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Alexander D.M. Kokini, Krishnakumar Kizhatil, Simon John; Determining Neuronal Elements of IOP Regulation Using Mice. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1600. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The nervous system is important in controlling intraocular pressure (IOP), however the precise mechanisms of neural control need further evaluation. The mouse enables functional experiments and so we are characterizing limbal innervation in mice. To understand neuronal functions regulating IOP, we are using modern molecular techniques and fluorescent reporter mice to map limbal neurons and determine pressure dependent neuronal activity.
We used a whole-mount procedure of the anterior mouse eye to study the innervation of the entire limbus in 3D. The Prox1-GFP mouse strain was used to label Schlemm’s canal (SC). The Thy1-YFP strain was used to identify sensory neurons. Antibodies to detect sympathetic (tyrosine hydroxylase), parasympathetic (vesicular acetylcholine transporter, choline transporter), nitrergic (neuronal nitric oxide synthase), and axons (neurofilament) have been used. 3D limbal images generated using confocal microscopy and Imaris were used to map and identify the number and type of neurons and nerve termini in SC and trabecular meshwork (TM). Neuronal activity in response to pressure elevation was assessed using fluorescent neuronal activation sensors (GCaMP). To do this, eyes were cannulated and held at pressures of 13 and 33 mmHg for 30 minutes followed by fixation, immunolabeling and confocal microscopy.
We have identified sympathetic nerve terminals and possibly non-nociceptive sensory terminals terminating in SC and TM. The TH labeled termini have varicosities and the YFP labeled termini appear as bouton like structures. Using GCaMP calcium sensor mice, we have identified neurons that are activated in the limbus in response to elevated pressure. These neurons are present in the SC and associated TM region.
We are constructing a detailed 3-dimensional (3D) map of the neuronal innervation of the AQH drainage structures to facilitate understanding of the neural control of IOP. Both SC and TM are highly innervated with activation of a subset of neurons in response to experimental elevation of IOP.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
Figure 1: Pattern of limbal innervation in a mouse eye.
Figure 2: Sympathetic and sensory nerves terminate in SC and TM
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