Abstract
Purpose :
Tropomyosin receptor kinase B (TrkB) is an effective target for neuroprotection in experimental glaucoma models. Supplying agonist ligands, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4), by protein or gene therapy in glaucoma models has been shown to protect retinal ganglion cells (RGCs). However, long-term protection is hindered by TrkB down-regulation and truncated TrkB isoforms. Previous experiments have demonstrated when both TrkB and BDNF are expressed and given to the eye before optic nerve injury, RGC death is delayed. For this experiment, we are using a two-vector approach to confirm the necessity of both the receptor and ligand for the long-term neuroprotection of RGCs.
Methods :
Adeno-associated viruses (AAVs) were made by our Viral Vector Technologies AAV Production Core. For in vitro protein quantification, HEK-293 cells were transduced with AAVs. Supernatant and lysate were collected three days afterward for ELISAs. Adult C57BL/6J mice or adult Sprague Dawley rats were used. The eyes were intravitreally injected with AAV2 vectors expressing TrkB, proBDNF, NT4, or a combination of AAVs. Three weeks after AAV injection, optic nerve injury was performed. Two or four weeks after injury, animals were sacrificed, eyes were collected, retinas were dissected, immunofluorescently stained for Brn3a and flat-mounted to count the RGC number.
Results :
In vitro transduction of HEK-293 cells after 3 days of AAV treatment shows increased expression of TrkB, BDNF, or NT4. In vivo treatment of the combination of AAV2-TrkB with either AAV2-proBDNF or AAV2-NT4 significantly increases the survival of RGCs up to 78% two or four weeks after severe optic nerve injury compared to up to 46% in single AAV in both mice and rats.
Conclusions :
Intravitreal administration of the combination of separate vectors encoding AAV2-TrkB and either AAV2-BDNF or AAV2-NT4 shows a significant neuroprotective effect after optic nerve injury in mice and rats, greater than achieved with ligand- or receptor-providing vectors alone.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.