Abstract:
The eyes give us the ability to see, to interact with our environment and with other people being fundamental for an independent and social live. Vision loss leads to a great impairment of our live, including the loss of autonomy and self- determination. Generally, various neurodegenerative diseases have an impact on the optic nerve leading to damage and cell death of the RGC and their axons forming the optic nerve. This results in the deterioration of vision up to the complete vision loss. One of the most common causes of blindness worldwide is the glaucoma. Predominantly in elderly people an elevated intraocular pressure mechanically damages the axons of the RGC ending in RGC death. Unfortunately, due to the lack of apparent symptoms until advanced disease stages, treatment is often late and insufficient. Currently, the only possible treatment available is decreasing the intraocular pressure which can lead to a slowdown in diseases progress. Consequently, the treatment possibilities are very limited for the glaucoma and other neurodegenerative diseases resulting in vision impairment or blindness of the patient.
Currently, the pathophysiology of the optic nerve as well as treatment possibilities are studied in animal models predominantly using for instance the optic nerve crush model. Animal models have certain limitations compared to human models. A limitation of the animal model is that due to numerous differences between animals and humans predominantly in the biology and in genetics, the results obtained in animal models are hardly transferable to patients. As an example, treatments tested in mice are uncertain to work in humans the same way. This leads to the conclusion that human models of the optic nerve are necessary to overcome these limits and to obtain an increased comparability and significance of the results.
In this thesis, the main goal was to develop a new human in vitro model of the optic nerve using the assembloid technique. The created and characterized optic nerve assembloids (ONA) are based on hiPSC derived organoids: a retinal organoid represents the retina and a thalamic organoid mimicking the target areas of the retinothalamic fibers in the thalamus, both connected by a 3D matrigel based sphere containing astrocytes. This “nerve-like compartment” is formed by the RGC axonal projections which grow out from the RGC layer of the RO and pass through the 3D matrigel based sphere into the TO. Additionally, the RGC axonal projections are ensheathed by astrocytes and form synapses to the thalamic neurons. This leads to the conclusion that the development of the RGC and the outgrowth of the RGC axonal projections can be studied in the ONA model. On top of that, the ONA model can be produced patient derived in order to perform drug testing in the setting of individualized medicine.
Furthermore, the nerve crush model was established in the ONA model providing the opportunity to study the pathophysiology of neurodegenerative diseases of the optic nerve and the glaucoma. The pathological changes take place particularly in the unmyelinated part of the optic nerve which is represented by the ONA model. RGC death as well as astrocyte activation and the inflammatory response were analyzed after the optic nerve crush treatment. The immune system was represented through the integration of microglia cells into the 3D matrigel based spheres which allows to study the immune reaction after crush treatment.
In summary, results obtained in the ONA crush/cut model showed a significant increase in the expression of Caspase 3 and BRN3a double positive cells indicating the RGC loss particularly at D1 after cut treatment in immunostainings. Besides, in the RNA expression level analysis a trend towards downregulation of RGC markers like NEFM was found. In addition to that, astrocyte activation was shown via a significant increase of the RNA expression levels of various astrocyte markers like GFAP, S100b and SLC1A3 and a trend towards a higher expression of the astrocyte marker GFAP was visible in the immunostaining analysis. Furthermore, different inflammation markers like NFkB1, CD44 and APOE showed a significant increase in their expression levels after the crush/cut treatment.
As the summarized results from the ONA model are overall comparable to those described in optic nerve crush and glaucoma studies in animal models, the ONA can be used to reduce or replace the animals needed in this research field. Additionally, benefits from the ONA model compared to the animal model are the human biology and that the ONA model can be produced in a high number needing less space and care and is easier to handle. Despite the variability within the ONA batches this provides further advantages particularly for drug testing in the glaucoma and neurodegenerative optic neuropathy research field.
Assembloid