A Surviving Intact Branch Stabilizes Remaining Axon Architecture after Injury as Revealed by In Vivo Imaging in the Mouse Spinal Cord  Ariana O. Lorenzana,

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A Surviving Intact Branch Stabilizes Remaining Axon Architecture after Injury as Revealed by In Vivo Imaging in the Mouse Spinal Cord  Ariana O. Lorenzana, Jae K. Lee, Matthew Mui, Amy Chang, Binhai Zheng  Neuron  Volume 86, Issue 4, Pages 947-954 (May 2015) DOI: 10.1016/j.neuron.2015.03.061 Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Acute Axon Degeneration after Highly Localized Laser Axotomies (A–J) Selected two-photon images from a time-lapse series taken every ∼5–15 min showing acute degeneration via fragmentation and retraction following an ascending branch axotomy. BP, branch point (blue arrow); M, main branch; A, ascending branch; D, descending branch; Lsr, laser axotomy (red arrow). Rostral is always to the right. Acute times shown are in hours and minutes (h:mm); “Injury” is the time point immediately after axotomy. Note that in (H)–(J) the tip of the proximal segment resembles retraction bulb-like structures described in Kerschensteiner et al. (2005). Scale bar = 100 μm. (K–P) Acute degeneration profiles of individual axonal segments clustered around the injury location (ascending, descending, or main branch) and the axonal segment (proximal or distal). See text for descriptions of colored arrows. In (K), (M), and (P), a branch point breach is indicated by a solid line splitting into one solid line and one dotted line of the same color. See also Movie S1 and Figures S1 and S2. Neuron 2015 86, 947-954DOI: (10.1016/j.neuron.2015.03.061) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 The Branch Point Is a Barrier to Retrograde but Not Anterograde Degeneration at Both Acute and Subacute Time Points (A–D) Degeneration following a descending branch axotomy placed at ∼50 μm from the branch point. (E–H) Degeneration following an ascending branch axotomy placed between 100 and 150 μm from the branch point. Montaged images are presented. Yellow arrows, landmarks to track axons through multiple imaging sessions; white arrow, uninjured axon. Scale bar = 100 μm. (I–N) Degeneration distance is plotted against the distance between axotomy site and branch point. See text for data interpretation. Note that the data points marked by open circles in (K) and (L) are the same data points marked by solid circles in (I) and (J), respectively. Orange arrowhead, two data points coincidentally superimposed on one another. Neuron 2015 86, 947-954DOI: (10.1016/j.neuron.2015.03.061) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Injury Location-Specific Effects on Axon Regeneration at the Subacute Time Point (A–C′) An example of axon regeneration via branching and elongation observed on day 5 after a main branch axotomy. Montaged images are presented. Green arrows, newly regenerated axons; brown arrows, Wallerian degeneration; magenta arrowheads, structures with growth cone appearance. Scale bar = 100 μm in (A)–(C) and 50 μm in (C′). (D) Regeneration frequency on day 5 after axotomy. ∗∗∗p < 0.001, chi-square and Fisher’s exact test. A, ascending; D, descending; M, main branch; Db, double branch axotomy; LD, long distance (on ascending branch). (E and F) Quantification of regeneration in terms of axonal growth (E) including maximum, total, and average per branch, and branch numbers (F) for primary (1°) and secondary (2°) branches with the same color coding as in (D). Error bars represent ± SEM. See also Figure S3. Neuron 2015 86, 947-954DOI: (10.1016/j.neuron.2015.03.061) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Dynamic Axonal Responses to Injury with Alternating Phases of Regeneration and Pruning over a Chronic Period (A–D′) An example of main branch axotomy that led to dynamic regeneration, pruning, and remodeling up to the 6-month time point. Note axon regeneration (including a U-turn) observed at 5 days, pruning at 5 weeks, and new axon regeneration at 6 months. Montaged images are presented. Solid green arrows, new axonal growth; hollow green arrows, previous axonal growth that sustained; orange arrowheads, varicosities. Scale bar = 100 μm in (A)–(D) and 50 μm in (D′). (E) New axon regeneration and pruning at each observation time point for individual axons (not including degeneration on day 0). Axons #1 and #2 were from the same mouse. (F) Net axon growth per branch. ∗p < 0.05, Student’s t test, n = 3. Error bars represent ± SEM. See also Figure S4. Neuron 2015 86, 947-954DOI: (10.1016/j.neuron.2015.03.061) Copyright © 2015 Elsevier Inc. Terms and Conditions