Femtosecond laser nanoaxotomy lab-on-a-chip for in vivo nerve regeneration studies

Abstract

A thorough understanding of nerve regeneration in Caenorhabditis elegans requires performing femtosecond laser nanoaxotomy while minimally affecting the worm. We present a microfluidic device that fulfills such criteria and can easily be automated to enable high-throughput genetic and pharmacological screenings. Using the 'nanoaxotomy' chip, we discovered that axonal regeneration occurs much faster than previously described and surprisingly the distal fragment of the severed axon regrows in the absence of anesthetics.The understanding of the biological mechanisms of nerve regeneration and degeneration after injury holds the key to developing novel therapies for human neurodegenerative diseases. These processes can be studied in model organisms by severing axons in a controlled manner, and then observing their regrowth and functional recovery. The ideal predisposition of the nematode Caenorhabditis elegans for such studies recently became accessible by the demonstration of precise nanoaxotomy using ultrafast laser pulses 1 . However, the side effects that the chemicals used to immobilize the worms for laser nanoaxotomy might have on nerve regeneration are difficult to evaluate, unless nanosurgery can be performed in vivo without anesthetics. The environment in which both surgery and monitoring are performed should have a minimal impact on the studied organism and its biological processes. To achieve this goal, we designed a microfluidic device that allows us to sever axons in C. elegans using ultrashort laser pulses with the same high precision as we demonstrated previously 1,2 while monitoring the subsequent axonal regeneration activity.Several microfluidic devices and microelectromechanical systems (MEMS) have recently been developed for C. elegans including, Petri dish-based microfluidics 3 , microfluidic traps 4,5,6 , the "CD-worm" 7 , a shadow imaging platform 8 , microfluidic maze structures 6,9 , a cantilever force MEMS sensor 10 and a platform to capture and sort worms 11 . However no demonstration of nanosurgery on-a-chip has been reported so far.Correspondence should be addressed to A.B. (ben-yakar@mail.utexas.edu The integrated microfluidic device we designed presents several unique features that are critical for the success of in vivo nerve regeneration studies: the worms are held directly against the glass cover for ideal focusing and precise nanosurgery; the trap is adjustable to the size of the worms allowing immobilization of worms at various developmental stages (L4 to adult size); and the system integrates feeding modules and thus allows long term follow-up studies of the axotomized worms as well as their sorting and screening.The high-throughput microfluidic system integrates two separate modules (Fig. 1a), a trapping module for nanosurgery and time-lapse imaging ( Fig. 1c-e) and a feeding module for recovery of the operated worms (Fig. 1b). Follow-up imaging of injured axons and their regrowth is performed using the same trapping module. Depending on the outcome of the imaging session,...

Large-scale in vivo femtosecond laser neurosurgery screen reveals small-molecule enhancer of regeneration

“…1 A and D). This fully constrains the animal motion for imaging and surgery (12,19). Once the animal is immobilized, the microscope and camera configuration automatically switches to high-resolution acquisition.…”

“…Precise laser axotomy and imaging at the cellular level require orientation and immobilization of animals. Traditional immobilization methods using anesthetics, such as sodium azide, levamisole, and tricaine/tetramisole, have significant and/or uncharacterized effects on nematode physiology, which may affect the regeneration process (12). In addition, anesthetics need several minutes to take effect, and recovery of nematodes from anesthesia requires exchange of media without losing animals, all of which hinder high-throughput screening.…”

C. elegansas a genetic model to identify novel cellular and molecular mechanisms underlying nervous system regeneration

“…To substitute the conventional way of immobilizing worms, such as anesthesia on agar pads, microfluidic devices immobilize worms in a chemicalfree manner through thermal or mechanical mechanisms. [71][72][73] By trapping the worm in a cooling liquid or a pressurized chamber, microfluidic devices effectively avoid the adverse effect of anesthesia on axon regeneration and minimize the time for worms to recover after laser surgery. 74 Furthermore, in combination with the interface to multi-well plates containing compounds or an RNAi library, microfluidic devices allow high-throughput screens for novel genes involved in axon regeneration and drugs that interact with axon regeneration in live animals.…”

At the cutting edge: applications and perspectives of laser nanosurgery in cell biology

“…Some of the limitations of this technique have been overcome and show how this technique could be even further exploited when for example it is integrated with new developments in other fi elds. Recently, some studies have successfully performed laser nanosurgery on C. elegans in microfl uidic environments (Allen et al , 2008 ;Guo et al , 2008 ;Zeng et al , 2008 ) opening the interesting possibility to achieve a much higher throughput for laser ablation and thus to perform experiments at even genome-wide scales ( even allow high-throughput applications of laser nanosurgery at the single cell level (Figure 5 A) as has been suggested for entire organisms in microfl uidic chambers (Ben -Yakar and Bourgeois, 2009 ). Furthermore, particular patterns could be used to segregate organelles in the cell periphery, therefore allowing their precise removal.…”