A Rapid, Quantitative Method for Assessing Axonal Extension on Biomaterial Platforms

Cregg, Jared M.; Wiseman, Sherri L.; Pietrzak-Goetze, Nicole M.; Smith, Martyn R.; Jaroch, David; Clupper, D. C.; Gilbert, Ryan J.

doi:10.1089/ten.tec.2009.0108

Cited by 18 publications

(14 citation statements)

References 20 publications

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“…For example, plug-ins enable ImageJ users to manually trace microscope images for quantifying branch-like cellular morphologies. 6,7 More semi-automated algorithms rely on image segmentation to quickly derive surface area, perimeter length, and lacunarity measurements of tissue structures or cell growths. [8][9][10][11][12][13] However, these methods are based on 2D image analysis, which approximates 3D cellular architecture with 2D planar projections.…”

mentioning

confidence: 99%

Three-Dimensional Image Quantification as a New Morphometry Method for Tissue Engineering

Rytlewski

Geuss

Anyaeji

et al. 2012

Tissue Engineering Part C: Methods

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Morphological analysis is an essential step in verifying the success of a tissue engineering strategy where the presence of a desired cellular phenotype must be determined. While morphometry has transitioned from observational grading to computational quantification, established quantitative methods eliminate information by relying on two-dimensional (2D) analysis to describe three-dimensional (3D) niches. In this study, we demonstrate the validity and utility of 3D morphological quantification using two common angiogenesis assays in our fibrin-based in vitro model: (1) the microcarrier bead assay with human mesenchymal stem cells and (2) the rat aortic ring outgrowth assay. The quantification method is based on collecting and segmenting fluorescent confocal z-stacks into 3D models with 3D Slicer, an open-source magnetic resonance imaging/computed tomography analysis program. Data from 3D models are then processed into biologically relevant metrics in MATLAB for statistical analysis. Metrics include descriptive parameters such as vascular network length, volume, number of network segments, and degree of network branching. Our results indicate that 2D measures are significantly different than their 3D counterparts unless the vascular network exhibits anisotropic growth along the plane of imaging. Additionally, the statistical outcomes of 3D morphological quantification agreed with our initial qualitative observations among different test groups. This novel quantification approach generates more spatially accurate and objective measures, representing an important step toward improving the reliability of morphological comparisons.

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mentioning

confidence: 99%

Three-Dimensional Image Quantification as a New Morphometry Method for Tissue Engineering

Rytlewski

Geuss

Anyaeji

et al. 2012

Tissue Engineering Part C: Methods

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“…As the explant sizes are not all the same, the area is measured for each explant by subtracting the area of the explant body from the total area of outgrowth (Figure 2H), and then normalize the area of outgrowth by expressing it as a ratio of outgrowth area:explant body area.Longest migration length can also be measured by using a line drawing tool as perviously described (Cregg et al , 2010). First, the explant epicenter must be identify (yellow bars; Figure 2G), then a series of bars are draw across the entire area of outgrowth intersecting the explant epicenter (blue bars; Figure 2I).…”

Section: Discussionmentioning

confidence: 99%

“…Longest migration length can also be measured by using a line drawing tool as perviously described (Cregg et al , 2010). First, the explant epicenter must be identify (yellow bars; Figure 2G), then a series of bars are draw across the entire area of outgrowth intersecting the explant epicenter (blue bars; Figure 2I).…”

Section: Discussionmentioning

confidence: 99%

Explant Methodology for Analyzing Neuroblast Migration

et al. 2017

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The subventricular zone (SVZ) in the mammalian forebrain contains stem/progenitor cells that migrate through the rostral migratory stream (RMS) to the olfactory bulb throughout adulthood. SVZ-derived explant cultures provide a convenient method to assess factors regulating the intermediary stage of neural stem/progenitor cell migration. Here, we describe the isolation of SVZ-derived RMS explants from the neonatal mouse brain, and the conditions required to culture and evaluate their migration.

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“…Images were analyzed for neurite outgrowth area and neurite length using ImageJ (Fiji Distribution, National Institute of Health, Bethesda, MA), as shown in Figure 1. (26, 27)…”

Section: Methodsmentioning

confidence: 99%

Adjuvant neurotrophic factors in peripheral nerve repair with chondroitin sulfate proteoglycan-reduced acellular nerve allografts

Boyer¹,

Sexton²,

Rodriguez-Feo³

et al. 2015

Journal of Surgical Research

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Background Acellular nerve allografts are now standard tools in peripheral nerve repair due to decreased donor site morbidity and operative time savings. Preparation of nerve allografts involves several steps of decellularization and modification of extracellular matrix to remove chondroitin sulfate proteoglycans (CSPGs), which have been shown to inhibit neurite outgrowth through a poorly understood mechanism involving RhoA and ECM-integrin interactions. Chondroitinase ABC (ChABC) is an enzyme that degrades CSPG molecules and has been shown to promote neurite outgrowth following injury of the central and peripheral nervous systems. Variable results following chondroitinase ABC treatment make it difficult to predict the effects of this drug in human nerve allografts, especially in the presence of native extracellular signaling molecules. Several studies have shown cross-talk between neurotrophic factor and CSPG signaling pathways, but their interaction remains poorly understood. In this study, we examined the adjuvant effects of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) on neurite outgrowth post-injury in CSPG-reduced substrates and acellular nerve allografts. Materials and Methods E12 chicken DRG explants were cultured in medium containing ChABC, ChABC + NGF, ChABC + GDNF or control media. Explants were imaged at 3 d and neurite outgrowths measured. The rat sciatic nerve injury model involved a 1-cm sciatic nerve gap that was microsurgically repaired with ChABC pre-treated acellular nerve allografts. Prior to implantation, nerve allografts were incubated in NGF, GDNF or sterile water. Nerve histology was evaluated at 5d and 8wk post-injury. Results The addition of GDNF in vitro produced significant increase in sensory neurite length at 3 d compared to ChABC alone (P < 0.01), while NGF was not significantly different from control. In vivo adjuvant NGF produced increases in total myelinated axon count (P < 0.005) and motor axon count (P < 0.01), while significantly reducing IB4+ nociceptor axon count (P < 0.01). There were no significant differences produced by in vivo adjuvant GDNF. Conclusions This study provides initial evidence that CSPG-reduced nerve grafts may disinhibit the pro-survival effects of NGF in vivo, promoting motor axon outgrowth and reducing regeneration of specific nociceptive neurons. Our results support further investigation of adjuvant NGF therapy in CSPG-reduced acellular nerve grafts.

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A Rapid, Quantitative Method for Assessing Axonal Extension on Biomaterial Platforms

Cited by 18 publications

References 20 publications

Three-Dimensional Image Quantification as a New Morphometry Method for Tissue Engineering

Three-Dimensional Image Quantification as a New Morphometry Method for Tissue Engineering

Explant Methodology for Analyzing Neuroblast Migration

Adjuvant neurotrophic factors in peripheral nerve repair with chondroitin sulfate proteoglycan-reduced acellular nerve allografts

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