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Glaucoma Dialogue IGR 18-3
Comment
Comment by Harry Quigley on:
71509 Vitamin B3 modulates mitochondrial vulnerability and prevents glaucoma in aged mice, Williams PA; Harder JM; Foxworth NE et al., Science, 2017; 355: 756-760
See also comment(s) by Keith Martin • Louis Pasquale • Derek Welsbie • Pete Williams & Simon John •This report is deals with an important and timely subject. Readers should assess several important aspects of its methods and conclusions. First, NAM treatment may behave differently in DBA/2J mice and it should be tested in other glaucoma models for three reasons.
First, NAM treatment may behave differently in DBA/2J mice and it should be tested in other glaucoma models
1. While the authors claim to study "how increasing age and high IOP interact to drive neurodegeneration", the study does not test age as a variable. DBA/2J mice that develop abnormal IOP lose retinal ganglion cells (RGC) around 6-12 months of age. No data are presented here comparing younger to older animals with similar IOP exposure - that requires comparing effects of IOP elevation and RGC protection in young compared to old mice. We, and others, have studied young versus older mouse RGC susceptibility to experimental IOP elevation, showing different effects of age in different strains of mice.
2. DBA/2J mice have dramatic degeneration of anterior ocular structures and the consequent inflammation is integral to this model, but not shared by human glaucoma or other rodent models that elevate IOP. The fact that higher doses of NAM prevented IOP elevation suggests that the inflammatory/degenerative features of DBA/2J may lead to different outcomes from other glaucoma models if the treatment blocks its unique features, but not general RGC protection pathways.
3. DBA/2J mice have a highly variable IOP profile and variable time to onset of RGC damage. Thus, it is difficult to study the time course of neurodegeneration, which is better modeled in other high IOP models (e.g., laser treatment, bead injection or hypertonic saline exposure), where the injury initiation is clearly defined in time. If NAM treatment is confirmed as useful in additional models, it will be possible to dissect its true pathway of protection. We cannot yet know from the present work whether the localization of the beneficial effect, if any, is at the cell body, the axon, both or neither.
There are other methodological issues.
1. Mouse RNA data were divided into four groups with the claim that "As disease progressed, there was an increase in transcript abundance that was most pronounced for mitochondrial reads." No evidence was provided that the transcript groups had different amounts of RGC loss or were matched to some level of progression, only that they could be segregated into groups.
2. Axon loss was assessed only qualitatively and the methods were insufficiently described in order to allow determination if there was any real effect. Paraphenylenediamine (PPD) was described as a "sensitive stain for damaged axons", while in fact it non-specifically labels many aspects of nerve tissue and is not specific for injury, which was judged subjectively. If axons are dead and leave no clear debris, which is often the case in mice, the method mistakes the nerve as normal. More accurate methods to assess axon loss are demonstrated in many papers, involving sensitive counts of the actual axon density in randomized images multiplied by nerve area. Quantitative methods show that the variability among mice in axon loss often requires 30 mice or more in a group to show effects at the level seen here, where there were only 8 mice in a group. In the critical Fig 2 C, one cannot tell which groups are being compared.
3. The claim that there is a "loss of axonal transport" with NAM treatment is not documented by any quantitative data, nor a presentation of the method used.
Prior to this paper, there were already many investigators publishing important links between mitochondrial function and glaucomatous damage. This paper will hopefully stimulate more interest in that field to corroborate or modify its findings.
