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Stretch Injury of Human Induced Pluripotent Stem Cell Derived Neurons in a 96 Well Format

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ABSTRACT

Traumatic brain injury (TBI) is a major cause of mortality and morbidity with limited therapeutic options. Traumatic axonal injury (TAI) is an important component of TBI pathology. It is difficult to reproduce TAI in animal models of closed head injury, but in vitro stretch injury models reproduce clinical TAI pathology. Existing in vitro models employ primary rodent neurons or human cancer cell line cells in low throughput formats. This in vitro neuronal stretch injury model employs human induced pluripotent stem cell-derived neurons (hiPSCNs) in a 96 well format. Silicone membranes were attached to 96 well plate tops to create stretchable, culture substrates. A custom-built device was designed and validated to apply repeatable, biofidelic strains and strain rates to these plates. A high content approach was used to measure injury in a hypothesis-free manner. These measurements are shown to provide a sensitive, dose-dependent, multi-modal description of the response to mechanical insult. hiPSCNs transition from healthy to injured phenotype at approximately 35% Lagrangian strain. Continued development of this model may create novel opportunities for drug discovery and exploration of the role of human genotype in TAI pathology.

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Injury with 2 mm stage displacement alters synaptic density.(A) Representative image of uninjured iCell neurons stained with MAP2, Synaptophysin, and Hoechst 33342. Synaptophysin staining was punctate and distributed across soma and along neurites. (B) Injury did not change the ratio of synaptophysin positive cell area to total cell area (n = 12). (C) Injury did not significantly change the ratio of synaptophysin positive neurite area to total neurite area, although there was a modest downward trend (n = 12). (D) Injury significantly increased the ratio of synaptophysin positive soma area to total soma area (n = 12, *=t-test with significance criterion Bonferroni corrected to p < 0.05/3, bars = standard error).
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f5: Injury with 2 mm stage displacement alters synaptic density.(A) Representative image of uninjured iCell neurons stained with MAP2, Synaptophysin, and Hoechst 33342. Synaptophysin staining was punctate and distributed across soma and along neurites. (B) Injury did not change the ratio of synaptophysin positive cell area to total cell area (n = 12). (C) Injury did not significantly change the ratio of synaptophysin positive neurite area to total neurite area, although there was a modest downward trend (n = 12). (D) Injury significantly increased the ratio of synaptophysin positive soma area to total soma area (n = 12, *=t-test with significance criterion Bonferroni corrected to p < 0.05/3, bars = standard error).

Mentions: iCell Neurons stained positively for synaptophysin. (see Fig. 5). Injury with 2mm stage displacement did not significantly alter the synaptophysin density in cells overall. However, when the somatic and neuritic compartments were considered in isolation, injury was found to significantly increase somatic synaptophysin density. Injury caused a downward trend in neuritic synaptophysin density, but this trend was not statistically significant.Synaptophysin labeling of synapses revealed the conventional punctate organization (see Fig. 5A). However, it was not appropriate to count synaptophysin-positive puncta per unit length of neurite in this system because the injury induced a beaded morphology in neurites that caused uniform stain distribution to appear punctate. Therefore, synapse distribution was quantified by measuring the ratio of synaptophysin positive cell area to total cell area. Stretch injury did not alter this ratio (Fig. 5B). To further investigate this finding, the cell domain was split into somatic and neuritic domains and the ratio of synaptophysin positive area to total area was computed for each domain. There was a downward trend in the synaptophysin positive area of the neurites, although this trend was not statistically significant (Fig. 5C). By contrast, there was a statistically significant increase in the synaptophysin positive area of the soma (Fig. 5D, t-test with significance criterion Bonferroni corrected to p < 0.05/3).


Stretch Injury of Human Induced Pluripotent Stem Cell Derived Neurons in a 96 Well Format
Injury with 2 mm stage displacement alters synaptic density.(A) Representative image of uninjured iCell neurons stained with MAP2, Synaptophysin, and Hoechst 33342. Synaptophysin staining was punctate and distributed across soma and along neurites. (B) Injury did not change the ratio of synaptophysin positive cell area to total cell area (n = 12). (C) Injury did not significantly change the ratio of synaptophysin positive neurite area to total neurite area, although there was a modest downward trend (n = 12). (D) Injury significantly increased the ratio of synaptophysin positive soma area to total soma area (n = 12, *=t-test with significance criterion Bonferroni corrected to p < 0.05/3, bars = standard error).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037451&req=5

f5: Injury with 2 mm stage displacement alters synaptic density.(A) Representative image of uninjured iCell neurons stained with MAP2, Synaptophysin, and Hoechst 33342. Synaptophysin staining was punctate and distributed across soma and along neurites. (B) Injury did not change the ratio of synaptophysin positive cell area to total cell area (n = 12). (C) Injury did not significantly change the ratio of synaptophysin positive neurite area to total neurite area, although there was a modest downward trend (n = 12). (D) Injury significantly increased the ratio of synaptophysin positive soma area to total soma area (n = 12, *=t-test with significance criterion Bonferroni corrected to p < 0.05/3, bars = standard error).
Mentions: iCell Neurons stained positively for synaptophysin. (see Fig. 5). Injury with 2mm stage displacement did not significantly alter the synaptophysin density in cells overall. However, when the somatic and neuritic compartments were considered in isolation, injury was found to significantly increase somatic synaptophysin density. Injury caused a downward trend in neuritic synaptophysin density, but this trend was not statistically significant.Synaptophysin labeling of synapses revealed the conventional punctate organization (see Fig. 5A). However, it was not appropriate to count synaptophysin-positive puncta per unit length of neurite in this system because the injury induced a beaded morphology in neurites that caused uniform stain distribution to appear punctate. Therefore, synapse distribution was quantified by measuring the ratio of synaptophysin positive cell area to total cell area. Stretch injury did not alter this ratio (Fig. 5B). To further investigate this finding, the cell domain was split into somatic and neuritic domains and the ratio of synaptophysin positive area to total area was computed for each domain. There was a downward trend in the synaptophysin positive area of the neurites, although this trend was not statistically significant (Fig. 5C). By contrast, there was a statistically significant increase in the synaptophysin positive area of the soma (Fig. 5D, t-test with significance criterion Bonferroni corrected to p < 0.05/3).

View Article: PubMed Central - PubMed

ABSTRACT

Traumatic brain injury (TBI) is a major cause of mortality and morbidity with limited therapeutic options. Traumatic axonal injury (TAI) is an important component of TBI pathology. It is difficult to reproduce TAI in animal models of closed head injury, but in vitro stretch injury models reproduce clinical TAI pathology. Existing in vitro models employ primary rodent neurons or human cancer cell line cells in low throughput formats. This in vitro neuronal stretch injury model employs human induced pluripotent stem cell-derived neurons (hiPSCNs) in a 96 well format. Silicone membranes were attached to 96 well plate tops to create stretchable, culture substrates. A custom-built device was designed and validated to apply repeatable, biofidelic strains and strain rates to these plates. A high content approach was used to measure injury in a hypothesis-free manner. These measurements are shown to provide a sensitive, dose-dependent, multi-modal description of the response to mechanical insult. hiPSCNs transition from healthy to injured phenotype at approximately 35% Lagrangian strain. Continued development of this model may create novel opportunities for drug discovery and exploration of the role of human genotype in TAI pathology.

No MeSH data available.