Femtosecond laser technique opens new opportunities for research on neural regeneration

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Femtosecond laser technique opens new opportunities for research on neural

regeneration 16 Dec 2004

In a breakthrough for research on nerve regeneration, a team of scientists

has reported using femtosecond laser pulses to precisely cut individual axons of

nerves in the roundworm Caenorhabditis elegans, one of the most versatile and

widely used experimental organisms for genetic and biomedical research. The

nerves severed by this precision technique regrew within 24 hours, often with

complete recovery of function.

The project was a collaboration between applied physics researchers at

Stanford University led by Adela Ben-Yakar and biologists at the University of

California, Santa Cruz, led by Yishi Jin and Chisholm.

The team's findings give researchers an experimental system in which they

will be able to investigate in great detail the genetic and molecular factors

that control whether or not damaged nerves can regrow, said Chisholm, an

associate professor of molecular, cell, and developmental biology at UCSC.

" This technique will enable us to find the genes that are important in

allowing an axon to regenerate. In the worm, we can do systematic screening of

large numbers of genes, and of drugs and other small molecules as well, to ask

how they affect the process of regeneration, " Chisholm said.

The researchers reported their findings in a paper published in the

December 16 issue of the journal Nature. The first author of the paper is Mehmet

Fatih Yanik, a Stanford graduate student in applied physics, who worked with

Ben-Yakar to develop the laser nanosurgery setup used in the study. Ben-Yakar

initiated a femtosecond laser nanosurgery project two years ago at Stanford and

is currently an assistant professor of mechanical engineering at the University

of Texas at Austin. The other coauthors include Jin, a professor of molecular,

cell, and developmental biology at UCSC and a Medical Institute

investigator; Hulusi Cinar, a postdoctoral researcher in Jin's lab; and Hediye

Nese Cinar, a postdoc in Chisholm's lab.

The Cinars met Yanik through personal connections and initially discussed

possible collaboration about a year ago. Yanik later told them about the

femtosecond laser and how other researchers had begun using it in biological

systems to surgically destroy extremely tiny structures.

" When Yanik described to me what this instrument can do, I immediately

thought of my work on the nervous system of C. elegans and came up with a nerve

regeneration experiment we could do together, and I designed the experiments, "

said Hulusi Cinar.

The technique uses extremely short pulses of intense laser light to focus

energy in a very small volume. When properly focused, the energy delivered by

the laser pulses breaks down chemical bonds at the targeted site, vaporizing the

tissue within a tiny volume without causing side effects such as heating of

surrounding tissue, Yanik said.

The duration of the laser pulses used in the study was 200 femtoseconds (a

femtosecond is a millionth of a billionth of a second), and the pulses were

delivered at a rate of one thousand per second. The delicate axons severed by

the procedure, with no apparent damage to surrounding tissue, were on average

just 0.3 microns, or 300 nanometers, in diameter (a nanometer is one billionth

of a meter).

" I am very excited about the merging of this new technology into

biological research. I didn't know anything about femtosecond laser technology

until the physicists explained it to me, " said Jin, who has spent years

investigating the development of the worm's nervous system. " Now there is a lot

to do--it has opened up the potential to address questions that we have never

been able to address before. "

In their experiments, the researchers cut a nerve that runs from one side

of the worm's body to the other. The nerve inhibits contraction of muscles on

one side of the body while the muscles on the other side are contracting. It

functions during the alternating contractions of muscles on either side of the

body that enable the worm to wiggle backward with a smooth wavelike motion.

Hulusi Cinar and Jin have been studying so-called " shrinker " mutants that lack

this ability due to a genetic defect.

" A normal worm, when you touch its head, will move backward in a smooth

motion. In the shrinker mutants, the muscles on both sides contract

simultaneously, so they don't move back, " Cinar said. " So these neurons were a

good target for surgery because we knew that when they are knocked out you get a

well-defined behavioral effect, and it's straightforward to see if their

function has been recovered through regeneration. "

Nese Cinar designed a movement assay to evaluate the behavioral effects in

the worms and evaluated the worms in the experiments in a " blinded " manner, not

knowing which ones had received the surgery.

" Without such functional assays, any anatomically observed regeneration

could be explained in various ways, such as bleaching and recovery of the marker

protein used to label the neurons, " she said.

The nanosurgery, performed by Yanik at Stanford on anesthetized juvenile

worms, could be completed in about 10 minutes per worm once the equipment was

set up. Although regeneration of peripheral nerves is nothing new, Chisholm said

he was still surprised by the rapid recovery of function in the worms. Most of

the severed axons regrew within 12 to 24 hours after the laser surgery.

Preliminary observations indicated that after an axon is cut, the nerve cell

sprouts a new axon from the severed end that regrows to reach the target muscle.

In some cases, however, it appears that the two severed ends reattach, Chisholm

said.

" Clearly there is a lot more biology going on here that we need to

explore. This just opens up a lot of exciting things to study, " he said.

C. elegans, an almost microscopic nematode or roundworm, has become an

extremely important system for biomedical research. Geneticists have identified

thousands of genes in the worm that have counterparts in humans. A relatively

simple organism with a short generation time, reproducing in just three days, it

is easy to study in the laboratory. It has many of the same basic physiological

and anatomical features, such as muscles and nerves, found in more complex

animals. And it is even transparent, making it easy to see things like nerves

inside its body. The researchers who pioneered the use of C. elegans for

biomedical research received the 2002 Nobel Prize for physiology and medicine.

Now this excellent model system for biomedical research is available for

studying nerve regeneration. One of the fundamental questions researchers want

to answer is why nerve damage in the central nervous system--the brain and

spinal cord--is usually permanent.

" In humans, peripheral nerves will regrow, but in the central nervous

system the regrowth of axons is inhibited by the local environment. That's why

spinal cord injuries are so serious. We want to find out why a severed axon will

regrow in some situations and not in others, " Chisholm said.