Moya-Díaz et al., 2014 - Electroablation: a method for neurectomy and localized tissue injury. BMC Developmental Biology   14:7 Full text @ BMC Dev. Biol.

Fig. 1

Electroablation setup and dependence of tissue damage on amperage. (a-c) Experimental setup for electroablation in zebrafish larvae (b) and adults (c). (a) Current pulses are generated by a precision current source (black arrow), which is connected to the sample by the microelectrode and a ground wire. The microelectrode is held by a micromanipulator (black arrowhead) and zebrafish are visualized under a fluorescence microscope (white arrowhead). (b) Close-up view of experimental setup for electroablation in larvae. The microelectrode (white arrow) enters the agarose through one side and a ground wire is connected to the other side (black arrow). Zebrafish larvae are mounted in a drop of agarose dissolved in E3 (black arrowhead) in the central depression of the acrylic plate (white arrowhead). (c) Adult zebrafish (black arrowhead) are positioned in an acrylic plate (white arrowhead) with a larger depression and with a small amount of E3. The microelectrode (white arrow) touches the caudal fin and the ground wire is immersed in the E3 medium (black arrow). (d-f) Merge of acridine orange (green) and bright field images showing progressive expansion of acridine orange stain as amperage increases. Pulses of different amperages (5 – 25 μA) were applied for 1 second, and acridine orange stain was performed 2 hpi. (g) Quantification of acridine orange fluorescence shows a direct relationship with applied amperage. Average fluorescence intensity was measured within a 50 μm radius surrounding the electroablation site. Data was normalized for each larva by the average fluorescence intensity measured in an adjacent uninjured area. Values are presented as a normalized average ± SEM from 15–21 larvae per condition, from three independent experiments. Scale bar, 50 μm.

Fig. 2

Neurectomy, degeneration and regeneration of the posterior lateral line (pLL) nerve. (a) Degeneration of a neurectomized pLL nerve after electrical injury. Transgenic TgBAC(neurod:EGFP) larvae with a labeled pLL nerve were neurectomized by applying a 17 μA pulse of current for 1.5 seconds. At 5 hpi a neurectomized larva was mounted in agarose for time-lapse imaging for 7 hours (anterior to the left). Fragmentation of the detached nerve fragment proceeds by a breakdown of the axons into many small sections simultaneously throughout the axotomized nerve (arrowheads). Note the intact contralateral pLL nerve which is visible (above the electroablated nerve, slightly out of focus) in all the images. (b) Regeneration of an axotomized pLL nerve. Transgenic TgBAC(neurod:EGFP) larvae was treated and imaged as before. In this case, the neurectomized larva was mounted for imaging for 6 hours starting at 12 hpi to examine regeneration of the pLL nerve. Note the progressive regeneration of the pLL nerve by elongation of the remaining axon stumps (arrows) as degeneration of the distal part of the axotomized pLL nerve has concluded. Scale bar, 50 μm; times in hh:mm:ss.

Fig. 3

Ablation and regeneration of a single posterior lateral line neuromast. (a-c)Tg(cxcr4b:mRFP) fish show labeling of posterior lateral line neuromasts and interneuromastic cells. (a) Lateral view of 72 hpf control larva showing intact lateral line neuromasts (arrowheads) connected by interneuromastic cells (arrows). (b) Two 8 μA pulses of 2 seconds each were applied to the L3 posterior lateral line neuromast. Complete dissapearance of the neuromast’s cells is observed (arrowhead) while adjacent neuromasts (L2 and L4) and interneuromastic cells remain intact. (c) The same larva is shown at 48 hpi, showing the regenerated L3 neuromast. Scale bar, 100 μm.

Fig. 4

Extent of damage inflicted to tissues by neuromast electroablation. Transgenic Tg(-8.0cldnb:lynEGFP) fish, which express EGFP in lateral line cells and epithelial cells of the skin, were stained with BODIPY. Stained larvae were then mounted in agarose and subjected to electroablation of the L3 neuromast. Confocal images of electroablated and uninjured control larvae were acquired 20 minutes after injury with 1.5 μm of separation between z-axis optical slices. (a) Sequential z-axis confocal images of an uninjured larva are shown. The intact neuromast (green) exhibits a rosette-like structure and BODIPY-TR (red) allows visualization of underlying muscular tissue. (b) Images acquired as in (a) of a larva with an electroablated neuromast showing local loss of cells in the skin, disconnection of interneuromastic cells, destruction of neuromast cells and complete loss of the rosette-like structure. BODIPY-TR staining also shows a gap in muscular tissue to a depth of 22.5 μm from the skin surface; deeper sections appear unperturbed. Values indicate distance froom skin surface towards the inside of the larva in μm. Scale bar, 50 μm.

Fig. 5

Neutrophil recruitment induced by nerve neurectomy. (a-f) A larva obtained from a cross of TgBAC(neurod:EGFP) and Tg(lyz:DsRED2) transgenic fish labels the posterior lateral line nerve in green and leukocytes in red. (a) Control larva showing an intact lateral line nerve and few leukocytes near the nerve. One hour after lateral line nerve neurectomy (b), leukocytes migrate and accumulate at the lesion site (b and c). From 6 hpi, the number of recruited leukocytes decreases and resolution of inflammation takes place. (d-f). The number of recruited leukocytes continues to decrease as the lateral line nerve regenerates. (g) Quantification of average fluorescence intensity within a 50 μm radius surrounding the neurectomy site (panel b, dotted circle) shows leukocyte recruitment and subsequent inflammation resolution. Data are presented as average ± SEM from 17 larvae per condition from two independent experiments. Comparisons were performed by using a repeated measurement two-way ANOVA, with Bonferroni’s post test. ***, p < 0.001; ns, p > 0.05. Scale bar (a-f) 100 μm. hpi, hours post-injury; a. u., arbitrary units.

Fig. 6

Neuromast electroablation induces an inflammatory response. Compound transgenic Tg(cxcr4b:mRFP; mpx:GFP) fish harboring neutrophils expressing green fluorescent protein and neuromasts labeled in red were used to study neutrophil inflammation induced by neuromast electroablation. Two 2 second 8 μA pulses were applied to induce damage to the pLL neuromast. (a-c) The trunk of a larva subjected to neuromast electroablation is shown with anterior to the left. (a) Immediately after electroablation, most neutrophils are present in the caudal hematopoietic tissue (CHT). (b) At 2 hpi, a large number of neutrophils have specifically migrated to the site of damage. (c) By 7 hpi, the number of neutrophils at the site of electroablation has diminished, suggesting resolution of inflammation is taking place. (d) Quantification of mean fluorescence in a 50 μm radius around the site of electroablation (panel a, dotted circle) as a measure of neutrophil recruitment. Data are presented as average ± SEM from 12 larvae per condition and two independent experiments. Comparisons were performed by using a repeated measurement two-way ANOVA, with Bonferroni’s post test. ***, p < 0.001; ns, p > 0.05. Scale bar (a-b) 100 μm. hpi, hours post-injury; a. u., arbitrary units.

Fig. 7

Electroablation applied to spinal cord injury in larvae and tissue damage in the caudal fin of adult fish. (a-c) 72 hpf TgBAC (neurod:EGFP) transgenic larvae were subjected to spinal cord injury. (a) The intact spinal cord before electroablation is shown. The dotted lines show the outline of the larva. (b) A 1 second 25 μA pulse was applied in the spinal cord (arrowhead), leaving a gap and rendering larvae unable to move their tails. (c) The regenerated spinal cord at 5 dpi. (d-i) Damaging of different tissues in caudal fin of adult fish by application of a 2 second 25 μA pulse of current. (d-f) Induction of neutrophil recruitment in the caudal fin of adult TgBAC(mpx:GFP) transgenic fish. A large number of neutrophils recruited to the site of damage is observed by 2 hpi (f). (g-i)Tg(flia:EGFP) transgenic adult fish in which blood vessels are labeled in green were subjected to electroablation in a single blood vessel (h), leaving a gap in it, as shown in (i) (see inset for higher magnification). (j-l) Caudal fin neuromast electroablation in sqet20 transgenic fish, which possess neuromasts labeled in green. (j) Intact fin neuromasts are shown. Within the line of five neuromasts shown in the inset, note positioning of the microelectrode over the second pair of neuromasts (k). (l) After electroablation, the pair of neuromasts has been destroyed and a gap in the GFP pattern can be observed at that position (see inset for details). Note that the electrode is easily observable during electroablation facilitating its positioning (e, h and k). Scale bars: a-c, 100 μm; d-l, 500 μm; and i, j, and l insets 100 μm. dpi, days post-injury.

Fig. S1

Recruitment of macrophages to the site of neurectomy. Compound transgenic fish, Tg(mpeg1:EGFP; neurod:TagRFP), labeling macrophages in green and the pLL nerve in red, were subjected to pLL neurectomy by application of a 17 μA pulse for 1.5 seconds. A temporal series of images (only green channel shown) shows macrophage infiltration into the site of axotomy starting 20 minutes after electroablation. Scale bar, 200 μm. Times expressed in hh:mm:ss.

Acknowledgments:
ZFIN wishes to thank the journal BMC Developmental Biology for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ BMC Dev. Biol.