All surgical and animal care protocols were approved by the Temple University School of Medicine’s Institutional Animal Care and Use Committee and performed per the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Sprague–Dawley rats (65–75 days, 200–224 g; Harlan Laboratories) were housed two per cage, on a 12 h light-dark cycle with food and water provided ad libitum. Animals were allowed 7 days of acclimatization prior to any experimental procedure. At the time of surgery, animals ranged in weight from 225 to 250 g. To prevent infection after surgery, animals received an IP injection of 0.5 ml Cefazolin (10 mg/ml). All animals received a subcutaneous injection of 2–3 ml of 0.9% NaCl solution to prevent dehydration following surgery. Analgesia was provided by twice daily oral administration of Rimadyl tablets (1 mg) for 3 days beginning immediately postoperatively.

AAV2-GFP-P2A-mitoDsRed was generated by subcloning the mitoDsRed from the original adenovirus vector (Nasr et al., 2008) into MluI and XhoI sites downstream of the P2A of pAM-CBA-eGFP-P2A. To generate AAV2-eGFPLC3-P2A-mitoDsRed the GFP coding region was removed from AAV2-GFP-P2A-mitoDsRed plasmid and replaced with eGFPLC3 (Addgene: #22405). Purified AAV plasmid was packaged using the helper-free method as reported previously (Ayuso et al., 2010; Liu et al., 2014). In brief, HEK293T cells at 70–80% confluency were transfected with two packaging plasmids, one carrying AAV rep and cap, the other with AAV helper functions, and the transgene using a polyethylenimine method (PEI; polyethylenimine, linear, MW 25k, Warrington, PA). Three days post transfection, cell supernatant, and lysates were harvested. 40% PEG 8000 was added to precipitate crude virus for 2 h. AAV samples were double-ultracentrifuged in a cesium chloride gradient with isolated viral fractions dialyzed in 0.1 M PBS/0.5% Sorbital overnight (Ayuso et al., 2010; Liu et al., 2014). Purified fractions were added to HEK293T cells to verify function. Quantitative real-time PCR of purified viral fractions was done to determine viral titer. The viral titer of AAV2 was 1.2 × 10¹³ GC/ml.

At the completion of each experiment, all animals were euthanized by injection of Fatal-Plus (Dearborn, MI, United States) and perfused with saline (0.9% NaCl) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.5). The brain and spinal cord were promptly dissected. Spinal cord samples were dissected with dorsal roots intact for identification of each spinal level. Samples were post-fixed in 4% PFA overnight at 4°C. Tissue samples were then transferred to a series of graded sucrose (10%, 20%, and 30%) where they remained for the following 24 h or until the brain section sunk to the bottom of the container. A 3 cm section of the spinal cord with the lesion at the center was removed from the whole spinal cord. The spinal cord samples were serially sectioned, sagittally at 30 μm intervals using a Leica CM3050S cryostat. Sections were collected and placed into cryoprotectant solution, then stored at −20°C until processed for histology.

To amplify the GFP and DsRed signals, sections were permeabilized in 0.3% Triton X-100 with 5% normal goat serum to block non-specific binding sites. Samples were then incubated with chicken-anti-GFP primary antibody (1:1,000; #GFP-1020; Aves Labs Inc., Tigard, OR, United States) and rabbit-anti-DsRed primary antibody (1:500; #632496; Takara Bio USA, Inc., Mountain View, CA, United States) overnight at 4°C. The next day, samples were incubated with donkey-anti-chicken-AlexaFluor 488 sary antibody (1:400, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, United States) and goat-anti-rabbit-AlexaFluor 594 sary antibody (1:400, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, United States) then mounted on glass slides.

Imaging was performed using AxioVision software (Carl Zeiss Microscopy, Thornwood, NY, United States). A 100× /1.3 N/A objective with a ~300 nm focal plane was used to generate Z-stacks using 250 nm Z-intervals through 30 μm of tissue sections. In order to maximize the power of the morphological analysis, 1 × 1 camera binning was used to generate the highest spatial resolution in the images. Max-intensity 2D projections were then generated using sets of sections from the Z-stack that contained mitochondria.

Chicken dorsal root ganglia (DRG) were dissected at embryonic day 7 (E7) or day 14 (E14; SPF eggs obtained from Charles River Laboratories). At this developmental stage, it is not possible to determine the sex, and both sexes were used at presumably a 50/50 ratio. Whole explants were cultured (3–4 explants/dish), or the DRG neurons were dissociated and transfected (as explained below; 1.5 explants/dish). The culturing substrata were previously coated with polylysine [Sigma; Catalog number (Cat#) P9011; 100 μg/ml in borate buffer], for 4 h and following 3× washing with phosphate-buffered saline (PBS) with 25 μg/ml laminin (Life Technologies Cat# 23017-015) in PBS overnight, all incubations were performed at 39°C. Explants and dissociated neurons were cultured in a defined F12H medium (Gibco; Cat#21700075) supplemented with NGF (20 ng/ml). For live imaging experiments, explants or dissociated neurons were plated in glass-bottom dishes.

For the preparation of dissociated neurons, sensory ganglia were incubated in Ca²⁺–Mg²⁺-free PBS (CMF-PBS), for 10 min at 37°C. Ganglia were then spun down for 1 min, and the supernatant was removed. Ganglia were then treated with 0.25% trypsin (Fisher Scientific Cat# MT25005CI), for 10 min at 37°C and spun down for 1 min. Ganglia were then pipette triturated 30 times in F12HS10 media (F12H medium supplemented with 10% fetal bovine serum: Fisher; Cat#MT350111CV) and then spun down for 4 min. The supernatant was removed and cells were transfected as described below.

Rat hippocampal neurons were dissociated from dissected E18 rat hippocampi as previously described (Thomas et al., 2012). Neurons were cultured in Neurobasal media supplemented with B27 (Thermo Fischer Scientific) in glass-bottomed dishes coated with polylysine [Sigma; Catalog number (Cat#) P9011; 100 μg/ml in borate buffer] and laminin (Life Technologies Cat# 23017-015; 25 μg/ml) as described above.

Adult rat DRG dissociated cultures were prepared as described previously (Pacheco et al., 2020).

For transfection of plasmids into neurons, 40 chicken DRGs were dissociated as described above, and after F12HS10 was removed neurons were suspended in 100 μl nucleofector solution (Lonza Cat# VPG-1002) and gently resuspended through trituration. The DRG cell suspension was transferred to a nucleofector cuvette containing 10 μg of Plasmid DNA and electroporated using an Amaxa Nucleoporator (program G-13). The electroporated solution was then immediately transferred to a tube containing F12H media as described above prior to plating.

For labeling mitochondria to determine mitochondrial fission rates, neurons were transfected with pDsRed2-Mito (Clontech Cat# 632421) as explained above. To measure mitochondrial length, mitochondria were labeled with mitochondria targeted dyes, which were prepared according to the manufacturer’s directions. Labeling was performed through incubation for 30 min with MitoTracker green (50 nM, Molecular Probes, Cat# M7514) or Mitoview 633 (20 nM, Biotium^®, Heyward, USA; Cat# 70055-T). Following labeling, the dye-containing medium was removed and cultures were washed three times with culturing medium not containing any dye. Imaging was performed at least 30 min after the dye was removed to allow cultures to acclimate.

For experiments in which DRG explants were used, explants were plated in glass-bottom dishes as described above and cultured for 48 h. Dishes were placed on the microscope stage for 10 min prior to severing. Axons were severed by dragging a No. 11 scalpel blade across the axon perpendicular to its direction of growth.

For experiments in which dissociated, transfected neurons were used, neurons were cultured in glass-bottom dishes as described above for 24 h, with the exception of neurons transfected with GCaMP6, which were cultured for 72 h (the time at which expression of GCaMP6 was optimal). Dishes were placed on the stage 10 min prior to severing. Severing was performed as previously described (Gallo, 2004). Briefly, only axons that exhibited a “healthy” uniform caliber axon were used in single-axon-severing experiments. The severing of axons was performed approximately 200 μm from the axon tip. For severing, borosilicate glass capillaries (AM Systems, Carlsborg WA; catalog # 625500) were pulled to fine tips using a Pul-1 micropipette puller (World Precision Instruments, Sarasota FL). Capillary needles were mounted on a mechanical micromanipulator (Leica Inc., Bannockburn, IL). The tip was then brought into contact with the substratum and axons severed by maintaining the position of the tip steady while moving the stage relative to the tip. The stage was moved manually in a continuous swift motion resulting in the uniform severing of axons.

Neurons were imaged using a Zeiss 200 M microscope equipped with an Orca-ER camera (Hamamatsu) in series with a PC workstation running Zeiss Axiovision software for image acquisition and analysis. Cultures were placed on a heated microscope stage (Zeiss temperable insert P with objective heater) for 10 min at a constant 39°C before and during imaging. Imaging was performed using a Zeiss Pan-Neofluar 100_objective (1.3 N.A.), 2 × 2 camera binning, and minimal light exposure. For the quantification of mitochondria length, mitochondria labeled with MitoTracker green (Chicken sensory neurons) or Mitoview 633 (rat hippocampal neurons), were imaged at 30 min intervals over a 180 min timeframe. For mitochondrial fission and attempted fission rate quantification, neurons were co-transfected with pDsRed2-Mito and either pEYFP-C1-Drp1 or pEYFP-C1-Drp1K38A [previously generated in our lab (Armijo-Weingart et al., 2019)]. Neurons were then imaged for 250 frames with a 3 s interframe interval. For analysis of changes in cytosolic and mitochondrial calcium, neurons were transfected with either GCaMP6 or mitoGCaMP2. Neurons were then imaged for 16 frames with a 1 min interframe interval.

Imaging was performed as described in the tissue imaging and live imaging sections above. The length of mitochondria in the axon was measured using ImageJ software. Mitochondria length was determined by a line, segmented if the mitochondrion was not strictly linear, running from one end to the other of the mitochondrion. Occasionally, mitochondria in axons overlapped as clearly evidenced by the additive signal of their fluorescence in the region of overlap. Overlapping mitochondria were excluded from the analysis to ensure only mitochondria with clearly defined ends were used for quantification.

Instances of fission were determined manually by the user through frame-by-frame analysis. Using the settings described in the live imaging section each pixel in the resulting images has a radius of 0.12 μm. A completed fission event was defined as when the fluorescence intensity of the mitochondrion at the site of apparent fission had decreased to the background level of the axonal segment not containing mitochondria and the two resultant mitochondria were separated by over 5 pixels representing 0.6 μm. Attempted fission events were defined as mitochondria undergoing constrictions but not continuing into a full fission event, as the constriction sites returned to exhibit the prior width and intensity.

All analyses were performed using Zeiss Axiovision software. For GCaMP6, regions of interest (ROIs) were drawn around the cut axon tip and 50 μm proximal to the cut. For mitoGCaMP2, ROIs were drawn around axonal mitochondria proximal to the cut site. Image acquisition was performed using excitation filters of 405 nm and 488 nm (Need specifications on Filters). Fluorescence time courses were computed as the mean pixel intensity of all the pixels within the ROI. At each timepoint, fluorescence intensity for both excitation filters (F₄₀₅ and F₄₈₈) was calculated as mean fluorescence within the ROI, F, relative to the background F₀ (F − F₀). Data is reported as a ratio of F₄₈₈/F₄₀₅.

All data were analyzed using Instat software (GraphPad Software Inc.). The software determines the normalcy of data sets using the Kolmogorov-Smirnov test. All data sets were determined to be non-normally distributed, therefore non-parametric analysis was used (Mann-Whitney test). For multiple comparison tests within experimental designs, non-parametric Dunn’s post hoc tests were used. For data sets representing categorical data falling into bins the Fischer’s exact test was used on the raw data, although the data are expressed as percentages for ease of appreciation. One or two tailed p values are reported based on whether the hypothesis did or did not dictate the directionality of the expected change in mean or median, respectively. All graphs were generated using Prism (GraphPad Software Inc.). Sample sizes and qualitative statistical presentation are denoted in figure legends or figures.

To assess axonal mitochondrial morphology following spinal cord injury, the mitochondria of CST neurons of adult Sprague–Dawley rats were labeled with mitochondrially targeted DsRed (mtDsRed) 4 weeks before performing dorsal column lesions at the C4-C5 level. Labeling was achieved by injecting adeno-associated virus co-expressing GFP and mitoDsRed (mitoDsRed/GFP/AAV) in the forelimb and hindlimb somatomotor cortex (Figures 1A,B). The mitochondria in non-injured axons at the C4-C5 level had a median length of 3.15 μm (Figure 1C). Mitochondria in C4-C5 CST axons severed following dorsal column lesion were analyzed in 100 μm bins from the site of injury as a function of time following injury. At 2 h following injury, mitochondria closest to the site of injury exhibited decreased length, while mitochondria more proximal along the injured axon maintained longer lengths although still decreased relative to controls (Figure 1C). Very small mitochondria in the vicinity of the injury site persisted for up to 3 days following injury (Figure 1C). Recovery of median mitochondrial length began by 1 week after injury with normal lengths almost fully restored by 2 weeks (Figure 1C; see Supplementary Figure 1 for data point distributions of the medians presented in Figure 1C).

Mitochondria in the <0.5 μm range are considered dysfunctional (Reddy, 2014; Galluzzi et al., 2016; Hu et al., 2017). In uninjured axons, less than 0.5% of mitochondria were shorter than 0.5 μm and only 4.4% of mitochondria fell within the 0.5–1.0 μm range. Herein, mitochondria with length <0.5 μm are defined as fragments, while mitochondria 0.5–1.0 μm are considered to be in the low normal range for length. By 2 h following injury, the percentage of mitochondria in the submicron range dramatically increased resulting in 53% of mitochondria displaying lengths <0.5 μm and 32% with length between 0.5 and 1.0 μm (Figure 1D). The presence of increased mitochondrial fragments persisted until 3 days following injury before returning to non-injury control levels by 1-week post-injury (Figure 1D). Fragmented mitochondria were identified throughout the distal region of severed axons and the axon tips. However, the length of mitochondria within retraction bulbs, defined as the distal end of the axon exhibiting a bulbous rounded morphology, relative to those in non-retraction bulb segments of axons, continued to exhibit sub-μm fragmentation up to at least 2 weeks (Figures 1E,F).

Fragmented dysfunctional mitochondria are removed through mitophagy1 (Ashrafi and Schwarz, 2013). To determine if the mitochondrial fragmentation along CST axons observed after spinal cord injury correlates with increased mitophagy, we examined the association of mitochondria with the autophagocytic marker LC3 (Maday, 2016; Maday and Holzbaur, 2016). Adult Sprague-Dawley rats were injected with AAV co-expressing GFP-LC3 and mitoDsRed into the somatomotor cortex as described above. Four weeks after injections, the rats received a dorsal column lesion at the C4-C5 level to sever labeled CST axons. Line scans of fluorescent intensities from randomly selected axons were analyzed to measure the degree of LC3 association with mitochondria. Within the C4-C5 level of non-injured rats, GFP-LC3 florescence (Figures 2A,B, green line) was mostly uniformly distributed with few regions showing increased pixel intensity associated with DsRed labeled mitochondria (Figure 2A, red line). Prior to the injury, we observed that only 7.1% of axonal mitochondria were associated with localized increases in LC3. At 2 h after injury, 18.5% of mitochondria were associated with localized increases in the levels of LC3 (Figures 2C,F; hereafter referred to as LC3+ mitochondria). The increase in LC3+ mitochondria reached its peak 1 day after injury (31.8% LC3+ mitochondria) but continued until 3 days after injury (22.5% LC3+ mitochondria) before returning to non-injury control levels 7 days after injury (10.1% LC3+ mitochondria; Figure 2D). The percentage of LC3+ mitochondria was not dependent on the distance from the injury site, as the percentage of LC3+ mitochondria was the same at all distances from the injury site at each time point (Figure 2E). These observations indicate that there is an increase in mitophagy that correlates with mitochondrial fragmentation up to 3 days following spinal cord injury. To determine if the association of GFP-LC3 accumulation along axon with mitochondria might be due to volumetric or non-specific changes, we considered the association of GFP accumulation with mitochondria. For this analysis we focused on 1 day after the injury as at this time point reflects the peak value of the percentage of LC3+ mitochondria (Figure 2D). Using the same criteria and analysis as for GFP-LC3, at 1 day after injury 8.1% of mitochondria (n = 20 axons, 148 mitochondria) were found associated with accumulation of GFP. These data indicate that the accumulation of GFP-LC3 noted in the injury conditions is not non-specific.

We next sought to assess the role of Drp1 mediated fission in the injury-induced mitochondrial shortening observed in vivo. mDivi-1 is a cell and blood-brain barrier permeable pharmacological inhibitor of Drp1 that blocks mitochondrial fission both in vitro and in vivo (Lackner and Nunnari, 2010; Li et al., 2016; Smith and Gallo, 2017). Adult Sprague-Dawley rats were injected with mitoDsRed/AAV in the somatomotor cortex 4 weeks prior to CST severing at the C4-C5 level. mDivi-1 or vehicle was delivered via IP injections at 12 h before injury, and again immediately post-injury for the 1 day survival group, similar to published in vivo protocols (He et al., 2016). Mitochondria were then analyzed at 2 h and 1-day post-injury in the distal 0–300 μm of axons. mDivi-1 treatment decreased injury-induced mitochondrial fragmentation at 2 h and 1 day after injury. At 2 h, mDivi-1 treatment resulted in mitochondria with intermediate lengths, ranging between the non-injury and injury conditions (Figure 3A). However, the 1-day treatment group exhibited mitochondrial lengths not significantly different than non-injured controls (Figure 3A). Analysis of the percentage of mitochondria in the fragmented range (<0.5 μm) and lower end of the normal range (0.5–1 μm) revealed that mDivi-1 treatment prevented fragmentation at both 2 h and 1 day (Figure 3B).

The fragmentation of axonal mitochondria was rapid and persistent in retraction bulbs even at longer times after injury. In order to determine whether mitochondria fragmentation could be a component of the mechanism that generates or supports retraction bulbs, the effects of mDivi-1 treatment on bulbs at the end of injured axons was examined. Adult Sprague-Dawley rats were injected with mtDsRed/GFP/AAV into the somatomotor cortex 4 weeks prior to the severing of CST axons at the C4-C5 level. The rats received IP injections of either mDivi-1 or vehicle the day before injury and once per day for 7 days after injury. Six weeks following injury, the animals were euthanized, and the spinal cord segments were analyzed. Vehicle injected controls showed cavitation at the injury site, axonal retraction, and numerous retraction bulbs at the distal tips of injured CST axons (Figure 3C). Consistent with a prior report (Li et al., 2016), animals treated with mDivi-1 showed a decreased lesion volume with lesioned axons adjacent the lesion site. In addition, there was a 73% decrease in the number of retraction bulbs observed at axon tips (Figure 3D).

In order to perform mechanistic studies not amenable to in vivo examination and to determine whether the response of axonal mitochondria to axon severing may be cell-intrinsic within the cut end of axons, we sought to set up in vitro model systems for examining mitochondrial response to axonal injury. We investigated mitochondrial morphology after in vitro axonal injury using neurons from two species and representing early embryonic, late embryonic, and adult stages: (1) Embryonic day (E)7 and E14 chicken DRG explants, reflective of developmental stages when sensory axons are undergoing extension and have reached their proximal targets respectively (Figures 4A–D). (2) E18 rat dissociated hippocampal neurons (Figure 4E) and (3) adult rat DRG dissociated neurons (Figure 4F).

Mitochondria within embryonic chicken sensory axons were labeled with MitoTracker Green dye and axons were severed using a scalpel blade after 48 h in culture when axons attained lengths between 1–1.5 mm. The mitochondrial length was monitored using live imaging. Prior to scalpel axotomy, axonal mitochondria showed median lengths of 1.63 μm and 2.41 μm in E7 and E14 neurons, respectively (Figures 4A–D), consistent with the prior demonstration of increased length in E14 neurons relative to E7 neurons (Armijo-Weingart et al., 2019). Following axotomy, both E7 and E14 axons showed a significant reduction in mitochondrial length 30 min after injury. Mitochondrial length began to recover between 120 min and 150 min post-injury before returning to pre-injury levels 180 min following injury (Figures 4C,D). The recovery from injury was faster in E7 (120 min) than E14 axons (180 min). E18 hippocampal neurons were cultured for 3 days, the time point at which axons attained lengths between 300 and 400 μm (stage III of in vitro development). Axonal mitochondria were labeled with Mitoview 633. Mitochondrial length decreased 30 min after injury and did not recover fully until at least 180 min (Figure 4E). Similar analysis using adult rat DRG neurons with mitochondria labeled using MitoTracker Green showed that lengths were decreased one hour after injury and the decrease in length persisted until sometime after 180 min (Figure 4F).

We next tested whether injury-induced mitochondrial shortening in sensory axons is Drp1 dependent in vitro. E14 chicken DRG explants were incubated with either DMSO (control) or 20 μM mDivi-1 for 30 min prior to scalpel blade axotomy. Mitochondria within sensory axons were labeled with MitoTracker green. Following axotomy mitochondrial size was monitored for 180 min. As with untreated neurons (Figure 4D), DMSO treated neurons demonstrated prominent mitochondrial shortening 30 min after injury before gradually returning to pre-injury lengths by 180 min. Neurons treated with mDivi-1 did not show mitochondrial shortening at any time point following axotomy (Figures 5A,B). Additionally, we tested whether injury-induced mitochondrial shortening in hippocampal neurons is Drp1 dependent in vitro. Axonal mitochondria length was measured in E18 hippocampal neurons (as in Figure 4E) treated with either DMSO or 20 μM mDivi-1 30 min before severing using a micro-needle as previously described (Gallo, 2004). The mitochondrial length was monitored using live imaging. Prior to axotomy, DMSO treated neurons displayed axonal mitochondria with a median length of 1.86 μm. The median mitochondrial length was significantly reduced at 30 min after axotomy. By 180 min following injury, median mitochondrial length was still significantly smaller than before axotomy but was trending toward recovery of pre-axotomy length (Figures 5C,D). Neurons treated with 20 μM mDivi-1 showed no change in median mitochondrial length over the 180-min monitoring period (Figure 5C). These observations indicate that injury-induced mitochondrial shortening in vitro is a Drp1 dependent process.

To further test the hypothesis that mitochondria shortening is due to Drp1 mediated fission, we expressed a dominant-negative Drp1 mutant (Drp1K38A) that lacks GTPase activity and is, therefore, unable to carry out mitochondrial fission (Pitts et al., 1999; Barsoum et al., 2006). E14 chicken DRGs were transfected with either YFP-Drp1 (control) and mitoDsRed or YFP-Drp1K38A and mitoDsRed. Single axon axotomy was performed using a micro-needle as done previously (Gallo, 2004). Because transfection with Drp1K38A allows for unopposed mitochondrial fusion, mitochondria become elongated (20–30 μm or larger) in sensory neurons by 24 h of expression (Armijo-Weingart et al., 2019). Thus, comparison of mitochondrial length after axotomy between control and Drp1K38A expressing neurons is not a reasonable analysis. We, therefore, measured the number of fission events and attempted fission events per 10 microns of mitochondria per min for each group monitored over a 15-min period. As previously described (Armijo-Weingart et al., 2019), a fission event was defined as when the fluorescence intensity of the mitochondrion at the site of apparent fission had decreased to the background level of the axonal segment not containing mitochondria and the two resultant mitochondria were separated by over 5 pixels representing 0.6 μm (Figure 5E). Attempted fission events were defined as mitochondria that underwent apparent constrictions but did not continue into a full fission event, as the constriction sites returned to exhibit the prior width and intensity (Figure 5E). It should also be noted that overexpression of WT Drp1 does not increase mitochondrial fission events at baseline (Armijo-Weingart et al., 2019). Axotomy resulted in increased fission and attempted fission events in control YFP-Drp1 expressing neurons (Figures 5F,G). The increases in fission and attempted fission events in response to axotomy were abolished by expression of YFP-Drp1K38A (Figures 5F,G).

Following axotomy, there is an influx of calcium into the axon, which is important in the formation of either a retraction bulb or a new growth cone (Bradke et al., 2012). We first transfected E7 chicken DRG neurons with GCaMP6, a cytosolic, ratiometric calcium-sensing protein (Chen et al., 2013). Individual axons were severed using a micro-needle, as above, and calcium changes were monitored over a 15-min period with live imaging. As expected axotomy caused a sustained influx of calcium resulting in a two-fold increase in GCaMP6 fluorescence (Figures 6A,B). To determine whether intra-mitochondrial calcium levels are increased following axotomy, E7 chicken DRG neurons were transfected with a mitochondrially tagged calcium-sensing protein, mitoGCaMP2. We monitored mitochondrial calcium levels for 15 min following axotomy, observing a steady increase in mitochondrial calcium culminating in a three-fold increase in mitoGCaMP2 fluorescence at 15 min post axotomy (Figures 6C,D). To determine if this increase in mitochondrial calcium was dependent on injury-induced fission, we pretreated neurons with either DMSO or 20 μM mDivi-1 as above. Blocking fission following axotomy had no effect on mitochondrial calcium demonstrating that this was not a fission-dependent process (Figure 6C). Given the increase in cytosolic calcium after axotomy, we next addressed if injury-induced mitochondrial fission may be dependent on an increase in mitochondrial calcium. Calcium is taken up into mitochondria through the mitochondrial calcium uniporter (MCU) present on the mitochondrial inner membrane (Gunter and Gunter, 1994). RU360 is an established inhibitor of calcium fluxes through MCU (Matlib et al., 1998; Zhou et al., 1998; Vanderluit et al., 2003). E7 chicken DRG explants were treated with either 10 μM RU360 or vehicle and mitochondria were labeled with MitoTracker Green 30 min prior to scalpel axotomy. Following axotomy, mitochondrial size was monitored for 30 min using live imaging. Application of RU360 had no effect on mitochondrial size in the absence of axotomy during the 30-min imaging period (Figure 6E). At 30 min after axotomy, vehicle treated neurons displayed a significantly reduced median mitochondrial length when compared to mitochondrial length prior to axotomy, while neurons treated with 10 μM RU360 displayed no change in median mitochondrial length (Figure 6E). These observations indicate that ion fluxes through MCU are a required component of the mechanism that drives injury-induced mitochondrial fission in vitro. In order to determine whether mitochondrial ion fluxes through MCU could also have a role in the in vivo response of axonal mitochondrial to spinal cord injury, adult Sprague-Dawley rats were injected with mtDsRed/GFP/AAV into the somatomotor cortex 4 weeks prior to the severing of CST axons at the C4-C5 level. Immediately following injury, gel foam soaked in either vehicle or 10 mM RU360 was placed at the site of injury. The animals were euthanized either 2 h or 24 h following injury and mitochondrial size as a function of time after injury and distance from the site of injury was analyzed as previously described. Treatment with RU360 resulted in longer mitochondria after injury compared to time-matched controls (Figure 6F). However, in all cases, the length of mitochondria was also shorter from that of uninjured controls (Figure 6F). Animals treated with RU360 showed greatly attenuated fragmentation in the vicinity of the injury site (0–100 micron) at either 2 h or 24 h post-injury (red percentages and p values in Figure 6F), indicating that mitochondrial ion fluxes through MCU are also a required component of the mechanism driving injury-induced mitochondrial fission in vivo.