Download Neurosurgery: Functional Regeneration after Laser Axotomy

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brief communications
*Division of Neuroanatomy and Behaviour,
Institute of Anatomy, University of Zurich,
8057 Zurich, Switzerland
†Division of Psychiatry Research, University of
Zurich, 8008 Zurich, Switzerland
‡Animal Welfare and Ethology Group, Institute of
Veterinary Physiology, University of Giessen,
35392 Giessen, Germany
e-mail: [email protected]
1. Van Praag, H., Kempermann, G. & Gage, F. H. Nature Rev.
Neurosci. 1, 191–198 (2000).
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Behav. Genet. 29, 41–46 (1999).
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Assembly and Conference & 7th FELASA Symposium, SECAL,
Madrid 207–210 (1999).
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1670–1672 (1999).
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(2002).
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Lab. Anim. 37, 300–313 (2003).
Supplementary information accompanies this communication on
Nature’s website.
Competing financial interests: declared none.
Neurosurgery
Functional regeneration
after laser axotomy
nderstanding how nerves regenerate is
an important step towards developing
treatments for human neurological
disease1, but investigation has so far been
limited to complex organisms (mouse and
zebrafish2) in the absence of precision techniques for severing axons (axotomy). Here
we use femtosecond laser surgery for axotomy in the roundworm Caenorhabditis
elegans and show that these axons functionally regenerate after the operation. Application of this precise surgical technique should
enable nerve regeneration to be studied in
vivo in its most evolutionarily simple form.
Femtosecond laser pulses provide high
U
peak intensities that reduce the energy
threshold for tissue removal (ablation)3 and
enable laser surgery to be carried out with a
low-energy source4,5. (For methods, see supplementary information.) We successfully
cut single axons inside C. elegans by using
pulse energies of 10–40 nanojoules at the
specimen and tightly focused, 200-femtosecond, near-infrared laser pulses. This
results in the vaporization of axon volumes
of about 0.1–0.3 femtolitres, assuming an
average axon diameter of 0.3 micrometres
(see supplementary information). Dyefilling of axotomized neurons confirmed that
the observed axon gaps are not due to photobleaching, but to physical disconnection of
the axons (see supplementary information).
The minimum energy used is consistent
with measured optical breakdown thresholds in transparent materials3,6. At these low
energies,we would expect mechanical effects
due to plasma expansion and shock waves to
be significantly reduced5,6 with respect to
other laser-surgery techniques that require
much higher energies (for example,0.4 microjoules with 0.5-nanosecond pulses7).The use
of pulses at a low repetition rate (1 kilohertz, 10 microwatts average power) should
reduce heat accumulation and extended
thermal damage to the environment. We
were able to cut individual processes within
a few micrometres of each other without
damaging the nearby processes (see supplementary information).
The D-type motor neurons in L4 larvalstage worms were selected as targets for
laser surgery. These neurons have ventral
cell bodies and extend circumferential axons
towards the dorsal side; they form synapses
to body muscles8. We cut the circumferential
axons (labelled with green fluorescent protein9) at their mid-body positions, leaving
the rest of the axon intact (Fig. 1a). Both ends
of the severed axons initially retracted.
Among 52 operated axons (in 11 worms),
Axon regrowth (%)
60
50
40
30
20
10
0
Behavioural
response (%)
b
100
80
60
40
20
0
a
c
Before
After
6h
12 h
24 h
None Partial Full
3h
12 h
24 h
Figure 1 Femtosecond laser axotomy in Caenorhabditis elegans worms using 100 pulses of low energy (40 nanojoules) and short duration
(200 femtoseconds) and a repetition rate of 1 kilohertz. a, Fluorescence images of axons labelled with green fluorescent protein before,
immediately after, and in the hours following axotomy. Arrow indicates point of severance. Scale bar, 5 m. b, Statistics of axon growth 24 h
after axotomy, based on fluorescence images (n52 axons). c, Time-course analysis of backward motion of worms following axotomy.
Seventeen worms were scored blindly at different time points (for criteria, see supplementary information). Improvement in backward
motion was graded as four levels from ‘shrinker’ behaviour (dark red) up to ‘wild type’ behaviour (yellow) in the hours following axotomy.
54% regrew towards their distal ends within
12–24 hours (Fig.1b).Axons that showed partial, aberrant, or no regrowth within 24 hours
did not show further improvement over
longer observation times (up to 36 hours).
To evaluate functional recovery associated with nerve regeneration, we tested the
behaviour of operated worms as it related to
motor-neuron function. Loss of D-neuron
function results in simultaneous contraction of dorsal and ventral body muscles
(‘shrinker’ phenotype)10, which prevents
backward locomotion. Operated worms
(17 in total) showed this expected ‘shrinker’
phenotype immediately after axotomy
(15 axons per worm), whereas sham-operated animals (6 in total) moved like wildtype worms.
Remarkably, the locomotion of operated
worms improved, approaching that of the
wild type within 24 hours of surgery
(Fig. 1c), indicating that the regenerated
axons were functional (see movie in supplementary information). By contrast, the
shrinker phenotype caused by laser ablation
of D-neuron cell bodies did not recover after
48 hours (results not shown). The correlation of axonal regrowth with behavioural
recovery in C. elegans indicates that these
nerves must have regenerated.
Femtosecond laser axotomy is a new
technique that can be performed with 100%
efficiency, sub-micrometre precision and
high speed. As simple organisms such as C.
elegans have amenable genetics, application
of the femtosecond laser axotomy technique
we describe here should help in the rapid
identification of genes and molecules that
affect nerve regeneration and development.
Mehmet Fatih Yanik*, Hulusi Cinar†,
Hediye Nese Cinar†, Andrew D. Chisholm†,
Yishi Jin†‡, Adela Ben-Yakar*§
*Department of Applied Physics, Ginzton Laboratory,
Stanford University, Stanford, California 94305, USA
†Department of MCD Biology, Sinsheimer
Laboratories, University of California, Santa Cruz,
California 95064, USA
‡Howard Hughes Medical Institute, University of
California, Santa Cruz, California 95064, USA
§Department of Mechanical Engineering,
University of Texas at Austin, Austin,
Texas 78712, USA
e-mail: [email protected]
1. Horner, P. J. & Gage, F. H. Nature 407, 963–970 (2000).
2. Bhatt, D. H., Otto, S. J., Depoister, B. & Fetcho, J. R. Science 305,
254–258 (2004).
3. Perry, M. D. et al. J. Appl. Phys. 85, 6803–6810 (1999).
4. Tirlapur, U. K. & König, K. Nature 418, 290–291 (2002).
5. Shen, N. Thesis, Harvard Univ. (2003).
6. Vogel, A. et al. Appl. Phys. B 68, 271–280 (1999).
7. Colombelli, J., Grill, S. W. & Stelzer, E. H. K. Rev. Sci. Instrum.
75, 472–478 (2004).
8. White, J. G., Southgate, E., Thomson, J. N. & Brenner, S.
Phil. Trans. R. Soc. Lond. B 314, 1–340 (1986).
9. Huang, X., Cheng, H. J., Tessier-Lavigne, M. & Jin, Y. Neuron 34,
563–576 (2002).
10. McIntire, S. L., Jorgensen, E., Kaplan, J. & Horvitz, H. R. Nature
364, 337–341 (1993).
Supplementary information accompanies this communication on
Nature’s website.
Competing financial interests: declared none.
NATURE | VOL 432 | 16 DECEMBER 2004 | www.nature.com/nature
822
©2004 Nature Publishing Group
Supplementary Information
“Axon regeneration in C. elegans after femtosecond laser axotomy”
M. F. Yanik, H. Cinar, H. N. Cinar, A. D. Chisholm, Y. Jin, and A. Ben-Yakar
Methods:
The worms were maintained on standard NGM agar plates at 20oC. Axotomy is
performed on L4 stage worms. For axotomy and following fluorescence imaging of
axons, the worms were anesthetized for short periods (15 minutes) on agarose pads with
5 µl phenoxypropanol per 1ml agarose. Sham operated worms were also subject to the
same procedure, but the laser beam was focused 5-10 µm distance away from the axons
instead of focusing on them.
The femtosecond laser axotomy technique is quite rapid. It takes less than a minute to
find, position, focus and cut an axon. Including the time to prepare slides, anesthetize
worms, cut 15 D-type axons, and recover axotomized worms, the entire process is
completed within 10 minutes per worm. We found that with 40nJ pulse energy, the
damage spot size is optimal so that we can focus the laser on the target easily and
perform axotomy rapidly. We can cut axons with 100% success rate.
Behavioral Studies:
We scored behavioral recordings of axotomized and sham operated juIs76 animals for
backward movements. By using criteria such as, number of backward waves upon each
touch to the head, presence or absence of spontaneous backward movement, deviation
from wild type sinusoidal movement, each movie was given a general score. All the
movies were scored blindly for both experimental conditions and recording time. The
scorings at multiple times were always consistent. Worms were recorded at (or around) 3,
12, 24, 36, 48, 54, and 60 hours after axotomy. 6 worms were sham operated and showed
wild type behavior. 17 worms were axotomized and scored as follows:
Score 1: No backward movement, animal shows shrinker behavior.
Score 2: Animal can move back up to two waves. It uses segmental muscle contractions
instead of sinusoidal ones.
Score 3: Animal can move backwards more than two waves, but uses segmental moves
instead of sinusoidal ones.
Score 4: Animal can move backward four or more waves by using sinusoidal muscle
contractions - WT behavior.
before
after
Mercury Lamp
Objective Lens
NA=1.4
Tube Lens
ICCD
Filters
Shutter
Relay
Lenses
Regenerative Amplifier
200fs, 1kHz, 10-40nJ pulses
at the sample
Scanners
Ti-Sapphire Laser λ ~ 800nm
200fs, 80MHz, 100pJ pulses
at the sample
Supplementary Figure 1: Experimental set-up for femtosecond laser axotomy. A tunable TiSapphire mode-locked laser (Coherent, “Mira 900”) produces 200 fs short pulses with a repetition
rate of 76 MHz and 5 nJ energy. For axotomy, a regenerative amplifier (Positive Light,
“Spitfire”) seeded by the Ti-Sapphire laser generates 1 mJ energy, 200 fs short pulses at 1 kHz
repetition rate. The laser energy on the specimen can be precisely varied using two attenuators.
Each attenuator involves an electronically controlled rotating half-wave plate that rotates the
polarization of the laser beam and a cube beam splitter in which the intensity of the transmitted
light depends on its polarization. A fast mechanical shutter is used to select the desired number of
pulses for the ablation experiments. The laser beam is tightly focused inside the sample through
an oil-immersion high numerical aperture objective lens (Zeiss 64×, NA=1.4). The fluorescence
imaging system consists of a mercury lamp, and an FITC filter set. The excitation filter and the
emission filter transmit wavelength ranges of 460-500 nm and 510-560 nm, respectively. The
dichroic mirror reflects wavelengths smaller than 510 nm. A cold mirror is used to transmit the
laser beam but reflect the fluorescence emission towards either an intensified camera (Roper
Scientific, “PI-Max”) for two-photon imaging or an interline transfer CCD camera (Princeton
Instruments, “Micromax”) for epifluorescence imaging. The resulting emission from the focal
spot is collected by the tube lens and detected by the CCD camera (6.5 µm pixel size and 2×2
binning). A filter blocks the scattered laser light. A camera exposure time of 300 to 600 ms is
used. Simultaneously with laser axotomy, the system can also perform two-photon imaging with
a pair of piezo-driven mirrors that scan the laser beam. A relay-lens system with a pair of lenses
(focal lengths of 75 mm & 160 mm) are used to image the center of the scanning mirrors to the
back aperture of the objective lens to prevent laser beam displacement during scanning. This
relay lens system also enlarges the laser beam to fill the back aperture of the objective lens and to
achieve diffraction limited focusing. The inset on the right upper corner shows an example of
femtosecond laser axotomy with 10 nJ pulse energy (200 fs duration, 400 pulses at 1 kHz rate).
Fluorescence images of a D-neuron axon before and 10 minutes after axotomy are shown. The
rightmost image shows a photo-disrupted region with 1.2 µm length. This indicates that a volume
of a few femtoliters is ablated at the focal point. Assuming an axon diameter of 0.3 µm, we
estimate that a volume of the axon less than 0.1 femtoliter (1.2 µm × π (0.15 µm)2 ) is ablated.
Scale bar of the inset is 5 µm. The ventral part of the worm is towards the bottom of the image.
Dye-filling Studies to Assess Axotomy, Specificity and Damage Extent:
In order to test whether the observed axon gap after axotomy is simply due to
photobleaching or not, we have stained phasmid neurons with DiO-green before cutting
the dendritic processes that connects the cell body to the sensory ending. After cutting the
dendrites of neurons, we have stained with DiI-red. While the axotomized neurons
showed no red fluorescence (Supplementary Figure 2), their distal ends and the
unoperated neurons were filled with DiI-red. Thus, although outside-connected distal
stumps can take up the dye, they cannot pass it to the neurons due to physical
disconnection.
In DiO/DiI staining experiments, we can specifically cut one of the dendrites among two
nearby dendrites (only few microns apart) while leaving the other dendrite completely
intact (Suppl. Fig. 2). This experiment shows that not only the damage extent is less than
a few microns, but also we can selectively cut nerve processes.
a
b
Supplementary Figure 2. Axotomy of PHAR phasmid neurons. Prior to axotomy, two nearby
phasmids have uptaken DiO-green dye. (a) After axotomy of one of the two nearby dendrites,
they are visualized with FITC (green) filter. The other dendrite is not axotomized. (b) Worms are
incubated in DiI-red dye. The phasmid whose dendrite was cut is not visible with red optical filter
indicating that its dendrite is physically disconnected and the phasmid cannot update DiI-red dye.
The other phasmid that was not axotomized fluoresces red. The scale bar is 5µm. The arrow is
indicating the axotomized part of the dendrite. The tail of the worm is towards the right bottom
corner of the image.
GFP fluorescence photobleaching and recovery studies:
We have also performed controlled photobleaching experiments with our setup by
exposing cell bodies to laser pulses with 1-2 nJ energy at 1.0 kHz repetition rate. We
have completely photobleached both touch and motor neurons, and observed that
fluorescence signal could recover from complete photobleaching within 3 hours. The
recovery duration is consistent with previously reported photobleaching studies using
Argon Ion laser based confocal microscopy [11], which show that completely
photobleached neurons are able to synthesize and recover GFP fluorescence within 3
hours. Furthermore, immediately after we photobleach all motor neurons, the worms can
still move backwards exactly like the wild type. In the regime of the pulse energies (1040 nJ) we used in axotomy, the neurons never recover GFP fluorescence even after 6
hours, indicating that a permanent damage occurs instead of simple photobleaching. The
worms also cannot move backwards.
Furthermore, during photobleaching, the entire neuron loses its fluorescence intensity
instead of the local laser spot that is being photobleached. This indicates that GFP
molecules rapidly diffuse in and out of the photobleached spot, and that only a localized
region cannot stay as photobleached. This is also another indication that the local gap
observed after axotomy is not simply photobleached.
Aberrated Growth Cases:
For the 10-40 nJ energies we use for axotomy, we observe cases where aberrant growth
or no-regrowth occurs, which is also suggestive of successful axotomy. If our
observations were simply photobleaching, no aberrant growth should have been
observable. In fact, operated axons mostly show a "de tour" extension.
Movie:
Behavioral response of an axotomized worm. The movie includes two parts: Part I shows
an axotomized worm 3 hours after the axotomy. The worm can move forward like wild
type. When we touch to its head, it attempts to move backwards, but it cannot move,
instead its body segments shrink. Part II shows the same worm 24 hours after the
axotomy. It is now able to move backwards with sinusoidal body motions when we touch
to its head.
All References including Manuscript & Supplementary Information:
1. Horner, P. J., Gage, F. H. Nature 407, 963-970 (2000).
2. Bhatt, D. H., Otto, S. J., Depoister, B., Fetcho, J. R. Science 305, 254-258 (2004).
3. Perry, M. D. et. al. J. Appl. Phys. 85, 6803-6810 (1999).
4. Tirlapur, U. K., König, K., Nature 418, 290-291 (2002).
5. Shen, N. (supervisor Mazur, E.) Ph. D. Thesis, Harvard University (2003).
6. Vogel, A. et. al. Appl. Phys. B 68, 271-280 (1999).
7. Colombelli, J., Grill, S. W., Stelzer, E. H. K. Rev. Sci. Inst. 75, 472-478 (2004).
8. White, J. G., Southgate, E., Thomson, J. N., Brenner, S.,Phil. Trans. Royal Soc.
London. Series B, Biol. Scien. 314, 1-340 (1986).
9. Huang, X., Cheng, H. J., Tessier-Lavigne, M., Jin, Y. Neuron 34, 563-576 (2002).
10. McIntire, S. L., Jorgensen, E., Kaplan., J., Horvitz, H. R. Nature 364, 337-341
(1993).
11. Dwyer, N. D. et. al. Neuron 31, 277-287 (2001).
12. Venugopalan., V., Guerra III, A., Nahen, K., Vogel, A. Phys. Rev. Lett. 88, 07810314 (2002).