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Results: 6

1.
Fig. 6.

Fig. 6. From: Mechanisms of acute axonal degeneration in the optic nerve in vivo.

Time course and Ca-dependence of autophagosome accumulation in AAD. (A) Quantification of LC3 positive axonal punctae/mm2 in a native ON (no crush), in ON at 30, 120, and 360 min postcrush and at 360 min postcrush and treatment with a calcium inhibitor mix. **, P < 0.01; *, P < 0.05 vs. no crush, §, P < 0.05 vs. 360 min without calcium inhibitor mix. (B) Pseudoconfocal micrographs of representative areas immunostained against LC3 at 200 μm proximal and distal to the crush corresponding to the quantification in A. (Scale bar in B, 20 μm.)

Johanna Knöferle, et al. Proc Natl Acad Sci U S A. 2010 March 30;107(13):6064-6069.
2.
Fig. 4.

Fig. 4. From: Mechanisms of acute axonal degeneration in the optic nerve in vivo.

Time course of axonal degeneration following autophagy inhibition by 3-MA. (A) Development of axonal integrity ratios for DMSO-treated (open rectangle; n = 4) and 3-MA-treated (cross; n = 6) animals. Error bars represent SEM. Statistical differences between DMSO- and 3-MA-treated groups: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by ANOVA and Student's t test (two-tailed, heteroscedastic). Axonal changes proximal to crush at indicated time points (min after lesion) in an ON pretreated with 3-MA (B) or DMSO (C).

Johanna Knöferle, et al. Proc Natl Acad Sci U S A. 2010 March 30;107(13):6064-6069.
3.
Fig. 2.

Fig. 2. From: Mechanisms of acute axonal degeneration in the optic nerve in vivo.

Time course of acute axonal degeneration following ON crush. Overview of the region proximal (A) and distal (C) to the crush site. Red rectangles indicate evaluated axon part in B or D. Distance to the lesion site (tie) is indicated. Axonal changes proximal (B) and distal (D) to crush at indicated time points (min after lesion). (E) Development of axonal integrity ratios for axons distal (open circle; n = 5) and proximal (solid square; n = 6) to the lesion site. AD are composite micrographs. Error bars represent SEM. For statistical analysis ANOVA and Student's t test (two-tailed, heteroscedastic) were used. Differences are considered significant vs. uncrushed ON. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Johanna Knöferle, et al. Proc Natl Acad Sci U S A. 2010 March 30;107(13):6064-6069.
4.
Fig. 5.

Fig. 5. From: Mechanisms of acute axonal degeneration in the optic nerve in vivo.

Role of intraaxonal calcium levels ([Ca2+]i) for axonal degeneration. (A) [Ca2+]i before and after ON crush in the untreated ON. Single measurements (n = 3). (B) Exemplary images of an ON before and after crush (crush site marked by constricted tie). Time before/after crush indicated in seconds. (C) Time course of axonal degeneration after application of a calcium channel inhibitor mixture (amiloride 100 μM, amlodipin 10 μM, NBQX 1 mM; crosses; n = 5), a calcium ionophore (A23187, 100 μM; open rhomboids; n = 4), or untreated ON (control; solid squares; n = 6). Error bars represent SEM. Statistical differences indicated in relation to control group: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by ANOVA and Student's t test (two-tailed, heteroscedastic). Representative images of axonal changes proximal to crush at indicated time points (min after lesion) after application of the calcium inhibitor mix (D) or the calcium ionophore (E).

Johanna Knöferle, et al. Proc Natl Acad Sci U S A. 2010 March 30;107(13):6064-6069.
5.
Fig. 3.

Fig. 3. From: Mechanisms of acute axonal degeneration in the optic nerve in vivo.

Ultrastructural changes after ON crush. Overview of the ON ultrastructure in the unlesioned control ON (A and I), and 30 min (B and J), 120 min (C and K) and 360 min (D and L) after crush (all longitudinal sections). Local axonal swellings (black arrows) appear earlier proximal than distal to the crush and increase in number and size over time (AD and IL). Early structural defects between axoplasm and myelin sheath distal to the crush (K and L; gray arrows). Histograms of g-ratios of ON fibers after crush (in % frequency, bin width 0.025) (EH and MP). Development of the mean fiber and axoplasm diameter (Q) and the resulting g-ratio (R) over time. Mean g-ratio is given ±SD (EH and MP) or ±SEM (R). LC3-immunogold labeling for autophagosomes (black arrows) 360 min after crush (S and T). **, P < 0.01; ***, P < 0.001 (compared to unlesioned control of the corresponding side), two-tailed, heteroscedastic Student's t test. [Scale bars, 5 μm (AD and IL); 250 nm (S); 100 nm (T).]

Johanna Knöferle, et al. Proc Natl Acad Sci U S A. 2010 March 30;107(13):6064-6069.
6.
Fig. 1.

Fig. 1. From: Mechanisms of acute axonal degeneration in the optic nerve in vivo.

Visualization of retinal ganglion cell (RGC) axons in the optic nerve (ON) in vivo. (A) Scheme of experimental setup (not to scale). RGCs (red) are labeled by intravitreal injection of AAV vectors expressing dsRed and visualized by epifluorescence. (B) Map of the vector construct (AAV(1/2).hSYN.dsRed). (C) Flatmount of an AAV(1/2).hSYN.dsRed-injected retina showing specific RGC labeling in the upper quadrant (U) by targeted injection. Temporal (T), nasal (N), lower (L), and upper (U) quadrants of the retina. (D) Higher magnification of Inset shown in C demonstrating transfection efficiency and axonal labeling. Transverse (E and F) and longitudinal (G–K) sections of transfected ON showing localized transfection of axons (arrows), dsRed (red, E, G, and I), phosphorylated neurofilaments (green, F, H, and K). (IK) Magnification of Insets shown in G and H, arrows mark single transfected axons; (J) overlay of I and K. (L) Example of an imaging result showing several superficial RGC axons by epifluorescence. (M) Higher magnification of single axon in L. [Scale bars, 1 mm (C); 200 μm (D); 100 μm (EH); 50 μm (L and M).]

Johanna Knöferle, et al. Proc Natl Acad Sci U S A. 2010 March 30;107(13):6064-6069.

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