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bioRxiv2016

Differential Canalograms Detect Outflow Changes from Trabecular Micro-Bypass Stents and Ab Interno Trabeculectomy

The increasing prevalence of glaucoma, a leading cause of blindness, makes the development of safer and more effective treatment more urgent. Recently introduced microincisional glaucoma surgeries that enhance conventional outflow offer a favorable risk profile but can be unpredictable. Two paramount challenges are the lack of an adequate surgical training model for new surgeries and the absence of pre-or intraoperative guidance to sites of reduced flow. To address both, we developed an ex vivo training system and a differential, quantitative canalography method to assess outflow enhancement by trabecular micro-bypass (TMB) implantation or by ab interno trabeculectomy (AIT). TMB resulted in insignificant (p>0.05) outflow increases of 13{+/-}5%, 14{+/-}8%, 9{+/-}3%, and 24{+/-}9% in the inferonasal, superonasal, superotemporal, and inferotemporal quadrants. AIT caused a 100{+/-}50% (p=0.002), 75{+/-}28% (p=0.002), 19{+/-}8%, and 40{+/-}21% increase in those quadrants. AIT eyes had a 7.5 (p=0.01), 5.7 (p=0.004), 2.3, and 1.8-fold greater outflow enhancement than matching quadrants of paired TMB-implanted eyes. Quantitative canalography demonstrated that TMB, when successful, provided focal outflow enhancements, while AIT achieved a more extensive access to outflow pathways including and beyond the surgical site itself.

Physiology↗

bioRxiv2016

Gene Transfer to the Outflow Tract

Elevated intraocular pressure is the primary cause of open angle glaucoma. Outflow resistance exists within the trabecular meshwork but also at the level of Schlemms canal and further downstream within the outflow system. Viral vectors allow to take advantage of naturally evolved, highly efficient mechanisms of gene transfer, a process that is termed transduction. They can be produced at biosafety level 2 in the lab using protocols that have evolved considerably over the last 15 to 20 years. Applied by an intracameral bolus, vectors follow conventional as well as uveoscleral outflow pathways. They may affect other structures in the anterior chamber depending on their transduction kinetics which can vary among species when using the same vector. Not all vectors can express long-term, a desirable feature to address the chronicity of glaucoma. Vectors that integrate into the genome of the target cell can achieve transgene function for the life of the transduced cell but are mutagenic by definition. The most prominent long-term expressing vector systems are based on lentiviruses that are derived from HIV, FIV, or EIAV. Safety considerations make non-primate lentiviral vector systems easier to work with as they are not derived from human pathogens. Non-integrating vectors are subject to degradation and attritional dilution during cell division. Lentiviral vectors have to integrate in order to express while adeno-associated viral vectors (AAV) often persist as intracellular concatemers but may also integrate. Adeno-and herpes viral vectors do not integrate and earlier generation systems might be relatively immunogenic. Nonviral methods of gene transfer are termed transfection with few restrictions of transgene size and type but often a much less efficient gene transfer that is also short-lived. Traditional gene transfer delivers exons while some vectors (lentiviral, herpes and adenoviral) allow transfer of entire genes that include introns. Recent insights have highlighted the role of non-coding RNA, most prominently, siRNA, miRNA and lncRNA. SiRNA is highly specific, miRNA is less specific, while lncRNA uses highly complex mechanisms that involve secondary structures and intergenic, intronic, overlapping, antisense, and bidirectional location. Several promising preclinical studies have targeted the RhoA or the prostaglandin pathway or modified the extracellular matrix. TGF-{beta} and glaucoma myocilin mutants have been transduced to elevate the intraocular pressure in glaucoma models. Cell based therapies have started to show first promise. Past approaches have focused on the trabecular meshwork and the inner wall of Schlemms canal while new strategies are concerned with modification of outflow tract elements that are downstream of the trabecular meshwork.

Molecular Biology↗