Automated Axon Counting in Rodent Optic Nerve Sections with AxonJ

Zarei, Kasra; Scheetz, Todd E.; Christopher, Mark; Miller, Kathy Ann; Hedberg-Buenz, Adam; Tandon, Anamika; Anderson, Michael G.; Fingert, John H.; Abràmoff, Michael D.

doi:10.1038/srep26559

Cited by 35 publications

(37 citation statements)

References 38 publications

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“…5 and SI Appendix, Table S5). Our WT C57BL/6 data match those obtained via other automated counting approaches (59,60). Consistent with previous analyses (19), there was no significant difference in the number or density of RGCs in adult Atoh7 KO/+ and SE/+ retinas compared to WT, and KO/KO retinas had scant RGCs.…”

Section: Resultssupporting

confidence: 90%

TheAtoh7remote enhancer provides transcriptional robustness during retinal ganglion cell development

Miesfeld

Ghiasvand

Marsh-Armstrong

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The retinal ganglion cell (RGC) competence factor ATOH7 is dynamically expressed during retinal histogenesis. ATOH7 transcription is controlled by a promoter-adjacent primary enhancer and a remote shadow enhancer (SE). Deletion of the ATOH7 human SE causes nonsyndromic congenital retinal nonattachment (NCRNA) disease, characterized by optic nerve aplasia and total blindness. We used genome editing to model NCRNA in mice. Deletion of the murine SE reduces Atoh7 messenger RNA (mRNA) fivefold but does not recapitulate optic nerve loss; however, SEdel/knockout (KO) trans heterozygotes have thin optic nerves. By analyzing Atoh7 mRNA and protein levels, RGC development and survival, and chromatin landscape effects, we show that the SE ensures robust Atoh7 transcriptional output. Combining SE deletion and KO and wild-type alleles in a genotypic series, we determined the amount of Atoh7 needed to produce a normal complement of adult RGCs, and the secondary consequences of graded reductions in Atoh7 dosage. Together, these data reveal the workings of an evolutionary fail-safe, a duplicate enhancer mechanism that is hard-wired in the machinery of vertebrate retinal ganglion cell genesis.

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Section: Resultssupporting

confidence: 90%

TheAtoh7remote enhancer provides transcriptional robustness during retinal ganglion cell development

Miesfeld

Ghiasvand

Marsh-Armstrong

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…www.nature.com/scientificreports www.nature.com/scientificreports/ In our study PPD (paraphenylendiamine) stain was used for histologic preparation. PPD stain has the attribute of labeling the myelin sheath strongly 35 . Therefore we could ensure that primarily axons of interest were stained, allowing these to be counted by both the semi-automated and manual methods, while simultaneously ensuring a strong contrast between the myelin sheath and other structures prior to digital image preparation.…”

Section: Discussionmentioning

confidence: 99%

Rapid and Precise Semi-Automatic Axon Quantification in Human Peripheral Nerves

Engelmann

Ruewe

Geis

et al. 2020

Sci Rep

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We developed a time-efficient semi-automated axon quantification method using freeware in human cranial nerve sections stained with paraphenylenediamine (PPD). It was used to analyze a total of 1238 facial and masseteric nerve biopsies. The technique was validated by comparing manual and semiautomated quantification of 129 (10.4%) randomly selected biopsies. The software-based method demonstrated a sensitivity of 94% and a specificity of 87%. Semi-automatic axon counting was significantly faster (p < 0.001) than manual counting. It took 1 hour and 47 minutes for all 129 biopsies (averaging 50 sec per biopsy, 0.04 seconds per axon). The counting process is automatic and does not need to be supervised. Manual counting took 21 hours and 6 minutes in total (average 9 minutes and 49 seconds per biopsy, 0.52 seconds per axon). Our method showed a linear correlation to the manual counts (R = 0.944 Spearman rho). Attempts have been made by several research groups to automate axonal load quantification. These methods often require specific hard-and software and are therefore only accessible to a few specialized laboratories. Our semi-automated axon quantification is precise, reliable and time-sparing using publicly available software and should be useful for an effective axon quantification in various human peripheral nerves. Microscopic analysis of peripheral nerves is key for many clinical and research based projects. Peripheral nerves have been analyzed through multiple methods, which can generally be categorized into 'manual' , 'automated' and 'semi-automated' methods. Here, the terms for 'manual' and 'fully automated' morphometry will be used as previously described 1-4. 'Semi-automated' will be used synonymously with Urso-Baiardas 'interactive automated' approach; an automated method with the opportunity for manual preparation or alteration 3. In the past, no prime and uniform method could be found, that is simple, cost efficient and time sparing. Therefore, tendentially, small research collectives use manual methods for analysis 1,2,5. Attempts have been made by several research groups throughout medical and scientific research, to automate this process 3,6-8. Unfortunately, it is often found, that these methods are either highly specialized, thus accessible to only few expert laboratories, or, in the case of highly developed software and hardware, very costly 3,9. By example, Marina et al. have coined a method which, similar to this project, focuses on simpler semi-automated anaylsis 10. Other research groups such as Hunter et al. focus on highly specialized methods, which are able to produce a wide range of data and process numerous variables 11. The semi-automated quantification method proposed in this study was developed as part of a greater study on Human facial nerves, for which a time sparing, cost efficient and user-friendly method of axonal quantification was required. Patients with facial palsy, caused by dysfunction of the seventh cranial nerve, suffer emotional distress and are often socially isolated 1...

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“…For automated axon counts, AxonJ was used to count the entire cross sectional nerve area at a 63x magnification for 10 nerves in each experimental group. 28 …”

Section: Methodsmentioning

confidence: 99%

Nicotinamide treatment robustly protects from inherited mouse glaucoma

Williams

Harder

Cardozo

et al. 2018

Communicative & Integrative Biology

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Nicotinamide adenine dinucleotide (NAD) is a key molecule in several cellular processes and is essential for healthy mitochondrial metabolism. We recently reported that mitochondrial dysfunction is among the very first changes to occur within retinal ganglion cells during initiation of glaucoma in DBA/2J mice. Furthermore, we demonstrated that an age-dependent decline of NAD contributes to mitochondrial dysfunction and vulnerability to glaucoma. The decrease in NAD renders retinal ganglion cells vulnerable to a metabolic crisis following periods of high intraocular pressure. Treating mice with the NAD precursor nicotinamide (the amide form of vitamin B3) inhibited many age- and high intraocular pressure- dependent changes with the highest tested dose decreasing the likelihood of developing glaucoma by ∼10-fold. In this communication, we present further evidence of the neuroprotective effects of nicotinamide against glaucoma in mice, including its prevention of optic nerve excavation and axon loss as assessed by histologic analysis and axon counting. We also show analyses of age- and intraocular pressure- dependent changes in transcripts of NAD producing enzymes within retinal ganglion cells and that nicotinamide treatment prevents these transcriptomic changes.

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Automated Axon Counting in Rodent Optic Nerve Sections with AxonJ

Cited by 35 publications

References 38 publications

TheAtoh7remote enhancer provides transcriptional robustness during retinal ganglion cell development

TheAtoh7remote enhancer provides transcriptional robustness during retinal ganglion cell development

Rapid and Precise Semi-Automatic Axon Quantification in Human Peripheral Nerves

Nicotinamide treatment robustly protects from inherited mouse glaucoma

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