Changes to mitochondrial ultrastructure in optic nerve vulnerable to secondary degeneration in vivo are limited by irradiation at 670 nm

Abstract

BackgroundTraumatic injury to the central nervous system results in damage to tissue beyond the primary injury, termed secondary degeneration. Key events thought to be associated with secondary degeneration involve aspects of mitochondrial function which may be modulated by red/near-infrared irradiation therapy (R/NIR-IT), but precisely how mitochondria are affected in vivo has not been investigated. Secondary degeneration was modelled by transecting the dorsal aspect of the optic nerve in adult rats and mitochondrial ultrastructure in intact ventral optic nerve vulnerable to secondary degeneration investigated with transmission electron microscopy.ResultsDespite reported increases in fission following central nervous system injury, we saw no change in mitochondrial densities in optic nerve vulnerable to secondary degeneration in vivo. However, in axons, frequency distributions of mitochondrial profile areas showed higher cumulative probabilities of smaller mitochondrial profiles at day 1 after injury. Glial mitochondrial profiles did not exhibit changes in area, but a more elliptical mitochondrial shape was observed at both day 1 and 7 following injury. Importantly, mitochondrial autophagic profiles were observed at days 1 and 7 in optic nerve vulnerable to secondary degeneration in vivo. Citrate synthase activity was used as an additional measure of mitochondrial mass in ventral optic nerve and was decreased at day 7, whereas mitochondrial aconitase activity increased at day 1 and day 28 after injury in optic nerve vulnerable to secondary degeneration. R/NIR-IT has been used to treat the injured central nervous system, with reported improvements in oxidative metabolism suggesting mitochondrial involvement, but ultrastructural information is lacking. Here we show that R/NIR-IT of injured animals resulted in distributions of mitochondrial areas and shape not significantly different from control and significantly reduced mitochondrial autophagic profiles. R/NIR-IT also resulted in decreased citrate synthase activity (day 7) and increased aconitase activity (day 1) in optic nerve vulnerable to secondary degeneration.ConclusionsThese findings suggest that mitochondrial structure and activity of enzymes of the citric acid cycle are dynamically altered during secondary degeneration in vivo and R/NIR-IT may protect mitochondrial structure.

Shedding light on mitophagy in neurons: what is the evidence for PINK1/Parkin mitophagy in vivo?

“…One reason that in vivo studies of mitophagy in the mammalian brain are lacking is the limited availability of appropriate tools. Thus far, electron microscopy has been used successfully to visualise mitochondria in autophagosomes [15,99]; however, this method is both very time consuming and restricted by limited sampling of tissue. Other ways to detect mitophagy include coimmunostaining for mitochondria and autophagosomal markers, such as LC3 (e.g.…”

Reactive species and oxidative stress in optic nerve vulnerable to secondary degeneration

“…Image analysis was conducted on a single image using Image J/Fiji analysis software, setting constant arbitrary threshold intensities for all images in an analysis and semi-quantifying mean intensities of the whole ventral ON images and mean intensities and areas above the set threshold. Transmission electron microscopy (TEM) and quantification of mitochondrial autophagic profiles was conducted as described 24 (n=4 animals/time point), using a using a Philips CM-10 TEM (Eindhoven, The Netherlands) attached to an Olympus Megaview III camera (1376x1032 pixels), at an accelerating voltage of…”

“…RNS also include radical and non-radical species, such as nitric oxide ( • NO) and peroxynitrite (ONOO -). There is evidence that in ON vulnerable to secondary degeneration, mitochondrial dysfunction and oxidative stress occur early after injury 24,25 . However, the identities of the ROS and/or RNS that contribute, and the nature of oxidative damage that they cause, are not clearly understood.…”

Optical Detection of Early Damage in Retinal Ganglion Cells in a Mouse Model of Partial Optic Nerve Crush Injury

“…Secondary degeneration of RGCs, which has been found in pONC or transection models of different animals, 33–36 could be caused by the deleterious molecules released from the RGCs or glial cells damaged by the primary direct insults. First, oxidative stress, calcium overload, or mitochondrial dysfunction in the primarily injured RGCs could produce or regulate proapoptotic signals to neighboring RGCs, 33,35,37,38 causing the DNA fragmentation and cell disintegration. Second, retinal glial cells such as microglia, astrocytes, and oligodendrocytes also played a role in the secondary degeneration after pONC, 39,40 which may also contribute to the D change observed here; however, the specific mechanisms of these complex pathologies need further study.…”