Milestone II: “Identify where and how we will conduct live subject studies in which we will test the three components of EyeCell at the same time. We have already discussed this issue and have plans to conduct the studies with the help of Bryan Jones at the Moran Eye Center.”
After successful completion of Milestone I, we are now prepared to move the next phase of our studies, which includes in vivo experiments with small animals. The primary objective of this phase (comprising Milestones II & III) is to study the effects of our technological platform in regenerating the retinal pigment epithelium (RPE) and potentially other tissue structures following induced injury to the retina. This phase is key to establishing the safety and efficacy of our technological platform while continuing on the path to realizing in-human studies.
We plan to conduct our experiments with the help of Dr. Bryan Jones and Dr. Mary-Elizabeth Hartnett of the Moran Eye Institute. They have already been very influential in helping us plan our study and will play an important role going forward as we conduct the study with their help and expertise. After consulting with each of them, we have determined that a mouse model will be the most effective model for examining retinal degeneration and consequent regeneration. As stated in our contract, we plan to experiment on 10 animals. We plan to obtain the mice with the help of Dr. Hartnett, the University of Utah and the Moran Eye Center, which is also where we plan to conduct the experiment.
Unfortunately, there is no perfect way to study human age-related macular degeneration (AMD) in animal models. Due to the complex nature of the disease and the lengthy time it takes to be developed, AMD is typically induced in animals with the use of pharmacologic agents or other means to help degrade the retina in a shorter amount of time. One common method of degrading the retina is laser induced choroidal neovascularization (CNV)1. This method uses a focused laser to create an injury in the animal’s RPE and Bruch’s membrane that leads to angiogenesis – a hallmark pathway typically seen in AMD. This is an especially effective AMD model in mice and can be used to examine molecular mechanisms and genetic manipulations (most relevant to our study as we seek to find the change in genetic expression levels of specific genes as the result of bioelectric stimulation).
Because we will be using animal subjects, we will need to gain study approval from IACUC and plan to care and use the animals according the University of Utah guidelines (Guide for the Care and Use of Laboratory Animals). We also seek to follow the recommendations outlined by the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Once IACUC approval and the animals have been obtained, we will begin our studies, which will be similar in protocol to Dr. Hartnett’s recent publication on her work with retinal inhibition of CCR3 in which laser induced CNV and real-time qPCR were used2.
The study subjects will be 10 mice (Mus musculus) in which AMD pathways will be produced via laser induced CNV, which produces specific sites of injury in the subjects’ retina by focusing a laser with harmful frequencies on the retina. We will follow the protocol for this procedure as outlined by Lambert et. al3 and under the direction of Dr. Hartnett. Unlike many other AMD-inducing methods, this method does not require a long waiting period as the pathways commonly found in AMD (thus serving as an accurate model of the disease) will typically be made manifest within 7-14 days.
After the waiting period is over, we will proceed with bioelectric stimulation of the mouse eyes. Stimulation will be administered 2-3 times per week and will occur for several weeks in order to maximize the effects of stimulation. We plan to implant a small electrode similar to a catheter lead into the mouse retina which we will then tunnel out through the distal side of the mouse as close to the neck as possible (or other location in which the mouse cannot bite or otherwise damage the lead). From there, we can easily and painlessly connect our bioelectric stimulator to the mouse when we need to administer the stimuli.
Following the bioelectric stimulation, the mice will be sacrificed and the eyes enucleated. Retina tissue will then be isolated and homogenized from which we will perform mRNA extraction. Following mRNA extraction, complimentary DNA (cDNA) will be synthesized and real-time qPCR will be performed to examine the specific changes in gene expression levels. This will be the most important data as it will reveal if and by how much our targeted genes were upregulated due to bioelectrical stimulation. A statistical analysis will be conducted and a thorough report written. We will seek to publish the results of our studies which will be beneficial to our company, Dr. Hartnett and Dr. Jones, the Moran Eye Center, and ultimately USTAR and the state of Utah.
- Shah RS, Soetikno BT, Lajko M, Fawzi AA. “A Mouse Model for Laser-induced Choroidal Neovascularization.” J Vis Exp. 2015 Dec. 27. 106:e53502.
- Wang H, Han X, Gambhir D, Becker S, Kunz E, Liu AJ, Hartnett ME. “Retinal Inhibition of CCR3 Induces Retinal Cell Death in a Murine Model of Choroidal Neovascularization.” PLoS One. 2016 Jun. 16. 11(6):e0157748.
- Lambert V, Lecomte J, Hansen S, et al. “Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice”. Nat Protoc. 2013 Nov. 8(11):2197-211.