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Fig 1.

Focal compression of isolated primary hippocampal axons.

(A) Schematic illustration of axon loading environment and orientation within the AIM. The axon only region overlaps with a 20μm thick compression pad above the testing chamber. Microfluidics are used to control the compression pad and localize loading to the area underneath the pad (blue box). (B) A series of panoramic TEM images reconstructing the entire area of the axon under the compression pad at higher magnification. (C) A single TEM image for quantifying number, density, and spacing of the cytoskeletal structures. Scale bar = 500nm.

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Fig 2.

Representative TEM images of microtubules in axons.

Microtubules are indicated by black arrows (B, D). Axon diameter, number of microtubules, and spacing between microtubules were measured for each image along unit axon length, L, at L/4, L/2, and 3L/4. (A) In Control axons, microtubules are oriented along the principal axis of the axon. (C) In Crushed axons, microtubules appear disorganized and misaligned. (B, D) Inset of Control and Crushed axons showing diameter (D) and spacing (SMT) measurements for microtubules. (E) Nodal bleb (arrow head) of Crushed axon showing mitochondria in each bleb. (F) Inset of Crush axon showing microtubule breakage and rupture (arrows). Parts of fig are adapted from [32]. Image used with permission from the Federation of American Societies for Experimental Biology or The FASEB Journal (www.fasebj.org). Scale bars = 100nm (A-D); 500nm (E-F).

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Fig 3.

Microtubule quantification in axons.

Each circle represents a single measure for the Control (blue) or Crushed (red) groups. Power law fits (solid lines) are shown to estimate the relationship between axon diameter and microtubule metrics with 95% confidence intervals (dashed lines). (A-C) A strong dependency on axon caliber is observed for all microtubule measures for Control and Crushed axons; however the populations of Control and Crush axons are not distinguishable given the overlap of the confidence intervals between both groups. The smallest diameters measured are approximately 100nm and no data was taken for axons of a smaller caliber than this.

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Fig 4.

Examination of binned microtubule data.

Axon diameter bins are given along the X-axis and error bars are standard error mean for all plots. (A-C) A strong dependency on axon caliber is observed for the number of (NMT), linear density (), and spacing between (SMT) microtubules in Control and Crushed axons. NMT and are significantly lower for Crushed, while SMT is significantly higher for Crushed, across nearly all axon diameter. The observed differences between Control and Crushed are more apparent at larger axon diameters. *(p<0.05)

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Table 1.

Quantification of microtubules and neurofilaments following focal compression.

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Fig 5.

Representative TEM images of neurofilaments in axons.

6nm Au-nanoparticles, outlined in yellow, were used to measure areal density and spacing between neurofilaments for each image. (A-C) In Control axons, Au-nanoparticles are regularly spaced along the length of the axon and across the axon diameter. (D-F) In Crushed axons, Au-nanoparticles appear more heterogeneous in their distribution and spacing. (B-C, E-F) Insets of Control and Crushed axons showing areal density and spacing distribution for nanoparticles. There is a greater number and areal density in Control axons (C) than in Crushed axons (F). Scale bars = 100nm.

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Fig 6.

Neurofilament quantification in axons.

Each circle represents a single measure for the Control (blue) or Crushed (red) groups. Linear fits (solid lines) are shown to estimate the relationship between axon diameter and neurofilament metrics with 95% confidence intervals (dashed lines). (A-C) A strong dependency on axon caliber is observed for almost all neurofilament measures in Control and Crushed axons; the exception being neurofilament spacing for Crushed axons. The populations for Control and Crushed axons can be separated more readily for NNF and at larger axon calibers; however SNF populations are not distinguishable given the confidence interval overlap between Control and Crushed groups. No data was taken for axons of a caliber <100nm; therefore our linear fit is limited in applicability to the data range shown.

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Fig 7.

Examination of binned neurofilament data.

Axon diameter bins are given along the X-axis and error bars are standard error mean for all plots. (A, B) A strong dependency on axon caliber is observed for the number (NNF) and areal density () of Au-nanoparticles for antibodies labeled neurofilaments in Control and Crushed axons. NNF and are significantly lower for Crushed across all comparable axon calibers. (C) Spacing between Au-nanoparticles (SNF) showed a strong dependency for Control, but not for Crushed axons. SNF appears larger for Crushed than Control in nearly all axon diameters and remains approximately 70nm for all Crushed axons regardless of axon diameter. The observed differences between Control and Crushed are more apparent at larger axon diameters. *(p<0.05)

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