Deep Brain Visualisation and Surgery in Animal Research

Charles W. Scouten, Leica Microsystems

Cellular events deep in the living brain have been monitored with electrical probes, but visual observation has not been possible.Two new instruments now enable visual observation of cellular level events deep in the living brain. For the first time, the lasermicroendoscope Leica FCM1000 permits visualisation at the cellular level. A very small probe, 300 microns or greater, is insertedinto the brain, and laser microscopy allows observation of a small group of cells located at the tip. Movement of fluorescentlabelledsubstances into or out of cells, axonal sprouting after chemical or physical trauma, migration and fate of injected stemcells, micro vascularisation changes, and other features can be directly observed even as they are occurring. For effective use inbrain research, it must be possible to get the tip of the microendoscope accurately to a target site in the brain, and even to be ableto return to the same target at a later time.

Reaching any target with stereotaxis

The new small animal stereotaxic instrument LeicaAngle Two significantly improves accuracy, andenables the operator to reach any target site in thebrain from any chosen angle accurately and consistently.A stereotaxic instrument consists of aspecies-specific head holder mounted on a baseplate and used to orient the animal’s head in a definedposition, and a manipulator with movementaxes aligned with the head holder. The manipulatoris used to move a probe to selected targets in thebrain of the animal relative to chosen zero points,usually skull landmarks Bregma and Lambda, visiblelines where skull plates have grown together. These points overlie the brain at consistent positionsrelative to brain structures in rodents. They are ata crossover of the midline suture, and the anterior(Bregma) and posterior (Lambda) coronal suturesacross the skull perpendicular to the midline.

Precise orientation

The Angle Two manipulator features linear encoderson the three linear axes, and rotary encoders ontilt and rotation movements, five instrumented axesin all. These connect to a computer, into which a targetpoint in conventional atlas coordinates relativeto Bregma and assuming “skull flat” – Bregma andLambda at the same vertical coordinate– is enteredeither by typing, or by scrolling an onscreen atlas ofcoronal sections of the brain, and clicking on the desiredtarget point with the mouse.

The animal is then anaesthetised and installed inthe head holder, the skull landmarks exposed, andthe manipulator tilted and/or rotated to any chosencomplex angle. Moving the probe tip to Bregma, andclicking an onscreen button, informs the computeras to where Bregma is in the tilted and rotated andnon-orthogonal coordinates, and enables it to instantlycalculate how far to move along each linearaxis to reach the chosen target, given the manipulatortilt and rotation. The atlas display on screenshows the current position of the probe tip aboveor in the brain as the operator moves it toward thetarget. The user can see what structures are beingtraversed as the probe moves toward the target.

No time-consuming adjustment

Adjusting the animal’s head to skull flat is normallya trial-and-error and time-consuming process. Verysmall errors in head tilt give large errors in positionreached, especially if the probe is being lowered toa position deep in the brain. The Leica Angle Two includes the Virtual Skull Flat™ feature. Moving theprobe to Lambda, and clicking a button on screen,after showing the computer where Bregma is located,enables the computer to calculate the degreeof head tilt, and how that alters the target position.The path to the target is then recalculated given thehead tilt, and a mathematically correct target positionpresented. It is no longer necessary to achieveactual skull flat adjustment.