueous suppressants or gene therapy. Although most newly studied drugs offer a higher safety profile compared to antimetabolites, their efficacy is in most cases lower when compared to MMC. Current literature on animal models and potential drug candidates for GFS were summarized in this review. Future research should focus on refining current animal models (for example through the induction of glaucoma prior to undertaking GFS) and standardizing animal research to ensure a higher reproducibility and reliability across different research groups. Lastly, novel therapies need to be further optimized, e.g. by conducting more research on the dosage, administration route, application frequency, the option of creating combination therapies, or the development of drug delivery systems for sustained release of anti-fibrotic medication.

Repeatability, reproducibility, and agreement of three tonometers for measuring intraocular pressure in rabbits.

The aim of this study was to evaluate repeatability, reproducibility, and agreement of three commonly used tonometers in animal research (TonoLab, TonoVet, and TonoPEN AVIA) in a cohort of 24 rabbits. Additionally, the impact of sedation on IOP was investigated in 21 New Zealand White rabbits with the TonoVet tonometer. Repeatability was determined using the coefficient of variation (CoV) for two observers. For the TonoLab (6.55%) and TonoVet (6.38%) the CoV was lower than for the TonoPEN AVIA (10.88%). The reproducibility was highest for the TonoVet (0.2 ± 3.3 mmHg), followed by the TonoLab (0 ± 12.89 mmHg) and lowest for the TonoPEN AVIA (- 1.48 ± 10.3 mmHg). The TonoLab and TonoVet showed the highest agreement (r = 0.85, R2 = 0.73). After sedation, a significant IOP reduction (often > 25%) was observed. Our results show that among the three tonometers tested, the TonoVet tonometer is best for use in rabbits while the TonoLab should be avoided. The impact of sedation on IOP was substantial and should be taken into account during experimentation.

DNA damage in embryonic neural stem cell determines FTLDs' fate via early-stage neuronal necrosis.

The early-stage pathologies of frontotemporal lobal degeneration (FTLD) remain largely unknown. In VCPT262A-KI mice carrying VCP gene mutation linked to FTLD, insufficient DNA damage repair in neural stem/progenitor cells (NSCs) activated DNA-PK and CDK1 that disabled MCM3 essential for the G1/S cell cycle transition. Abnormal neural exit produced neurons carrying over unrepaired DNA damage and induced early-stage transcriptional repression-induced atypical cell death (TRIAD) necrosis accompanied by the specific markers pSer46-MARCKS and YAP. In utero gene therapy expressing normal VCP or non-phosphorylated mutant MCM3 rescued DNA damage, neuronal necrosis, cognitive function, and TDP43 aggregation in adult neurons of VCPT262A-KI mice, whereas similar therapy in adulthood was less effective. The similar early-stage neuronal necrosis was detected in PGRNR504X-KI, CHMP2BQ165X-KI, and TDPN267S-KI mice, and blocked by embryonic treatment with AAV-non-phospho-MCM3. Moreover, YAP-dependent necrosis occurred in neurons of human FTLD patients, and consistently pSer46-MARCKS was increased in cerebrospinal fluid (CSF) and serum of these patients. Collectively, developmental stress followed by early-stage neuronal necrosis is a potential target for therapeutics and one of the earliest general biomarkers for FTLD.

Pharmacokinetics and efficacy of a ketorolac-loaded ocular coil in New Zealand white rabbits.

Eye drops are considered standard practice for the delivery of ocular drugs. However, low patient compliance and low drug levels compromise its effectiveness. Our group developed a ketorolac-loaded ocular coil for sustained drug delivery up to 28 days. The aim of this study was to gain insight into the pharmacokinetics and efficacy of the ocular coil. The pharmacokinetics of the ketorolac-loaded ocular coil versus eye drops were tested in New Zealand White rabbits by repetitive sampling for 28 days. Efficacy of the ocular coil was also tested in New Zealand White rabbits. Ocular inflammation was induced where after the ocular coil was inserted, or eye drops, or no treatment was provided. The total protein concentration and cytokine levels were measured in tears, aqueous humor, and plasma at 4 h, 8 h, 24 h, 4 d, 7 d, 14 d, 21 d, and 28 d. Four h after inserting the ocular coil in the eye, ketorolac levels in aqueous humor and plasma were higher in the ocular coil group than in the eye drop group. Ketorolac released from the ocular coil could be detected up to 28 d in tears, up to 4 d in aqueous humor and up to 24 h in plasma. After inducing inflammation, both the ocular coil and eye drops were able to suppress prostaglandin E2, TNFα and IL-6 levels in aqueous humor and plasma as compared to the group that received no treatment. To conclude, the ocular coil facilitated a sustained release of the drug and showed similar therapeutic benefit in suppressing post-operative inflammation as eye drops.

Pipeline for the removal of hardware related artifacts and background noise for Raman spectroscopy.

Raman spectroscopy is a real-time, non-contact, and non-destructive technique able to obtain information about the composition of materials, chemicals, and mixtures. It uses the energy transfer properties of molecules to detect the composition of matter. Raman spectroscopy is mainly used in the chemical field because background fluorescence and instrumental noise affect biological (in vitro and in vivo) measurements. In this method, we describe how hardware related artifacts and fluorescence background can be corrected without affecting signal of the measurement. First, we applied manual correction for cosmic ray spikes, followed by automated correction to reduce fluorescence and hardware related artifacts based on a partial 5th degree polynomial fitting and Tophat correction. Along with this manuscript we provide a MatLabⓇ script for the automated correction of Raman spectra.•"Polynomial_Tophat_background_subtraction _methods.m" offers an automated method for the removal of hardware related artifacts and fluorescence signals in Raman spectra.•"Polynomial_Tophat_background_subtraction _methods.m" provides a modifiable MatLab file adjustable for multipurpose spectroscopy analysis.•We offer a standardized method for Raman spectra processing suitable for biological and chemical applications for modular confocal Raman spectroscopes.

Design of the ocular coil, a new device for non-invasive drug delivery.

Eye drops and ointments are the most prescribed methods for ocular drug delivery. However, due to low drug bioavailability, rapid drug elimination, and low patient compliance there is a need for improved ophthalmic drug delivery systems. This study provides insights into the design of a new drug delivery device that consists of an ocular coil filled with ketorolac loaded PMMA microspheres. Nine different ocular coils were created, ranging in wire diameter and coiled outer diameter. Based on its microsphere holding capacity and flexibility, one type of ocular coil was selected and used for further experiments. No escape of microspheres was observed after bending the ocular coil at curvature which reflect the in vivo situation in human upon positioning in the lower conjunctival sac. Shape behavior and tissue contact were investigated by computed tomography imaging after inserting the ocular coil in the lower conjunctival fornix of a human cadaver. Thanks to its high flexibility, the ocular coil bends along the circumference of the eye. Because of its location deep in the fornix, it appears unlikely that in vivo, the ocular coil will interfere with eye movements. In vitro drug release experiments demonstrate the potential of the ocular coil as sustained drug delivery device for the eye. We developed PMMA microspheres with a 26.5 ± 0.3 wt% ketorolac encapsulation efficiency. After 28 days, 69.9% ± 5.6% of the loaded ketorolac was released from the ocular coil when tested in an in vitro lacrimal system. In the first three days high released dose (48.7% ± 5.4%) was observed, followed by a more gradually release of ketorolac. Hence, the ocular coil seems a promising carrier for ophthalmic drugs delivery in the early postoperative time period.

Safety and Comfort of an Innovative Drug Delivery Device in Healthy Subjects.

Purpose: The aim of this study was to investigate safety and comfort of two versions of a placebo-microsphere filled ocular coil (straight and curved) in healthy subjects. Methods: The study was a single-center intervention study. One ocular coil was placed in the inferior conjunctival fornix for the intended duration of 28 days. Forty-two healthy adult subjects were included. At baseline, 30 minutes, 8 hours, 24 hours, 48 hours, 7 days, 14 days, 21 days, and 28 days after insertion, examinations were performed, including slit lamp evaluation to score ocular redness, intraocular pressure measurement, visual acuity, tear secretion test, and questionnaires. Results: The straight and curved ocular coils had a median retention time of 5 days and 12 days, respectively. After 48 hours, 57% and 81% subjects retained the straight and curved ocular coil, respectively. Four (19%) subjects with the straight coil and six (29%) with the curved coil completed the entire study period. Minor changes in ocular hyperemia were observed in both groups. On day 7, the straight coil was more comfortable than the curved coil with a visual analogue scale (VAS) score of 77 ± 21 compared to 94 ± 11 (P = 0.028), respectively. No other ocular adverse events were observed. Conclusions: Comfort and safety of the straight and curved ocular coil are high. Because the retention time is too short for long-term sustained drug release, the use in the perioperative or immediate postoperative period could prove to be more valuable. Translational Relevance: The ocular coil is a noninvasive, comfortable and safe short-term drug delivery device.

Validation of Computerized Quantification of Ocular Redness.

PURPOSE: To show feasibility of computerized techniques for ocular redness quantification in clinical studies, and to propose an automatic, objective method. METHODS: Software for quantification of redness of the bulbar conjunctiva was developed. It provides an interface for manual and automatic sclera segmentation along with automated alignment of region of interest to enable estimation of changes in redness. The software also includes the redness scoring methods: (1) contrast-limited adaptive histogram equalization (CLAHE) in red-green-blue (RGB) color model, (2) product of saturation and hue in hue-saturation-value (HSV), and (3) average of angular sections in HSV. Our validation pipeline compares the scoring outcomes from the perspectives of segmentation reliability, segmentation precision, segmentation automation, and the choice of redness scoring methods. RESULTS: Ninety-two photographs of eyes before and after provoked redness were evaluated. Redness in manually segmented images was significantly different within human observers (interobserver, P = 0.04) and two scoring sessions (intraobserver, P < 0.001). Automated segmentation showed the smallest variability, and can therefore be seen as a robust segmentation method. The RGB-based scoring method was less sensitive in redness assessment. CONCLUSIONS: Computation of ocular redness depends heavily on sclera segmentation. Manual segmentation appears to be subjective, resulting in systematic errors in intraobserver and interobserver settings. At the same time, automatic segmentation seems to be consistent. The scoring methods relying on HSV color space appeared to be more consistent. TRANSLATIONAL RELEVANCE: Computerized quantification of ocular redness holds great promise to objectify ocular redness in the standard clinical care and, in particular, in clinical trials.

In vitro and in vivo datasets of topically applied ketorolac tromethamine in aqueous humor using Raman spectroscopy.

This article includes datasets acquired by Raman spectroscopy from in vivo and in vitro ocular samples collected from the dataset from Bertens and Zhang et al., "Confocal Raman spectroscopy: Evaluation of a non-invasive technique for the detection of topically applied ketorolac tromethamine in vitro and in vivo" (Bertens and Zhang, et al.). Detection of ketorolac tromethamine in pig eyes was performed in vitro and rabbit eyes in vivo. Extracted aqueous humor samples from pig and rabbit eyes were measured in vitro using a cuvette. This manuscript shows the spectral Raman data without pre-treatment or analysis from ocular tissues and provides further information towards aqueous humor research via alternative data processing methods. Furthermore, the raw data enclosed may be used for future aqueous humor investigations and pharmaceutical research.

Confocal Raman spectroscopy: Evaluation of a non-invasive technique for the detection of topically applied ketorolac tromethamine in vitro and in vivo.

Current information about the pharmacokinetics of an ocular drug can only be achieved by invasive sampling. However, confocal Raman spectroscopy bears the potential to quantify drug concentrations non-invasively. In this project, we evaluated the detection and quantification of ocular ketorolac tromethamine levels with confocal Raman spectroscopy after topical administration. Confocal Raman spectroscopy and high-performance liquid chromatography (HPLC) were compared in terms of sensitivity of detection. Enucleated pig eyes were treated with different concentrations of ketorolac. Hereafter, ketorolac concentrations in the aqueous humor of pig eyes were analyzed by confocal Raman spectroscopy and HPLC. Subsequently, twelve rabbits were treated with Acular™ for four weeks. At several time points, ketorolac concentrations in aqueous humor of the rabbits were measured by confocal Raman spectroscopy followed by drawing an aqueous humor sample for HPLC analysis. In ketorolac treated pig eyes, both ex vivo Raman spectroscopy as well as HPLC were able to detect ketorolac in a broad concentration range. However, in vivo confocal Raman spectroscopy in rabbits was unable to detect ketorolac in contrast to HPLC. To conclude, confocal Raman spectroscopy has the capacity to detect ketorolac tromethamine in vitro, but currently lacks sensitivity for in vivo detection.

Topical drug delivery devices: A review.

For the treatment and prevention of ocular diseases, most patients are treated with conventional drug delivery formulations such as eye drops or ointments. However, eye drops and ointments suffer from low patient compliance and low effective drug concentration at the target site. Therefore, new medical devices are being explored to improve drug delivery to the eye. Over the years, various delivery devices have been developed including resorbable devices, oval- and ring-shaped devices, rod-shaped devices, punctum plugs, contact lenses and corneal shields. Only a few devices (eg. Mydriasert®, Ozurdex®, Surodex®, Iluvien®, Lacrisert® and Retisert®) have made it to the market while others are being investigated in clinical trials. Altogether, there is a need for enhanced topical drug delivery. Only by working together (academia, industry and authorities) and by exploring parallel strategies (new drug delivery devices, enhanced drug formulations, better understanding of the pharmacokinetic properties), the therapeutic effect of drug treatments can be improved.

FcRγ-chain ITAM signaling is critically required for cross-presentation of soluble antibody-antigen complexes by dendritic cells.

The uptake of Ag-Ab immune complexes (IC) after the ligation of activating FcγR on dendritic cells (DC) leads to 100 times more efficient Ag presentation than the uptake of free Ags. FcγRs were reported to facilitate IC uptake and simultaneously induce cellular activation that drives DC maturation and mediates efficient T cell activation. Activating FcγRs elicit intracellular signaling via the ITAM domain of the associated FcRγ-chain. Studies with FcRγ-chain knockout (FcRγ(-/-)) mice reported FcRγ-chain ITAM signaling to be responsible for enhancing both IC uptake and DC maturation. However, FcRγ-chain is also required for surface expression of activating FcγRs, hampering the dissection of ITAM-dependent and independent FcγR functions in FcRγ(-/-) DCs. In this work, we studied the role of FcRγ-chain ITAM signaling using DCs from NOTAM mice that express normal surface levels of activating FcγR, but lack functional ITAM signaling. IC uptake by bone marrow-derived NOTAM DCs was reduced compared with wild-type DCs, but was not completely absent as in FcRγ(-/-) DCs. In NOTAM DCs, despite the uptake of ICs, both MHC class I and MHC class II Ag presentation was completely abrogated similar to FcRγ(-/-) DCs. Secretion of cytokines, upregulation of costimulatory molecules, and Ag degradation were abrogated in NOTAM DCs in response to FcγR ligation. Cross-presentation using splenic NOTAM DCs and prolonged incubation with OVA-IC was also abrogated. Interestingly, in this setup, proliferation of CD4(+) OT-II cells was induced by NOTAM DCs. We conclude that FcRγ-chain ITAM signaling facilitates IC uptake and is essentially required for cross-presentation, but not for MHC class II Ag presentation.