Electron Microscopes Shed New Light on Neural Prostheses

In the basement of the Huntington Medical Research Institutes building on Fairmount Avenue, two researchers sit in a darkened room, peering into the glass viewing port of a brand-new transmission electron microscope.  Above the console, a video image glows on a computer screen, bordered by graphical pull-down menus.  With the click of a mouse, a picture comes into focus.

Al Lossinsky, Ph.D., and Steve Manoonkitiwongsa, Ph.D. (Manoon, for short), are examining a fragment of nerve tissue taken near an implanted electrode.  They magnify the image to reveal incredible detail, down to the level of individual cells and the organelles within them.  But as they work, they're seeing something more:  the future of neural engineering at HMRI.

A Morgani M268D Digital Transmission Electron Microscope (TEM) was recently acquired by HMRI, along with an XL30 ESEM Scanning Electron Microscope (SEM), both made by FEI Corporation, a division of Philips.  Together, they will help launch a new research initiative, investigating how implanted electrodes interact with living tissue at the ultra-microscopic level.

The science of neural engineering - stimulating nerves with electrical impulses - at HMRI was pioneered by Robert H. Pudenz, M.D., and C. Hunter Shelden, M.D.  HMRI researchers have devised better and better neural prosthetic devices with improved biocompatible materials, more durable coatings, and more precise shapes that deliver electrical pulses efficiently and with less tissue damage.  Throughout the 1960s to '90s, an earlier TEM was used in implant studies at HMRI.  But it's the 21st century now.  Sophisticated new materials are available, and biological techniques are much more powerful.  Meanwhile, as our population ages, the demand for neural engineering is growing fast.

Blindness.  Hearing loss.  Parkinson's Disease.  Epileptic seizures.  Incontinence.  All are serious problems that devastate an individual's quality of life.  And all are being treated with implanted neural prostheses.  While some concepts like artificial vision are still in the early stages, others are proven technologies.  One of the most successful is the Huntington Helix vagus nerve stimulator, developed at HMRI.   The Helix now helps control epileptic seizures in 16,000 patients worldwide.  Like other neural prostheses, it can deliver instant relief without the side effects of drug therapy.

Under the direction of physicist Leo Bullara, technicians in the Neural Engineering Laboratory are working on tiny electrodes made from rare substances including platinum and iridium, the most corrosion-resistant material on earth.  Like spider's silk, these electrodes are fine as angel's hair and delicate as a whisper.  But besides being unobtrusive, implanted devices must transmit electrical impulses with the utmost reliability.  They must be durable yet flexible, and work continuously for 15 years without causing chronic inflammation, being walled off, dissolved, or otherwise deactivated by the body.  That's a tall order.

The human nervous system is an infinitely complex network of nerve cells, receptors and bundles that reach from microscopic fibers at the tips of the toes to the spinal cord and brain.  With an endless number of connections and lines that can malfunction, prostheses and implants are needed in a variety of configurations, sizes, materials, depths and arrays, with tips that can penetrate flesh and carry current without damaging the most delicate tissues.  Posters in Bullara's lab show their evolution and variety.  Peripheral nerve electrodes include flag bipolar, silicone and dacron backed cuffs, tripolar spiral, bidirectional, bipolar and multipolar designs.  Central nervous system, spinal cord and inner ear designs include the occipital cortex array, faceted tip, conical tip, monopolar shaft, disk, large area disk, cochlear nucleus surface array, cortical and spinal cord arrays.

"This is a pretty invasive technology," says Douglas McCreery, Ph.D., Director of the Neural Engineering Program.  "So the mechanical engineering and the electrochemistry must be precise and well defined.  We need to be able to resolve spatial details less than 1/100,000ths of an inch.  To perfect and maintain quality and consistency in design and fabrication, we need to be able to see details far beyond the limits of light microscopy.  For neuroengineering development, fabrication and testing, we need resolution that's possible only with electron microscopy."

The new microscopes were formally unveiled at a "first light" ceremony, attended by HMRI Executive Director William Opel, Ph.D., former Neural Engineering Program Director William F. Agnew, Ph.D., Board member Robert J. Mackin, Jr., Ph.D., and donor Herb Hezlep of the Hezlep Family Foundation.

Focus on the Future

Now, Dr. Lossinsky readies the SEM for a scan.  His sample is a tiny titanium screw that will be used to fasten a neural device securely to a subject's skull while its electrodes reach deep into the brain.  Such deep brain implants have been shown to provide relief from the tremors and spasticity of Parkinson's Disease once drug therapy loses its effectiveness.

He clicks on a vacuum pump that quietly purges air out of the sample chamber.  But it's no ordinary pump - it's a high-tech, ultra-clean turbomolecular pump, which the manufacturer agreed to adopt after urging from HMRI researchers.  In fact, finding the perfect pair of microscopes was itself a science.  Lossinsky made a total of 18 site visits to different manufacturers and their customers.  (A side benefit:  he brought real samples to test, and collected enough data to publish a paper.)  After careful consideration, the XL30 and its cousin the Morgani were finally purchased and installed.

Now the vacuum is ready.  A little water vapor is released back into the chamber, to ground the material electrically.  At the top of the chamber, an electron gun bathes the sample in energy, transmitted to a monitor as a recordable digital image.  At high magnification, the titanium's surface is a fantastic landscape of ridges, pits and variations invisible at all but the highest magnifications.  The XL30, a top-of-the-line device, even permits researchers to study samples under different environmental conditions.  In fact, unlike other scopes, some air or water vapor can be introduced into the chamber, allowing researchers to study cells (and even insects) while still alive.

As he scans the sample, he notes that these will be the first images of many neural engineering materials and designs ever captured at such high magnification. "We're still in an early era of implanting into the central nervous system," he says.  "For practices such as deep brain stimulation for Parkinson's, we're still at the frontier of knowledge."  Now, as new implants are tested, they can be studied at the finest levels of detail, enlarged up to 280,000 times.

But understanding how neural implants react to human tissue is only half the story.  For the other side - how the body reacts to the implants - Drs. Lossinsky and Manoon go to the Morgani Transmission Electron Microscope, a few doors down the hall, where histology and EM technologist Jess Chavez has prepared a sample of tissue taken from the vagus nerve of an implanted test subject.  Preparing material for the TEM is a special art.  Tissue must be fixed, processed, embedded into a plastic block, sliced into sections far thinner than a human hair, and painstakingly stained with exotic chemical and biochemical reagents.

Dr. Manoon carefully places the sample into the chamber.  Magnifying it 7,000 times, he can easily inspect the fast-conducting myelin fibers of the nerve.  The area where the electrode touched the nerve has become inflamed, as the body has begun to form a capsule around the device.  Researchers at HMRI have long recognized the problem of managing tissue inflammation.  "We could see this at low power with a light microscope," Lossinsky explains.  "The difference is that now, we can go in and see what's happening within single cells."

"Ultimately, you have to look at the cells themselves to understand the consequences of biochemical, molecular and physiological changes due to implantation and electrical stimulation," Manoon asserts.  And today, transmission electron microscopy does more than merely magnify images.  New high-tech methods such as immunocytochemistry, enzyme cytochemistry and molecular in situ hybridization can actually pinpoint biological processes going on within intact tissues and cells - like taking a snapshot of cell physiology in action.   While conventional molecular analyses can monitor levels of nucleic acids, proteins, enzymes and other substances in tissue extracts, they can't identify where these substances are located in intact cells and tissues.  Now, TEM can.

"HMRI is a world leader in neural engineering," says Lossinsky.  "Now, we will be applying the latest histological techniques to the development of neural prosthetics."

Leo Bullara adds, "This equipment is a godsend.  We have been striving to perfect some of our prostheses for 20 years.  This will help ensure that our designs are absolutely optimized for implantation."  HMRI is working to expand the field of neural engineering, perfecting a cochlear nucleus implant for profound deafness and an implant to control stress incontinence in seniors. Grant applications are being written, and additional funding is sought to capitalize on the advanced research opportunities afforded by the new electron microscopes.  "We're helping people, and that's very worthwhile," Bullara says.  "That's what keeps us coming in to work every day."

Reporter: Gregory M. Vogel