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Injuries to the HeadWe have all heard of athletes, particularlyhockey and football players, who receivednumerous head injuries during theircareers. These injuries are believed to leadto degenerative brain diseases similar toAlzheimer’s disease. Some of these athletesdied at an early age and postmortem diag-noses pointed to vascular injuries as apotential cause of the degeneration. Weknow that such injuries can cause a rangeof disorders in the brain, but we do notknow how.

The Right Tools Whether it’s Alzheimer’s disease, epilepsy,small strokes, brain cancer, or spinal cordinjury, dysfunction results from a distur-bance at the cellular level inside thecentral nervous system. To see at this scaleinside the brain—actually observe howindividual cells are affected, as well aswhere and when after an injury or in dis-ease development—is very difficult. Yet itis essential to see the cellular behavior inorder to comprehend the mechanisms thatlead to dysfunction. A major focus in my

lab is to understand the cellular-levelchanges that occur in the central nervoussystem and lead to these diseases.

Because of the technical challenge ofdoing such experiments, my lab developstools and techniques and uses them toanswer key scientific questions. We focusabout one third of our effort on develop-ment of novel techniques and two thirdson answering questions about neurologicaldisease states, often using advanced toolsthat we develop.

Optical Tools for Seeing HowDisease Changesthe BrainHow do injuries to small blood vessels in the brain contribute tobrain disease? Why don’t axons regrow after a spinal cord injury?What is the relationship between impaired brain blood flow andAlzheimer’s disease? To find out, we develop tools and techniquesthat let us see what is happening to individual cells inside the brainduring disease development in animal models.

Chris B. SchafferBIOMEDICAL ENGINEERING

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Disturbances in the BrainMedical scientists have known for sometime that injury to small blood vessels in the brain is associated with cognitivedecline and dementia in aging humans. Theyhave found in postmortem examinationsthat people who exhibited more severecognitive decline had more small-vesselinjuries. This kind of clinical data, however,does not help us to understand the mecha-nisms by which small-vessel injuries leadto brain cell dysfunction.

Getting at the Root of Microvascular StrokeTo uncover what’s happening at the cellularlevel after a microvessel injury, we need an intact circulatory system carrying bloodthrough the brain, so we do our experimentsin animal models—mostly mice. We causean injury by clotting or hemorrhaging a

little vessel and then directly observe howthe injury affects the cells in the brain inorder to help us understand how brain cellsbecome dysfunctional after such small vascular injuries.

We produce vascular injuries using intensepulses of light that are fantastically shortin duration, about 100 millionths of a bil-lionth of a second long. This short pulse of light can be used to injure the wall of a targeted blood vessel. For larger injuries,this causes the vessel to rupture and pro-duce a small hemorrhage. With a moresubtle injury, the bleeding is limited, butthe injury initiates clotting and a blockageis formed in the vessel.

We then use laser-based optical imagingtechniques to see how individual cells areaffected, how they die, how they lose func-tion, what new cells invade the injury site,and how they interact after the microvesselinjury. We can identify individual cells andsubcellular features and track how theychange over time, from minutes to monthsafter the injury over a spatial scale ofabout a millimeter and with micrometerspatial resolution. Our goals are to revealthe mechanisms underlying these brain dis-eases and identify therapeutic targets totreat the problem.

A Closer Look at Small HemorrhagesRecently my lab began looking keenly atsmall hemorrhages. We and others have pre-viously found that occlusion of small bloodvessels causes the death of nearby neuronsand other brain cells. For small hemorrhages,

however, the mechanism that leads to dysfunction appears to be not so simple. We were surprised to find that when we puta small hemorrhage in the brain, the nearbycells did not die. The neurons around the

small hemorrhage were unharmed andremained so for weeks, as long as wewatched. Inflammatory cells invaded thearea near the hemorrhage, but they did notseem to do anything catastrophic.

So whatever causes the dysfunction doesnot kill the cells. In preliminary data, wedo see that there is an elevation in the rateat which the neurons near the hemorrhagechange their pattern of wiring to eachother. This random or semi-randomrewiring of the neurons in the brain maydisrupt the brain’s normal function and isa potential cause of cognitive dysfunctionfollowing small hemorrhages.

Only by SeeingWe could have made this discovery onlythrough an imaging technique—we need tosee an individual synapse before producinga little injury. After we produce the injury,we need to see it again. We have to see itagain two days later, two weeks later, andsee if the same synapse is still there or if itwent away or if a new one formed, and atwhat rate synapses are lost and gained. Weneed the combination of advanced opticaltechnologies for imaging and the ability tomanipulate with cellular resolution in alive animal, having clear hypotheses aboutwhat structural and functional changes mayunderlie disease, in order to make advances.

In addition to studying the effect of small vascular lesions, their link to Alzheimer’s disease, and cellular dynamics after spinal cord injury, my lab also has projects that focus on studies of epilepsy, brain cancermetastases, blood cancer, neural prostheses,and the development of next-generation toolsfor biomedical research.

Fascinating! t

We image the brain of live anesthetizedmice after removing a section of the skullto gain optical access to the brain. Themice recover from the surgery, and we cancome back and reimage the same animalover time after an injury. We can find thesame blood vessel that we hemorrhaged.We can find the same dendrite that sitsnext to that blood vessel, and we caneven find the same dendritic spine—themicron-sized structure where neuronscommunicate with each other. We can finda one-micrometer region located withinthe brain of the mouse and tell whether itchanges day to day.

We’ve found that after some kinds of braininjury, new dendritic spines form and existingones die at a faster rate than in controls.This random rewiring could, even in theabsence of cell death, lead to cognitivedysfunction.

Research in Progress

Linking Small Strokes and Alzheimer’s DiseaseResearchers have shown clinically thatAlzheimer’s disease and microvascularinjuries in the brain are often associatedwith each other—they are comorbidities. Is it because microvascular injuries triggerAlzheimer’s or does Alzheimer’s pathologycause microvascular injuries—or both, orneither? Alzheimer’s disease is a clinicaldiagnosis. It could be that, in order toexhibit enough dementia to be classed ashaving Alzheimer’s disease, two independentdisease pathways are affecting the brain.

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In very recent work,we have found thatsmall vascular occlusions occur spontaneously at amuch higher rate inmice with Alzheimer’sdisease than in normalmice. Together with ourresults suggesting thatsmall strokes can driveamyloid-beta accumu-lation, this suggeststhat microvasculardysfunction and thepathology of Alzheimer’sdisease form a viciouscycle that mutuallyexacerbate each other.

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Short-pulse laser systems have been a consistent part of my research career. As an undergraduate, I worked on designingand building laser systems that produced femtosecond-duration laser pulses. These pulses were as short as 10-14

second in duration! I studied the physics of the interactions between these pulses and materials as a graduate student.When I became a postdoc, I realized that all I had studied and done could be applied to create what is essentially a fancylaser scalpel for biomedical research, and perhaps it could be used as a surgical tool.

I transitioned to biology-based research in order to apply some of the tools I had discovered. One aspect of my currentresearch is to develop high-fidelity research tools for studying diseases in animal models, so that we can uncover theunderlying mechanisms of disease. If we understand the mechanisms that lead to a disease, then we can identifytherapeutic targets for drug development, surgical strategies, or a broad variety of medical approaches for treatment or prevention.

Why this Research?

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To help sort this out, our experiments usemice genetically engineered to get Alzheimer’sdisease to see if more Alzheimer’s pathologyis triggered when we induce a small stroke.Alzheimer’s is caused by a buildup ofamyloid-beta, a small peptide that is pro-duced by neurons and cleared through thevasculature. It is the aggregated form ofthis peptide that causes neurons to losefunction and die.

We found that when we blocked a smallblood vessel in the brain, this triggered theaggregation of new amyloid beta in theimmediate vicinity of the clot. This makessense, because if we clot a blood vessel, it’slike plugging the drain that amyloid-betauses to get out of the brain. This leads toan increase in concentration of amyloid-beta, and therefore more aggregation. Ourdata suggest that cerebral vascular diseasecould be an initiating or exacerbating fac-tor in the development of Alzheimer’s.

Our finding is exciting because it suggestsnew preventative or treatment strategies forAlzheimer’s. We could target the vascularcomponent of Alzheimer’s disease

independently of other strategies beingdeveloped to target the amyloid-betaaggregation or neural dysfunction.

Building, Experimenting, and GettingIlluminating ResultsCreating the right tools to see is pivotal.For example, many studies of spinal cordinjury in animal models were limited bythe tools that were available, primarilybehavioral assays and postmortem histol-ogy. As a result, incorrect or ambiguousconclusions were drawn. Researchers didnot have the right tools to answer vitalquestions like, is an individual axon dyingback or growing after an injury? Recently,we developed a technique to image suchdynamics and quantify them.

It turns out that, for the study of spinalcord injury, the technique developmentthat was needed involved a surgical prepa-ration, not a fancy optical tool. We neededa good way to do surgery on a mouse sothat we could have optical access to thespinal cord and keep that access for days,weeks, and months.

With this new approach, we can image thedieback and potential regeneration of indi-vidual severed axons in the spinal cord ofthe same mouse over time and withmicrometer-scale spatial resolution. Ourtechnique will help us and other researchersin understanding spinal cord injury anddeveloping therapeutic strategies.

Seeing the InvisibleAxons, the neurons that conduct impulsesaway from a cell body to other cells, in thespinal cord are wrapped with many layersof cell membrane called myelin. Researchersbelieve that the loss of myelin surroundingindividual axons may be a factor in degen-eration after spinal cord trauma and thatthis loss impedes regeneration.

Myelin provides essential electrical insulationfor the axon. Without it, the signal that isconducted down the axon tends to fail. Insome spinal cord injuries, evidence suggeststhat the axons are not severed. They havelost myelin, and that’s why the conductionis not good. With poor conduction, theaxons do not communicate well with thecentral nervous system about what the

We label structures and cells of interest by injecting a fluorescent dye into the bloodstream, and all the blood vessels carry it, like angiography. We can add fluorescent dyes that give structuralinformation about where neurons are, determine if cells are dead or alive, and even discern if theability of a neuron to do its job has been altered as a result of disease. In the image, we visualizethe amyloid-beta aggregates that are characteristic of Alzheimer’s disease (green) and the bloodvessels (orange) in the brain of a live mouse.

Schaffer Lab

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muscles should do. In order to understandwhat happens to myelin and developstrategies to encourage remyelination, it isnecessary to be able to see the myelin in alive animal.

We have many fluorescent labels that canbe used to mark the cells and axons in thespinal cord, but labeling myelin for imagingis very difficult, because the fluorescent dyesthat can be added to myelin dramaticallydisrupt the structure. In recent work, wefound that a nonlinear optical techniquecalled third-harmonic generation (light ofone wavelength is converted into light onethird of the wavelength by interacting withthe sample) is very efficient whenever wehave a bold optical interface, like that

produced by myelin (mostly lipid) that wrapsaround an axon (mostly water-filled).

Using third-harmonic generation, we cansee inside a mouse’s spinal cord after aspinal cord injury and investigate howchanges in the amount or structure ofmyelin occurs. We want to use this toolultimately to study strategies for triggeringremyelination in injured axons, movingtoward therapies for spinal cord injury.

The Rewards of Seeing ClearlyOur research approach of developing toolsthat enable novel classes of experiments isespecially rewarding when we can comeinto a field with a key unanswered scientificquestion. Often the question is obvious,

but the right tool is not available to studyit. We can build a tool for the experiment,do the experiment, and get the whole fieldmoving again.

courses2.cit.cornell.edu/schafferlab

We use an imaging technique called two-photonexcited fluorescence microscopy, which was developed here at Cornell in the early 1990s in the lab of Watt Webb, Applied and EngineeringPhysics. It is a way to image fluorescently labeledobjects in three dimensions inside tissue, while atthe same time alleviating the problem of loss ofcontrast and resolution that is caused by scatter-ing of the fluorescent light. The image shows a 3-D rendering of blood vessels in the brain of alive mouse.

Two-Photon ExcitedFluorescence Microscopy

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