In glaucoma, although axonal injury drives retinal ganglion cell (RGC) death, little is known about the underlying pathomechanisms. To provide new mechanistic insights and identify new biomarkers, we combined latest non-targeting metabolomics analyses to profile altered metabolites in the mouse whole retina 2, 4, and 7 days after optic nerve crush (NC). Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry and liquid chromatography Fourier transform mass spectrometry covering wide spectrum of metabolites in combination highlighted 30 metabolites that changed its concentration after NC. The analysis displayed similar changes for purine nucleotide and glutathione as reported previously in another animal model of axonal injury and detected multiple metabolites that increased after the injury. After studying the specificity of the identified metabolites to RGCs in histological sections using imaging mass spectrometry, two metabolites, i.e., L-acetylcarnitine and phosphatidylcholine were increased not only preceding the peak of RGC death in the whole retina but also at the RGC layer (2.3-fold and 1.2-fold, respectively). These phospholipids propose novel mechanisms of RGC death and may serve as early biomarkers of axonal injury. The combinatory metabolomics analyses promise to illuminate pathomechanisms, reveal biomarkers, and allow the discovery of new therapeutic targets of glaucoma.
Measuring the optokinetic response (OKR) to rotating sinusoidal gratings is becoming an increasingly common method to determine visual function thresholds in mice. This is possible also through direct electrophysiological recording of the response of the neurons in the visual cortex to the presentation of reversing patterned stimuli, i.e. the pattern visually evoked potential (pVEP). Herein, we optimized the conditions for recording pVEPs in wild-type mice: we investigated the optimal depth (1, 2, or 3 mm) of the inserted electrode and the optimal stimulus pattern (vertical, horizontal, or oblique black and white stripes, or a checkerboard pattern). Visual acuity was higher when measured with the optimal pVEP recording conditions, i.e., with the electrode at 2 mm and a vertical-stripe stimulus (0.530 ± 0.021 cycle/degree), than with OKR (0.455 ± 0.006 cycle/degree). Moreover, in murine eyes with optic nerve crush-induced low vision, OKR could not measure any visual acuity, while pVEPs allowed the reliable quantification of residual vision (0.064 ± 0.004 cycle/degree). Our results show that pVEPs allow more sensitive measurement of visual function than the OKR-based method. This technique should be particularly useful in mouse models of ocular disease and low vision.
Our data suggest that Ecel1 induction is part of the retinal neuroprotective response to axonal injury in mice. These findings might provide insight into novel therapeutic targets for the attenuation of RGC damage, such as occurs in traumatic optic neuropathy.
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