Calcium chelation promotes microtubule regrowth and axonal recovery after laser-induced axonal injury

Calcium chelation promotes microtubule regrowth and axonal recovery after laser-induced axonal injury | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Calcium chelation promotes microtubule regrowth and axonal recovery after laser-induced axonal injury View ORCID Profile Ashish Mishra , Pooja Joshi , Pramod Pullarkat doi: https://doi.org/10.1101/2025.08.08.668418 Ashish Mishra 1 Raman Research Institute , C V Raman Avenue, Bengaluru, 560080, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ashish Mishra Pooja Joshi 1 Raman Research Institute , C V Raman Avenue, Bengaluru, 560080, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pramod Pullarkat 1 Raman Research Institute , C V Raman Avenue, Bengaluru, 560080, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: pramod{at}rri.res.in Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Axons that are damaged locally due to stretch or crush injury often undergo widespread and irrecoverable damage. Transient elevation of cytosolic free calcium across extended regions of the axon, and calcium mediated disintegration of cytoskeletal elements is one of the main causes of this neurodegeneration. When axonal damage involves complete transection of axons, any recovery has to be driven by the formation of fresh motile tips (growth-cones). However, axons could also undergo partial damage where the axonal plasma membrane retains its continuity while cytoskeletal elements undergo extensive disintegration triggered by elevated free calcium. We invoke such a condition using a partial laser ablation technique, and we show that complete axonal recovery, mainly mediated by microtubule regrowth, is possible in such cases when extracellular calcium is depleted. This method allows us to explore the dynamics of cytoskeletal damage after injury and the modes of recovery of microtubules and actin filaments. Apart from understanding the mechanisms of cytoskeletal damage and recovery, our results can have significant impact on devising treatment strategies for crush injuries to nerves. Introduction Neuronal cells are highly susceptible to damage caused by mechanical stress [ 1 ]. Impacts or sudden acceleration of the head can lead to concussion, diffuse axonal injury (DAI), or traumatic brain injury (TBI) [ 1 – 3 ]. Axons of the brain are particularly vulnerable as shear/stretch deformation of the brain leads to stretching of the axons that form intricate networks within the brain [ 4 , 5 ]. Injuries to the spinal cord or to the peripheral nerves are also common, either due to local compression or local stretch during accidents, excessive limb movement, dislocation, breech delivery, or during surgical manipulation [ 6 – 12 ]. Such injuries form a leading cause of death and debilitating disabilities, especially among the young [ 13 ]. Stretch or compression-induced damage to axons can occur either due to the direct disruption of axonal cytoskeletal elements or structural degradation triggered by molecular events [ 4 , 7 , 14 ]. Calcium influx into the axons is a key event that is well known to result in widespread axonal damage after injury [ 14 ]. This influx can occur via the opening of stretch-sensitive ion channels, reversal of Na + -Ca ++ exchanger, voltage-gated ion channels, or via pore formation in the axonal membrane [ 15 – 17 ]. Calcium entry can trigger a series of cascade events which drastically augment damage [ 14 ]. It triggers the release of calcium from major internal stores like the endoplasmic reticulum, and this calcium induced calcium release spreads rapidly along the axon away from the site of injury [ 18 , 19 ]. Following this short timescale molecular event, elevated calcium activates proteases like calpain. Calpain cleaves cytoskeletal proteins such as spectrin, neurofilaments, and microtubule-associated proteins, to initiate a slower and more widespread phase of cytoskeletal degradation within the axon [ 14 , 20 , 21 ]. Other mechanisms like mitochondrial failure and oxidative stress can amplify damage [ 22 , 23 ]. This degradation disrupts axonal transport, causes axonal swelling, and ultimately leads to complete degeneration of the axon [ 23 ]. Understanding calcium’s role offers insights into potential therapeutic strategies to limit secondary injury. Currently, treatment strategies targeting calcium dysregulation, calpain activation, and oxidative stress have emerged as promising approaches to mitigate neuronal damage in conditions like diffuse axonal injury and nerve stretch injury. However, a detailed understanding of the ultrastructural events that occur post-injury and potential recovery mechanisms post-treatment is still lacking. Current laboratory methods include using stretchable substrates [ 4 ], transection of axons, or using mechanical devices [ 24 – 27 ] or using lasers [ 28 , 29 ]. While such techniques are valuable in understanding the mechanisms of axonal regeneration, the complex morphological changes that ensue make it difficult to delineate specific calcium-dependent cytoskeletal processes that follow damage. Here, we present a laser ablation method which can induce local damage to the axonal cytoskeleton and trigger a calcium response while maintaining the axonal plasma membrane connectivity. We show that such damage results in a catastrophic loss of cytoskeletal support to the axon, resulting in complete axonal atrophy. However, axonal degradation can be mitigated or even reversed if extracellular calcium is chelated, thereby preventing calcium elevation within the axon. The partial laser ablation method we employ allows for detailed quantitative imaging of the cytoskeletal changes that follow localised damage with or without calcium chelation. We show that actin based structures and microtubules exhibit different degradation patterns post-ablation. More remarkably, a subset of axons exhibit complete recovery when extracellular calcium is chelated, with actin filaments and microtubules showing starkly different recovery patterns. Further, we show that recovery of actin filaments alone does not guarantee axonal recovery, whereas microtubule regrowth can lead to full recovery of damaged axons. Materials and Methods Cell culture medium L-15 medium (21083–027, Thermo Fisher Scientific) was made viscous using autoclaved methylcellulose (H7509-100 g; Sigma-Aldrich, Darmstadt, Germany) at a ratio of 100 ml to 0.6 g by stirring overnight at 4 ° C. This was then supplemented with 10% (v/v) heat-inactivated fetal bovine serum (10100; Gibco), 2% (v/v) 33.3 mM glucose (G6152; Sigma-Aldrich, St. Louis, MO), 20 ng per ml Nerve Growth Factor (13290-010; Invitrogen, Carlsbad, CA) and 10 µl/ml Penicillin-streptomycin-glutamine (10378-016, Gibco). Neuronal cell culture Fertilised Giriraja-2 chicken eggs were acquired from the Karnataka Veterinary, Animal and Fisheries Sciences University, Bangalore, India. Eggs were incubated at 37 ° C for 8-9 days and dissected in HBSS buffer (14025-092, Gibco) under a sterio microscope to isolate Dorsal Root Ganglia (DRGs). After extraction, DRGs were rinsed in the HBSS buffer lacking Ca ++ or Mg ++ (14025-092, Gibco) (14175-095, Gibco), incubated at 37 C with 0.5% Trypsin-EDTA (15400-054, Gibco) for 10 min, and then dissociated by gentle pipetting. The cells were seeded on a clean, uncoated glass coverslips. Cells were incubated at 37 ° C for 96 hr to allow for growth. It is known that by this stage, the membrane associated periodic skeleton is fully developed [ 30 ]. Before performing ablation experiments, neurons were incubated for 30 min in L-15 medium lacking methylcellulose but containing all the other supplements mentioned above. Laser ablation setup and imaging A home-built laser ablation setup was used to perform the partial ablation experiments. This setup consists of a 355 nm, 25 µJ, 350 ps pulsed laser (PowerChip PNV-M02510-100; Teem Photonics, Meylan, France) coupled to the side port of a Leica TCS SP8 confocal microscope using a custom filter-cube and focused on the sample using 40 × dry Phase Contrast objective. A custom-made 90 ° rotated filter cube having a UV-reflecting dichroic filter (T387lp-UF3, Chroma) was used to reflect the laser light into the objective. During and after ablation, Phase Contrast images were recorded using a CCD camera (DFC365 FX, Leica) at 15 fps initially and then at 6 fps to capture the initial fast and subsequent slower retraction process of the ablated axons. Calcium imaging and chelation To detect any elevation of calcium levels, cells were preloaded with Fluo-4 AM (F14217, Invitrogen) at 0.5 µM concentration and 20 min of incubation. Chelation of extracellular or intracellular calcium was done using EGTA (03777-10G, Sigma-Aldrich) at 5 mM or BAPTA AM (B6769, Invitrogen) at 10 µM, respectively. The EGTA stock solution was prepared in Millipore deionised water at a concentration of 100 mM. The working concentration of 5 mM was prepared in L15 medium lacking methylcellulose. The pH of the working solution was adjusted to within 7.2–7.3 by adding either NaOH or HCl and measured using a pH meter. BAPTA was prepared using DMSO as a solvent. The control experiment for Ca ++ imaging was done using DMSO as a vehicle. Images were acquired using a Leica TCS SP8 confocal system, keeping all the imaging parameters exactly the same before and after ablation. Visualization of cytoskeletal dynamics SPY555-Tubulin dye (SC203, Spirochrome, Switzerland) and SPY650-FastAct dye (SC505, Spirochrome, Switzerland) were used to stain microtubules and actin filaments, respectively. These dyes are specific to these biopolymers and do not label tubulin or actin unless they are in polymerized form. The neurons were incubated with the dye using L15 media lacking methylcellulose at a concentration of 1:1000 v/v for 30 min to 1 hour before starting the ablation experiments. EB3 Transfection and live-cell imaging To visualize the dynamics of microtubule plus-ends, primary dorsal root ganglia were dissected from 9-day-old chick embryos and transfected with the pCAG-mNeon-EB3 construct via electroporation using a Nepagene electroporator. After electroporation, the cells were plated onto uncoated glass coverslips and maintained in a neuronal growth medium. Following 4 days in vitro (DIV), live-cell imaging was performed using a Leica TCS SP8 confocal microscope in widefield fluorescence mode with a 63 ×/1.4 NA oil immersion objective. The mNeon fluorescence was detected using a GFP filter cube (excitation: 450-490 nm, emission: LP 515 nm). Time-lapse sequences were acquired at 5-second intervals to capture the dynamics of EB3-decorated microtubule plus-ends. Kymographs were generated from the time-lapse data using the KymoResliceWide plugin in FIJI/ImageJ software. Results Partial laser-ablation of axons Axonal transection studies using lasers are usually performed by locally ablating the axon using a pulsed laser of sufficient pulse energy. Here we use a method where an axon is ablated with a lower pulse energy than that is required for complete transection so that the mechanical continuity of the cytoskeletal components can be compromised without causing any catastrophic damage to the membrane. In such cases, one can observe two retraction fronts propagating away from the ablation point–one towards the growth-cone and the other towards the soma (see Fig. 1a and Video 001). Partial ablation events are easily distinguishable from complete transections as (i) a thin membrane connection is visible between the retracting fronts when imaged in phase contrast mode, and (ii) the two retracting ends remain more or less aligned along the initial axis of the axon. This is in contrast with complete transection, where the axonal segments exhibit pronounced buckling and the ends “fly-away” from the initial axonal axis (Video 002). To assess the extent of damage caused by the laser, we performed control experiment using paraformaldehyde-fixed axons. It such axons, no retraction response is observed and the laser induced damage is limited to less that 2 µ m (Suppl. Mat., Fig. S1, Video 003). Download figure Open in new tab Figure 1: Axonal response to partial laser ablation. (a) Sequence of phase contrast images showing the response of an axon to partial laser ablation. The white arrow indicates the point of ablation and the ablation was performed at time t = 0 s. Post ablation, the axon exhibits two retraction fronts–one propagating towards the soma and the other towards the growth-cone. A thin membrane tube spans the region between these two fronts. The scale bar is 10 µ m. (b) Plots showing the time evolution of the normalized contour lengths of the retracting segments (length of each retracting segment divided by its initial length). Data for individual segments are shown in faded colors and the average of all segments is shown using black circles. Note that the average gets skewed beyond t ≈ 200 s as faster retraction events end early. The gap in the data at around t = 56 s is due to the switching of the recording speed from 15 fps to 6 fps. Shaded region around the average plot represents the standard errors of the mean. The thinness of the tube between the retracting fronts of partially ablated axons suggests that all cytoskeletal components, along with much of the cytoplasm, retract toward either side post ablation. This will be discussed later with the help of fluorescence images. When cells are grown on untreated coverglass, without any adhesion-promoting treatments, most axons tend to be free of the glass surface except at the soma and the growth-cone ends. In such cases, the two retracting fronts of a partially ablated axon retract continuously until all the material is absorbed into either the soma or the growth cone. This can be seen by analyzing the normalized length of each segment and plotting it as a function of time, as shown in Fig. 1b . Partial laser-ablation causes Ca ++ elevation in axons In order to explore the possible role of free calcium in driving axonal retraction after partial ablation, we imaged intracellular free Ca ++ levels using Fluo4-AM dye. As can be seen from Fig. 2a , there is a sudden and significant increase in the intracellular Ca ++ level when the axon is ablated (Video 004). To identify the mechanisms of calcium elevation, we pretreated cells with either EGTA (5mM), which chelates extracellular Ca ++ , or with the cell permeable intracellular Ca ++ chelator BAPTA-AM (10 µ M). These treatments almost completely suppress Ca ++ elevation in ablated axons, and example images of an axon treated with EGTA is shown in Fig. 2b . The quantification for both treatments is shown in Fig. 2c . These data show that the elevation in Ca ++ is triggered by entry of extracellular Ca ++ , possibly at the ablation point, which triggers the release of Ca ++ from internal stores. Measurement of the Fluo4-AM fluorescence intensity shows that the Ca ++ elevation post-ablation is sudden and reaches peak intensity within a few seconds. This is followed by an exponential decay of free calcium levels as can be seen from the inset of Fig. 2c , with a decay time of 42.7 sec. In comparison to this ablation-induced response, mechanical indentation of soma of rat cortical neurons evoked a calcium response in the axon, with a decay time constant of approximately 24.1 sec [ 31 ]. The calcium level seems to decay to a value that is higher than the pre-ablation value but this could be because of the limited observation time in our experiments. Download figure Open in new tab Figure 2: Partial ablation causes Ca ++ elevation in axons. (a) Images of an axon labelled with the Ca ++ indicator Fluo4-AM taken before ablation (above) and immediately after ablation (below). A sudden increase in fluorescence intensity is seen in every ablated axon. The white arrow marks the point of ablation and the scale bar is 20 µ m. (b) Images of an axon incubated in medium containing EGTA and ablated as before. No significant increase in fluorescence is seen in this case. (c) Data showing the normalised fluorescence intensity (I(post)/ I(pre)) for control axons (n = 40), axons grown in medium containing EGTA (n = 47) and for axons treated with BAPTA AM (n = 9). I(pre) and I(post) are the intensities before and just after ablation, respectively. The concentrations used are indicated in the plot. The error bars are standard errors of the mean. (Inset) Plots showing the time evolution of intracellular free calcium for control axons and for axons in medium containing EGTA. Ablation was performed at time t = 0 s. The data points are the averages, and the shaded regions are standard errors of the mean for control axons (n = 5) and EGTA-treated axons (n = 7). A sharp increase in Ca ++ followed by a decay can be seen for the control axon, whereas the EGTA treated axon shows a nearly constant base value of intensity. For control axons, a fit to an exponential function is shown using a solid black line, which yields a Ca ++ decay time of τ = 42.7 sec. Chelation of Ca ++ allows for axonal recovery post ablation A very different morphological response is seen when axons are partially ablated in a medium free of extracellular Ca ++ . When free Ca ++ was chelated using EGTA (5mM), retraction was partial. More remarkably, a subset of axons (10/20 axons) recovered their diameter subsequent to an initial thinning down of their mid-section (see Fig. 3a ). This initial retraction occurred in both the proximal and distal axonal segments, but subsequent recovery happened mostly from the proximal side. This is quantified by calculating the contour length of each segment, and the data for proximal and distal sides are displayed in Fig. 3b . A subset of axons retracts partially after ablation and does not recover within the observation time of about 600 s. Example images and population data for these are shown in Fig. 3c,d , respectively. The retraction velocity is much less for axons in calcium free medium as compared to control axons, as can be seen in Fig. 3d . Download figure Open in new tab Figure 3: Recovery of damage seen in Ca ++ chelated medium. (a) A sequence of phase contrast images showing the development of a damaged region and subsequent axonal resealing. The cell body is to the left of the images and the white arrow indicates the point of ablation. Both proximal and distal axonal segments initially retract from the ablation point, but recovery mainly occurs from the proximal side. The scale bar is 10 µ m. (b) The normalized contour length versus time plot for the axonal segments shows the retraction and recovery for individual segments. The cell body side is shown using circles and the growth-cone side using squares. Segments from the same neuron are shown using the same color. (c) Phase contrast images of an axon which was partially ablated in Ca ++ chelated medium. In this case, the axon has retracted only partially, even after about 400 s. The scale bar is 10 µ m. (d) Normalized contour length for axons ablated in medium containing 5 mM EGTA. As can be seen from the data, these axons retract only partially, and the retraction velocity is much less than that for control axons. The blue squares show the averaged data, and the black circles are the averaged data obtained for control axons. Shaded regions along the average plots indicate the standard errors of the mean. The gap in the data at around t = 57 s is due to the switching of the recording speed from 15 fps to 6 fps. Axonal microtubules regrow and reseal after ablation in Ca ++ -free medium The resealing response seen in calcium chelated medium raises the question as to how the axonal cytoskeleton may be responding under such conditions. Microtubules are found in abundance in chick DRG axons, and so it is logical to check their contribution to the retraction and resealing processes. For this, we labelled microtubules using the cell permeable SPY555-tubulin dye which labels only polymerized tubulin. For control cells grown in medium containing calcium, we see that microtubule intensity recedes in both directions and all the way to the two extremities of partially ablated axons (see Fig. 4a ). This mirrors the retraction response seen in phase contrast images. No detectable fluorescence intensity is seen in the thinned down region, suggesting complete depolymerization of microtubules (see Fig. 4b ), as this region expands with time. A slight increase in intensity is observed at the boundaries of the retracting segments. Download figure Open in new tab Figure 4: Microtubule dynamics in partially ablated axons. (a) Sequence of fluorescence images of an axon labelled with the membrane permeable SPY tubulin 555 dye and partially ablated at the point shown by the white arrow. The dye labels only microtubules and not tubulin dimers. Microtubule intensity retracts along with the retraction front seen in phase contrast images. (b) Plots of the microtubule fluorescence intensity corresponding to the images shown in (a). No detectable intensity can be seen in the thinned down mid section of the axon–the dotted lines indicate background intensity. A slight accumulation of microtubules can be seen at the retraction fronts. (c) Fluorescence images showing the microtubule retraction in an ablated axon where calcium in the medium was chelated using 5 mM EGTA. In this case, microtubules retract only partially and then remain static for about 25 min (observation time). (d) Plots showing the time evolution of the intensity reduction in the thinned out regions for the axon shown in (c). (e , f) Image sequence and intensity plots for an axon that exhibited only partial recovery of microtubules when ablated in Ca ++ chelated medium. (g , h) Fluorescence images and intensity plots for an axon that showed complete recovery of microtubule intensity subsequent to an initial retraction phase when ablated in Ca ++ chelated medium. In all cases, recovery of microtubules correlated with recovery of diameter as seen from phase contrast images (see Suppl. Mat., Fig. S2). The scale bar is 20 µ m (same for all image panels). In all cases, intensity plots were constructed by smoothening the raw intensity data with a Gaussian filter ( σ = 1.4) in Matlab and then integrating along the axonal thickness. Next, we explored how microtubules behave when extracellular Ca ++ is chelated. Axons of neurons grown in medium containing 5 mM EGTA show three kinds of responses after partial ablation. Some axons showed an initial retraction of microtubule intensity after which the intensity evolution stops and remains stable for the rest of the observation time of about 25 min (see Fig. 4c,d ). Comparison of phase contrast and fluorescence images shows that the regions devoid of microtubules appear thin, while the parts where intensity is unaffected have normal caliber. Remarkably, in some cases, the intensity initially recedes, then stops receding, starts to recover, and eventually recovers either partially ( Fig. 4e,f ) or completely ( Fig. 4g,h ). The extent of microtubule recovery for complete resealing can be seen from the plot shown in Fig. 4f . The time evolutions of microtubule intensity for different axons mirror the observations of diameter measured for the same axons. This suggests that the microtubule content in axons is directly correlated with axonal diameter (see Suppl. Mat., Fig. S2). Next, we explored how the microtubule recovery happens in the subset of axons that showed recovery when maintained in calcium chelated medium. For this, we transfected neurons with mNeon tagged EB3 to visualize the plus-end dynamics of growing microtubules [ 32 ]. Note that in DRG axons microtubules are arranged with a nearly perfect polar order with their plus tip pointed towards the growth cone end. This can be seen in control axons where EB3 comets travel towards the growth cone end with only very few comets in the opposite direction (see Suppl. Mat., Fig. S3). During the post-ablation recovery phase, we observe that this bias is maintained, but the comet density near the recovering ends is higher, as can be seen from Fig. 5 (see Suppl. Mat., Fig. S4 for more examples). This suggests that recovery may be happening through regrowth of existing microtubule fragments with pre-established polarity rather than de novo nucleation and growth. In the latter case, one would have expected random polarity for nucleating tubules. Download figure Open in new tab Figure 5: Visualization of microtubule regrowth via EB3 in Ca ++ chelated media. (a) Selected frames from a time-lapse movie showing EB3 fluorescence intensity during axonal recovery following partial laser ablation (see Suppl. Video 005). The injury site is indicated by a red arrow. The cell body is to the left of the images. Scale bar:10 µ m. (b) A kymograph generated along the axon showing EB3 dynamics during the recovery phase. A red arrow and a dotted red line mark the injury site. The horizontal axis represents distance along the axon (scale bar: 10 µ m), and the vertical axis represents time (scale bar: 5min). (c) The same kymograph as in (b), with overlaid traces highlighting EB3 comet trajectories. Green lines represent anterograde growth, while magenta lines indicate retrograde ones. The proximal side of the injury demonstrates a higher concentration of tracks, which aligns with site-specific microtubule regrowth. Actin filaments too recover in calcium-chelated medium Compared to microtubules organization, axonal actin filaments form more diverse structures. Mature axons contain a membrane associated periodic skeleton [ 33 ] as well as other highly heterogeneous cortical actin structures like actin hot spots and trails [ 34 ]. Although less abundant, these structures can contribute significantly to axonal mechanics and retraction dynamics [ 30 , 35 – 38 ]. Note that the membrane associated actin-spectrin periodic skeleton is fully developed in the four days in culture chick DRG neurons we use [ 30 ]. To study the damage and possible recovery of actin filaments, we imaged actin using the cell permeable SPY650-FastAct dye which labels only polymerized actin. In control experiments, where Ca ++ is present in the medium, actin fluorescence is seen to recede completely as in the case of microtubules (see Fig. 6a ). Very prominent actin peaks can be observed at the retracting fronts on either side, suggesting significant accumulation of filaments at these locations ( Fig. 6b ). No actin filaments could be detected in the thinned mid section within the observation time of 30 min. However, when extracellular Ca ++ is depleted using EGTA, we observe recovery of actin filaments in the thinned out region. Different behaviors are observed in such axons. Unlike microtubules, actin filaments recovers in all axons maintained in Ca ++ chelated medium–irrespective of whether the diameter too recovers or not. In many cases, after an initial decrease and subsequent recovery, the intensity overshoots–exceeding its pre-ablation value as can be seen in Figs. 6c,d and is quantified in Fig. 7 . This excess actin eventually redistributes. The recovery pattern too is different from what was observed for microtubules under similar conditions. No clearly demarcated growing fronts are observable in the case of actin filaments. Instead, actin recovery is distributed all along the axon and can be heterogeneous, as can be seen in Fig. 6e,f . Another notable difference, when compared to microtubule recovery, is in the correlation between recovery of axonal diameter and actin filaments. In Fig. 6g we show an example of an axon which has fully recovered its diameter as well as actin filament intensity. However, in Fig. 6h , one can see a case where actin intensity has recovered fully, but the diameter failed to recover within the same time span. Download figure Open in new tab Figure 6: Recovery of actin filaments post ablation in Ca ++ chelated medium. (a) Sequence of fluorescence images of a control axon which was labeled using SPY650-FastAct dye which labels only polymerized actin and was partially ablated. The white arrow indicates the point of ablation. Post-ablation, a thin tube spans the region between the retracting edges (no visible in the fluorescence images). Actin filaments tend to accumulate at the retracting edges of each axonal segment. (b) Intensity plots showing the time evolution of actin fluorescence. Hardly any actin intensity is seen in the thinned out regions. The dotted lines represent the zero intensity level. (c) When partial ablation is performed in calcium chelated medium, actin intensity in the thin region initially decreases (t = 75 s), then recovers, often overshoots (t = 350 s), and finally redistributes (t = 3210 s). The blue arrow indicates filopodia-like structures that form at regions with enhanced actin intensity. (d) Quantification of the actin fluorescence post ablation and during recovery for the images shown in (c). The overshoot is clearly visible at t = 350 & 975 s. (e , f) Another example of an axon maintained in calcium chelated medium. In this case, retraction is much more extensive, making it easier to see how actin filaments recover in the region between the retraction fronts. Unlike microtubules, recovery of actin intensity is seen all along the thinned out segment (t = 725 & 1225 s). A slight overshoot can be seen in this case too (t = 4945 s). The corresponding intensity plots are shown in (f). To obtain all the intensity plots, the raw intensity data has been smoothened with a Gaussian filter ( σ = 1.4) using Matlab. The intensity is then integrated across the axonal thickness. (g , h) While all axons showed recovery of actin filament intensity, the recovery of actin filaments may or may not be correlated with the recovery of axonal diameter. In (g) we show the example of an axon which exhibited diameter recovery as well as actin recovery. Whereas, in (h) one can see that there is no recovery in diamter even though actin intensity has recovered all along the axon. The scale bar is 10 µ m. Download figure Open in new tab Figure 7: Comparison of post-ablation intensity of microtubules and actin filaments. The data show the ratio of pre-ablation intensity per unit axonal length I(max) to the maximum post-ablation intensity per unit length observed in the initially thinned out region I(0). Significant overshoot in intensity is observed in the case of actin, even when there is no diameter recovery. The error bars represent values within 1.5 times the interquartile range from the first and third quartiles, illustrating the range of non-outlier data. Discussion When control axons are ablated, a thin membrane segment develops at the ablation point and expands in either direction, leading to axonal atrophy. Fluorescence imaging shows that the parallel microtubule array or bundle within the axon depolymerizes progressively from the ablation point forming a receding front–analogous to the shortening of a burning pyrotechnic fuse. Acin filaments too disappear from the thinned out mid section but show significant accumulation near the retraction fronts. These cytoskeletal changes are schematically shown in Fig. 8a . Download figure Open in new tab Figure 8: Schematic of cytoskeletal retraction and recovery in Ca ++ free medium. (a) Diagrams showing the receding of microtubules (green) and actin filaments (red) post ablation. The red arrowhead indicates the point of ablation. Actin rings, seen commonly in axons, are not shown. (b) Schematic of a case where microtubules and actin filaments recover in calcium chelated medium. Growing regrowing microtubule tips are marked in light green, and are seen mostly in the proximal segment. (c) A case where only actin filaments reform in the thin section. In such cases, there is no recovery of diameter. The pattern of microtubule disruption is noteworthy, as it is known that axons contain labile and stable fractions of microtubules–the stable fraction remains unaffected for long periods under the action of microtubule disrupting drugs like Nocodazole [ 38 , 39 ]. When treated with Nocodazole, axonal microtubule bundles thin down, leaving a stable core along the axon [ 38 ]. In partially ablated axons, however, we see an expanding region of complete depolymerisation. These observations raise the question as to what causes a progressive decay of the microtubule array in the form of a receding front. We also observe that the action of the laser triggers a transient elevation of cytosolic Ca ++ in the entire axon within a few seconds ( Fig. 2a ). Chelation of extra-cellular calcium using EGTA or intra-cellular free calcium using BAPTA-AM not only suppresses Ca ++ elevation, but also renders microtubules more resistant to laser-induced degeneration. More remarkably, complete recovery of microtubules also occurs under such conditions. Hence, it is likely that microtubule depolymerization is driven at least partly by calcium induced mechanisms. Calcium is known to affect microtubule catastrophe frequency in in vitro experiments [ 40 – 42 ], and in cells caplain activation by calcium is known to degrade microtubules [ 14 , 19 ]. These observations raise a further question–why do microtubules depolymerise as a slow propagating front when Ca ++ elevation spreads all along the axon within a few seconds ( Fig. 2a )? Here, we speculate that collective effects arising from microtubule-microtubule interactions may be responsible for the observed dynamics. When microtubules across an axonal cross-section are cut by the laser, these microtubules are left with unstabilized ends containing GDP-tubulin and they depolymerize ( Fig. 9 ). The collapse of these microtubules leads to a reduction in the number of neighbours for overlapping microtubules which were not directly affected by the laser, making them unstable. This process continues as depicted in Fig. 9 . Download figure Open in new tab Figure 9: Proposed NNN-dependent microtubule bundle instability mechanism. Schematic showing a possible mechanism for the loss of microtubules within the bundle in the form of a retracting front. Microtubules with compromised stability are shown in light green. Microtubule-associated proteins that interlink microtubules are not shown. Microtubules that are directly cut by the laser become unstable and depolymerise. This leads to a reduction in the number of neighbours for adjacent microtubules, making them unstable–either due to MAP unbinding or due to severing enzymes like spastin and katanin gaining access to loosely packed tubules. This causes a cascade effect leading to a depolymerising front. The dependence of microtubule stability on the presence or absence of neighbours may arise for the following reasons. (i) Microtubules may be stabilised by direct Microtubule Associated Proteins (MAPs) based interaction with neighbours. (ii) Loss of neighbours may open up gaps in the array and hence easy access to microtubule severing enzymes like spastin and/or katanin. (iii) The tubulin post-translational modification (PTM) state of microtubules is altered when PTM modifying enzymes gain access when gaps open up. This number of nearest neighbour dependent (NNN-dependent) stability hypotheses may account for the catastrophic failure of the microtubule bundle through a cascading effect, as is illustrated in Fig. 9 . Remarkably, a fraction of axons show recovery of normal caliber when calcium is chelated. Imaging of microtubules shows that this requires complete recovery of microtubules (see Fig. 4g,h and the schematic 8(b)). Furthermore, imaging of EB3 comets shows that microtubule regrowth happens predominantly from the proximal segment. The maintenance of microtubule polarity during this regrowth indicates that microtubule recovery is most likely via regrowth of existing filaments and not through de novo nucleation [ 43 , 44 ]. Actin filaments too recover after the initial disruption when extra-cellular calcium is chelated. Unlike in the case of microtubules, the reformation of these filaments occurs all along the thinned regions and in several cases exceeds the initial levels (see Fig. 6e,f ). This recovery pattern is reminiscent of the recovery of actin filaments in cellular blebs, where fresh nucleation of filaments occurs from a free membrane soon after the expansion of a bleb, mediated by membrane-bound actin nucleator proteins [ 45 ]. Recovery of actin filaments occurs throughout the entire thinned down segment of all axons maintained in calcium chelated medium. This is in contrast to the formation of a polymerization front in the case of microtubules. Moreover, actin filament recovery alone is not enough for the recovery of axonal caliber (see Fig. 6h and the schematic 8c). In conclusion, we have developed a partial laser ablation technique that allows for relatively facile interrogation of cytoskeletal stability in injured axons. We show that when perturbed locally, the entire axonal microtubule bundle undergo catastrophic collapse via a propagating depolymerization/degradation front. This process is dependent on the injury-induced elevation in intra-cellular free Ca ++ . We show that cytoskeletal damage can be mitigated, and even complete axonal recovery post-injury can be promoted by chelation of Ca ++ . We also show that the recovery of axon caliber requires regrowth of microtubules. Moreover, we demonstrate key differences between the loss and recovery patterns of microtubule and filamentous actin. Finally, we propose a number of nearest neighbor dependent (NNN-dependent) microtubule stability hypothesis which can account for the observed microtubule depolymerization pattern. We believe that these studies open up new avenues in investigating the stability of axonal cytoskeleton, especially under injury mimicking conditions. Author Contributions A.M. and P.A.P. designed the research. A.M. and P.J. performed research and analysis. A.M., P.J. and P.A.P. interpreted results and wrote the article. Acknowledgments The authors thank the lab of Aurnab Ghose for sharing the pCAG-mNeon-EB3 construct. We acknowledge support through The Wellcome Trust DBT India Alliance (grant IA/TSG/20/1/600137) and through the Indo-Portugal grant, DBT, Gov. of India (DST/INT/Portugal/P-04 /2022). Funder Information Declared The Wellcome Trust DBT India Alliance , IA/TSG/20/1/600137 Indo-Portugal grant, DBT, Gov. of India , DST/INT/Portugal/P-04/2022 References [1]. ↵ Hill , C. S. , M. P. Coleman , and D. K. Menon , 2016 . Traumatic axonal injury: mechanisms and translational opportunities . Trends in neurosciences 39 : 311 – 324 . OpenUrl CrossRef PubMed [2]. Smith , D. H. , D. F. Meaney , and W. H. Shull , 2003 . Diffuse axonal injury in head trauma . The Journal of head trauma rehabilitation 18 : 307 – 316 . OpenUrl CrossRef PubMed Web of Science [3]. ↵ Johnson , V. E. , W. Stewart , and D. H. Smith , 2013 . Axonal pathology in traumatic brain injury . Experimental neurology 246 : 35 – 43 . OpenUrl CrossRef PubMed [4]. ↵ Tang-Schomer , M. D. , A. R. Patel , P. W. Baas , and D. H. Smith , 2010 . Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration . The FASEB Journal 24 : 1401 . OpenUrl CrossRef PubMed Web of Science [5]. ↵ Keating , C. E. , and D. K. Cullen , 2021 . Mechanosensation in traumatic brain injury . Neurobiology of disease 148 : 105210 . OpenUrl CrossRef PubMed [6]. ↵ Anthes , D. L. , E. Theriault , and C. H. Tator , 1995 . Characterization of axonal ultra-structural pathology following experimental spinal cord compression injury . Brain research 702 : 1 – 16 . OpenUrl CrossRef PubMed Web of Science [7]. ↵ Fournier , A. J. , J. D. Hogan , L. Rajbhandari , S. Shrestha , A. Venkatesan , and K. Ramesh , 2015 . Changes in neurofilament and microtubule distribution following focal axon compression . PloS one 10 : e0131617 . OpenUrl PubMed [8]. Yap , Y. C. , A. E. King , R. M. Guijt , T. Jiang , C. A. Blizzard , M. C. Breadmore , and T. C. Dickson , 2017 . Mild and repetitive very mild axonal stretch injury triggers cystoskeletal mislocalization and growth cone collapse . PloS one 12 : e0176997 . OpenUrl CrossRef PubMed [9]. Vialle , R. , C. Piétin-Vialle , M. Vinchon , S. Dauger , B. Ilharreborde , and C. Glorion , 2008 . Birth-related spinal cord injuries: a multicentric review of nine cases . Child’s nervous system 24 : 79 – 85 . OpenUrl PubMed [10]. Avis , D. , and D. Power , 2018 . Axillary nerve injury associated with glenohumeral dislocation: a review and algorithm for management . EFORT open reviews 3 : 70 – 77 . OpenUrl PubMed [11]. Aoki , M. , H. Takasaki , T. Muraki , E. Uchiyama , G. Murakami , and T. Yamashita , 2005 . Strain on the ulnar nerve at the elbow and wrist during throwing motion . JBJS 87 : 2508 – 2514 . OpenUrl CrossRef PubMed [12]. ↵ Sharp , E. , M. Roberts , A. Žurada-Zielińska , A. Zurada , J. Gielecki , R. S. Tubbs , and M. Loukas , 2021 . The most commonly injured nerves at surgery: A comprehensive review . Clinical Anatomy 34 : 244 – 262 . OpenUrl PubMed [13]. ↵ Karaboue , M. A. A. , F. Ministeri , F. Sessa , C. Nannola , M. G. Chisari , G. Cocimano , L. Di Mauro , M. Salerno , and M. Esposito , 2024 . Traumatic brain injury as a public health issue: epidemiology, prognostic factors and useful data from forensic practice . In Healthcare . MDPI , volume 12 , 2266 . OpenUrl [14]. ↵ Büki , A. , and J. Povlishock , 2006 . All roads lead to disconnection?–Traumatic axonal injury revisited . Acta neurochirurgica 148 : 181 – 194 . OpenUrl CrossRef PubMed Web of Science [15]. ↵ Wolf , J. A. , P. K. Stys , T. Lusardi , D. Meaney , and D. H. Smith , 2001 . Traumatic axonal injury induces calcium influx modulated by tetrodotoxin-sensitive sodium channels . Journal of Neuroscience 21 : 1923 – 1930 . OpenUrl Abstract / FREE Full Text [16]. Aydin , M.Ş. , S. Bay , E. N. Yiğit , C. Özgül , E. K. Oğuz , E. Y. Konuk , N. Ayşit , N. Cengiz , E. Erdoğan , A. Him , et al. , 2023 . Active shrinkage protects neurons following axonal transection . Iscience 26 . [17]. ↵ Khaitin , A. , 2021 . Calcium in neuronal and glial response to axotomy . International Journal of Molecular Sciences 22 : 13344 . OpenUrl PubMed [18]. ↵ Villegas , R. , N. W. Martinez , J. Lillo , P. Pihan , D. Hernandez , J. L. Twiss , et al. , 2014 . Calcium release from intra-axonal endoplasmic reticulum leads to axon degeneration through mitochondrial dysfunction . Journal of Neuroscience 34 : 7179 – 7189 . OpenUrl Abstract / FREE Full Text [19]. ↵ Ziv , N. E. , and M. E. Spira , 1995 . Axotomy induces a transient and localized elevation of the free intracellular calcium concentration to the millimolar range . Journal of neurophysiology 74 : 2625 – 2637 . OpenUrl PubMed Web of Science [20]. ↵ Vosler , P. , C. Brennan , and J. Chen , 2008 . Calpain-mediated signaling mechanisms in neuronal injury and neurodegeneration . Molecular neurobiology 38 : 78 – 100 . OpenUrl CrossRef PubMed Web of Science [21]. ↵ Ma , M. , 2013 . Role of calpains in the injury-induced dysfunction and degeneration of the mammalian axon . Neurobiology of disease 60 : 61 – 79 . OpenUrl CrossRef PubMed [22]. ↵ Pivovarova , N. B. , and S. B. Andrews , 2010 . Calcium-dependent mitochondrial function and dysfunction in neurons . The FEBS journal 277 : 3622 – 3636 . OpenUrl CrossRef PubMed [23]. ↵ Frati , A. , D. Cerretani , A. I. Fiaschi , P. Frati , V. Gatto , R. La Russa , A. Pesce , E. Pinchi , A. Santurro , F. Fraschetti , et al. , 2017 . Diffuse axonal injury and oxidative stress: a comprehensive review . International journal of molecular sciences 18 : 2600 . OpenUrl PubMed [24]. ↵ Gallo , G. , 2004 . Myosin II activity is required for severing-induced axon retraction in vitro . Experimental neurology 189 : 112 – 121 . OpenUrl CrossRef PubMed Web of Science [25]. Kerschensteiner , M. , M. E. Schwab , J. W. Lichtman , and T. Misgeld , 2005 . In vivo imaging of axonal degeneration and regeneration in the injured spinal cord . Nature medicine 11 : 572 – 577 . OpenUrl CrossRef PubMed Web of Science [26]. Blizzard , C. , M. Haas , J. Vickers , and T. Dickson , 2007 . Cellular dynamics underlying regeneration of damaged axons differs from initial axon development . European Journal of Neuroscience 26 : 1100 – 1108 . OpenUrl CrossRef PubMed Web of Science [27]. ↵ Shao , X. , R. You , T. H. Hui , C. Fang , Z. Gong , Z. Yan , R. C. C. Chang , V. B. Shenoy , and Y. Lin , 2019 . Tension-and adhesion-regulated retraction of injured axons . Biophysical journal 117 : 193 – 202 . OpenUrl PubMed [28]. ↵ Kunik , D. , C. Dion , T. Ozaki , L. A. Levin , and S. Costantino , 2011 . Laser-based single-axon transection for high-content axon injury and regeneration studies . PLoS One 6 : e26832 . OpenUrl PubMed [29]. ↵ Cengiz , N. , G. Öztürk , E. Erdoğan , A. Him , and E. K. Oğuz , 2012 . Consequences of neurite transection in vitro . Journal of neurotrauma 29 : 2465 – 2474 . OpenUrl CrossRef PubMed [30]. ↵ Dubey , S. , N. Bhembre , S. Bodas , S. Veer , A. Ghose , A. Callan-Jones , and P. Pullarkat , 2020 . The axonal actin-spectrin lattice acts as a tension buffering shock absorber . Elife 9 : e51772 . OpenUrl CrossRef PubMed [31]. ↵ Gaub , B. M. , K. C. Kasuba , E. Mace , T. Strittmatter , P. R. Laskowski , S. A. Geissler , A. Hierlemann , M. Fussenegger , B. Roska , and D. J. Müller , 2020 . Neurons differentiate magnitude and location of mechanical stimuli . Proceedings of the National Academy of Sciences 117 : 848 – 856 . OpenUrl Abstract / FREE Full Text [32]. ↵ Stepanova , T. , J. Slemmer , C. C. Hoogenraad , G. Lansbergen , B. Dortland , C. I. De Zeeuw , F. Grosveld , G. van Cappellen , A. Akhmanova , and N. Galjart , 2003 . Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein) . Journal of Neuroscience 23 : 2655 – 2664 . OpenUrl Abstract / FREE Full Text [33]. ↵ Xu , K. , G. Zhong , and X. Zhuang , 2013 . Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons . Science 339 : 452 – 456 . OpenUrl Abstract / FREE Full Text [34]. ↵ Leterrier , C. , P. Dubey , and S. Roy , 2018 . The nano-architecture of the axonal cytoskeleton . Nature Reviews Neuroscience 19 : 58 – 59 . OpenUrl [35]. ↵ Costa , A. R. , S. C. Sousa , R. Pinto-Costa , J. C. Mateus , C. D. Lopes , A. C. Costa , D. Rosa , D. Machado , L. Pajuelo , X. Wang , et al. , 2020 . The membrane periodic skeleton is an actomyosin network that regulates axonal diameter and conduction . Elife 9 : e55471 . OpenUrl CrossRef PubMed [36]. Mutalik , S. P. , J. Joseph , P. A. Pullarkat , and A. Ghose , 2018 . Cytoskeletal mechanisms of axonal contractility . Biophysical journal 115 : 713 – 724 . OpenUrl CrossRef PubMed [37]. Fan , A. , M. S. H. Joy , and T. Saif , 2019 . A connected cytoskeleton network generates axonal tension in embryonic Drosophila . Lab on a Chip 19 : 3133 – 3139 . OpenUrl CrossRef PubMed [38]. ↵ Datar , A. , J. Ameeramja , A. Bhat , R. Srivastava , A. Mishra , R. Bernal , J. Prost , A. Callan-Jones , and P. A. Pullarkat , 2019 . The roles of microtubules and membrane tension in axonal beading, retraction, and atrophy . Biophysical journal 117 : 880 – 891 . OpenUrl CrossRef PubMed [39]. ↵ Baas , P. W. , A. N. Rao , A. J. Matamoros , and L. Leo , 2016 . Stability properties of neuronal microtubules . Cytoskeleton 73 : 442 – 460 . OpenUrl PubMed [40]. ↵ Weisenberg , R. C. , 1972 . Microtubule formation in vitro in solutions containing low calcium concentrations . Science 177 : 1104 – 1105 . OpenUrl Abstract / FREE Full Text [41]. Weisenberg , R. C. , and W. J. Deery , 1981 . The mechanism of calcium-induced microtubule disassembly . Biochemical and biophysical research communications 102 : 924 – 931 . OpenUrl CrossRef PubMed Web of Science [42]. ↵ O’Brien , E. T. , E. Salmon , and H. P. Erickson , 1997 . How calcium causes microtubule depolymerization . Cell motility and the cytoskeleton 36 : 125 – 135 . OpenUrl CrossRef PubMed Web of Science [43]. ↵ Nguyen , M. M. , C. J. McCracken , E. Milner , D. J. Goetschius , A. T. Weiner , M. K. Long , N. L. Michael , S. Munro , and M. M. Rolls , 2014 . G-tubulin controls neuronal microtubule polarity independently of Golgi outposts . Molecular biology of the cell 25 : 2039 – 2050 . OpenUrl Abstract / FREE Full Text [44]. ↵ Yau , K. W. , P. Schätzle , E. Tortosa , S. Pagés , A. Holtmaat , L. C. Kapitein , and C. C. Hoogenraad , 2016 . Dendrites in vitro and in vivo contain microtubules of opposite polarity and axon formation correlates with uniform plus-end-out microtubule orientation . Journal of Neuroscience 36 : 1071 – 1085 . OpenUrl Abstract / FREE Full Text [45]. ↵ Charras , G. T. , C.-K. Hu , M. Coughlin , and T. J. Mitchison , 2006 . Reassembly of contractile actin cortex in cell blebs . The Journal of cell biology 175 : 477 – 490 . OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted August 12, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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Share Calcium chelation promotes microtubule regrowth and axonal recovery after laser-induced axonal injury Ashish Mishra , Pooja Joshi , Pramod Pullarkat bioRxiv 2025.08.08.668418; doi: https://doi.org/10.1101/2025.08.08.668418 Share This Article: Copy Citation Tools Calcium chelation promotes microtubule regrowth and axonal recovery after laser-induced axonal injury Ashish Mishra , Pooja Joshi , Pramod Pullarkat bioRxiv 2025.08.08.668418; doi: https://doi.org/10.1101/2025.08.08.668418 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7636) Biochemistry (17704) Bioengineering (13898) Bioinformatics (41967) Biophysics (21460) Cancer Biology (18599) Cell Biology (25525) Clinical Trials (138) Developmental Biology (13384) Ecology (19909) Epidemiology (2067) Evolutionary Biology (24326) Genetics (15613) Genomics (22512) Immunology (17740) Microbiology (40423) Molecular Biology (17191) Neuroscience (88645) Paleontology (667) Pathology (2835) Pharmacology and Toxicology (4825) Physiology (7646) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9825) Zoology (2271)