Your own thank you so
I know this is a diverse audience so

please don't hesitate to stop me if
I'm using lingo that maybe you don't

understand so today I'm that talked
to about the work that my dad and

my lab does using diffuse optics
which are kind of the general

name that we call
the technologies that we use and

specifically how we use it to develop
noninvasive biomarkers of brain injury.

With spin towards Key
after complications so

the physical principles that underlie the
techniques that we use are well known to

most of you whether you are familiar not
if you've ever taken a class flight and

you sign it on your hand what you
notice is that your hand glows red So

you're sending white light in but
only red lights coming out.

The other thing that you'll notice is
that unlike X.-Ray where you can make out

distinct features in your hand like
bones instead of your hand glows.

Because the light has
diffused through the tissue.

So the reason that your hand glows is
because tissue is highly scattering and

it leads to a random walk of
photons through the tissue.

And here I'm plodding in the.

Scattering coefficient for
tissue in the near infrared

where this reduced scattering coefficient
is the inverse of the random walk step

size that a photon takes in between
scattering events and you can see that

in this near infrared region it's on the
earth order of ten inverse centimeters so

what that means is that
a photon is going to.

Have an average random walk
step size of one millimeter So

when your light goes through your hand
which is say two centimeters thick it

scattering hundreds of times before
it makes it out to the other side.

In addition to being highly scattering
tissue is also highly absorbing so

here if we look at the absorption
spectrum as a function of wavelength

where this absorption coefficient new
A is the inverse of the average distance

that a photon is going to travel before
being absorbed we can see that on average

absorption is actually quite high for here
I'm putting the two main chroma fours that

are the two main things that absorb light
in brain tissue which are oxy and D.

oxy hemoglobin so absorption is quite
high in most of the visible spectrum but

in the near infrared there's this
window where absorption is low and

if we zoom in on that window so

just within the near infrared you can see
the absorption coefficient is on the order

of point one which is about one hundred
times lower than the scattering

coefficient which means that your
photons are going to scatter hundreds of

times before being absorbed leading to a
deep penetration of light through tissue.

So since I apply this principle to
the brain I often get asked whether light

penetrates through the skull and
the answer is Yes So here we took.

Laser pointer and put it on the interior
of a skull and you can see that that light

makes it out through the other side and
it's diffused through the skull.

So the goal of these diffuse optical
spectroscopy techniques in my lab

is to develop tools that exploit this
unique property of tissue to penetrate.

Deeply.

And specifically we use two tools
called Near Infrared spectroscopy and

diffuse correlations spectroscopy talk
a little bit more about them and a bit

we use these tools we apply them to
the brain using just a light source

on a detector we characterize the optical
properties of the brains the absorption

the scattering of as well as a diffusion
coefficient which I'll talk about later.

And then we relate these optical
properties to physiologically relevant

parameters that clinicians can use to
guide patient care at the bedside so

specifically we're able to quantify
oxygen saturation blood volume

blood flow as well as oxygen metabolism.

So today I'm just going to talk to
you about one of the diffuse optical

techniques that we use
which we call D.C.S.

or diffuse correlation spectroscopy D.C.S.

is used to measure the shear induced
diffusion So this is D B parameter.

Of the red blood cells that are flowing
through the tissue vasculature and

this diffusion coefficient
is directly proportional

to the blood flow in the tissue.

So just a little bit about how D.C.S.
works basically near infrared coherent

light source the light scatters multiple
times as it kind of treats the tissue and

some of the light is going to make
it back to the tissue surface so

the electric field at the tissue surface
is built from the superposition of

multiple scattered light fields that have
travelled along different paths to reach

the tissue surface then
they constructive and

destructively to form this
interference pattern.

If we just look at one of
these little speckles and

we track its intensity as a function
of time if all of the Scouters in your

tissue were static that intensity about
speckle is just going to be static

it's not going to change but
if there are moving scatters in the tissue

which there are because red blood
cells are highly scattering.

Then you're going to start to see
intensity fluctuations and the faster

the blood cells are moving in the tissue
the faster the lights going to fluctuate

so to characterize these
fluctuations in practice

what we do is we place a light detector
some distance away from our source and

we capture just one of these speckles and
we quantify in intensity autocorrelation

function which just you know from this
measured intensity data as a function of

time where the decay rate of this
intensity autocorrelation function

is related to the blood flow in
the tissue so just to give you kind of.

Intuitive sense of what's going on we
measure this autocorrelation function this

was data we took on a patient who we had
breathed room air for a little bit and

then breathe C O two carbon dioxide
is a Visa dilator in the brain so

it's going to increase your blood flow and
you can see that the curve that was taken

during the room air period where blood
flows lower decays longer than the curve

that is taken during the C O two period
where we know blood flow is increased so

this decay rate of this autocorrelation
curve is reflective of the blood flow in

the tissue and we have these simple models
that tell us how this this current.

We fit the curve to simple models in
order to extract a blood flow index

in the tissue.

This measured blood flow index
represents a bulk average of the tissue

underneath our optical sensor

that kind of you can think of it
as being a banana shaped region.

That propagates from the light source
to the light tech detector and

the farther apart we place from
our source from our detector

the deeper on average that banana pattern
is going to penetrate into the tissue.

So you know in theory we could go
all the way through the brain but

in reality we're limited by signal noise
so for most of our measurements in humans

at least probing mostly cortical like
this the superficial cortical tissue.

So just to give you a sense of what our
instrumentation looks like this is our

whole little lab on a car we've got our
D.C.'s box is measuring blood flow and

then we also have another technique that
we use to measure several other parameters

but I'll focus on this D.C.S. part the
important things with these techniques so

that they're completely noninvasive So
it's red light it's no more exposure to

radiation than you would get
like on a sunny day outside

we're highly portable you know our carts
on wheels wheel it all over the hospital.

We have very high temporal resolution
we can get down right now to

about fifty Hertz.

And an important distinction is that we're
actually sensitive to the microvascular

as opposed to the large like
feeding arteries in the brain where

we're really sensitive to what's
going on at the tissue level.

And not to make it sound like we're all
you know like this is the end all be

all technology we do have very low spatial
resolution because of this diffuse

the nature of the light propagation so
we certainly can't compete with something

like M.R.I. but because of our
portability we're able to do

measurements the bedside technique like
M.R.I. may not be able to cannot do.

So we custom design our optical sensors so
the patient interface portion of

this device we can make measurements
that the sensors that we can use for

spot measurements so say we just come and
measure a patient on a daily basis or

we can make sensors that we strap on to
the head and we do laundry monitoring for

you know we've we've measured patients for
up to seventy two hours so

armed with this tool now to measure
blood flow we've used to study flow

in the brain in both health and health and
disease in numerous clinical studies.

If they're there mostly pilots we've
probably measured maybe one hundred

fifty to two hundred patients today
across a wide range of disease states so

I'm just highlighting a couple
results here so we've been able to for

example quantify the result
the response to different

therapeutic interventions that clinicians
gives of this data was taken in.

Let's go in very very sick
babies who were outside

too much acid in their blood they give
them a drug called sodium by carbonate

to neutralize some of that acid and what
we saw is that depending on how much of

the tread they give you can see profound
increases in cerebral blood flow.

We've also done a lot particularly have
done a lot in healthy neonatal development

characterizing how blood flow changes.

You know during that neonatal period we
see differences between males and females

between right and left hemisphere between
different brain regions and a lot of this

work really hasn't been able to be done
before because taking a very healthy baby

and sedated them feeding them in them and
then M.R.I. is just not ethical right so

because we're all noninvasive and
can come to the bedside we've been able to

glean information about human development
that hasn't been seen before.

We also are and
this is a group where I did my Ph D.

Actually that has shown that
the blood flow technology can

be used as a biomarker of disease
severity So this is data taken in acute

ischemic stroke patients they basically
put two sensors on the brain one on.

The stroke come a spear or
that the hemisphere that had a stroke and

one over the normal It's not
really normal because they have a.

But they OK side of the brain and
they looked at how the brain

responded just to a simple postural
manipulation because there's a lot of

thought that with stroke you have impaired
regulation of flow in the brain and

what they saw was that with
increasing stroke severity you saw.

A cemetery and how the stroke versus
healthy side of the brain response so

suggesting that D.C.S.
can be used as a surrogate marker for

disease severity or maybe progression.

So going back to kind
of the main goal slide.

We've been able to start giving
clinicians the blood flow information

with the hopes of helping
to guide patient care.

But I think this is great in theory but
in reality oftentimes when we give

clinicians this information they don't
really know what to do with it so

this is especially true in
the critical care arena where all

of their patient management is
governed by trying to maximize

blood flow to the brain in order to
maintain adequate oxygen delivery and

preserve neuronal health and yet
they don't monitor the brain at all so

all they do is monitor her grade and blood
pressure and oxygen saturation and they've

never actually known what's going on the
brain so they're so excited when you can

tell that the blood flow has actually
dropped profoundly in this patient but

then when you say OK so now what do we
do they're kind of like I don't know.

Charted territory.

And so one of the things that we've been
working on lately is using pre-clinical

models of the diseases that we're studying
the clinic to try to elucidate mechanisms

that are driving changes in these blood
flow parameters that we're measuring and

then you know with the goal of hopefully
better understanding what's going on and

being able to give clinicians ideas of
how they can normalize blood flow and

hopefully improve outcome.

So for the remainder of the talk I'll
discuss recent work from my lab that's

been in the field of mild traumatic
brain injury and order to understand how

the blood flow data we get can actually
be one day used to guide patient care.

So just a general overview of mild
traumatic brain injury it's caused by

rapid acceleration of the head it disturbs
brain function in the absence of.

Structural debt structural
defamations know skull fractures.

The cognitive facts of concussion
appear immediately and

tend to resolve over time and we know
that repeated head injuries within some

vulnerable window can lead to worsened and
longer lasting Coggan of facts.

So just you know it's a significant
public health problem affects millions of

athletes per year just to put it in kind
of a local context in two thousand and

fourteen I pulled the numbers.

Eight hundred diagnose concussions
twenty five percent of those kids that

had a prior history of concussion and this
number is probably a gross underestimation

of the actual concussions in
the area given how poorly

under reported they are as well as you
know it's hard to diagnose some of these.

So what I'm interested in is although many
patients are going to recover relatively

clear quickly concussion
are to mild T.B.I..

Usually within a week there's about
ten to forty percent of patients who

are persistent long term consequences.

Like cognitive impairments can be seen
up to a year after concussion and

I'm sure you guys have heard in the news
lately we're now learning that repetitive

concussive and sub concussive injuries can
lead to really long term neurodegeneration

So there was the study that was
published last year by him as well.

They looked at post-mortem analysis
retired N.F.L. players and

eighty seven percent evidence of basically
like Alzheimer's like to mention dementia.

So our goal is to determine what is unique
about these patients who have these

persistent deficits and who go on to
develop long term nerve generation so

who are they who's most vulnerable
to subsequent concussions and and

who will have these long term effects why
are certain patients more susceptible and.

And I guess along why issue we know
that biomechanics of injury alone

are insufficient to predict outcome so
just because two people had the exact

same head injury they might not they might
have completely different outcomes and

then finally we want to help answer what
what can we do once we know once we've

identified who these most
susceptible patients are so

to answer these questions we translated
to a pre-clinical mild traumatic brain

injury model we use this
close head injury model that

mimics human concussion in that it
features kind of rapid head Excel or a.

Head about the neck.

It's a pretty simple model so if we take.

Them.

We have this acrylic board that we've
cut a hole out in the center and

we rested on top of it.

We grasp the mouse's tale and then we
drop a bolt on this guy to when the bolt

hits their head you know I saw your
face I know it's really a mild injury.

So there and that's by the way so
when the ball hits their head they

know it breaks the cam way they
they wrap and they rapidly rotate.

And this induces injury in the brain so
the consequences of this model

are really subtle we've done lots of
them you know has chemistry there's

no dear Ronald degeneration there's
no arc so no injury there's no D.N.A.

damage and there's no blood
brain barrier permeable A-T..

Deficit's And just to show that it's
not a problem with his chemistry

we've done these same studies
in more severe T.B.I. model and

you can see that we can actually detect
these predators It's just that this

this injury model is very mild and
I should point out that this was actually

after five hits we spaced them once daily
and we still are seen neuronal injury.

However what we do see in this model is
the emergence of cognitive deficits so

after a single close head injury
there's no cognitive deficits

the my surf actively find.

And we assess cognitive deficits
using Morris water mains for

those of you who aren't
familiar with Morris water maze

Basically you take the mouse
you put him in a tank of milk.

The water so we can't see what's
underneath it and you submerge a platform

just below the water's surface and
mice don't like water so

they swim around eventually they stumble
upon the platform and they sit on it and

you repeat this across multiple trials and

eventually they get really good
at using visible cues in the room

to find that platform even though
they can't actually see it.

So this is like kind of a typical
performance on one of these.

Trials So this is the latency is
the time to find the platform and

you can see as you give them more and

more trials in the tests they get better
and better at finding the platform.

However when you give increasing
the number of hits spaced once daily

cognitive deficits begin to emerge so
this is after five

five hits spaced once daily and you can
see that the injured group in these open

circles takes significantly longer to find
the platform they're just not good at it.

Remembering what's going on.

I just want to point out though that
these performance opposites that we see

on the group level.

You know are clearly statistically
significant but within this injured group

there's actually a substantial amount of
very bill just like in the human condition

so even though these mice were subjected
to a very consistent mechanical impact you

can see that there are some my like this
one on the top just like he can't get it.

But there's some my super
form just like sham So

what is the difference between
this guy versus the sky.

So next thing we done recently is looked
at a genetically modified all Summers

mouse where they develop
Alzheimer's plaques with age and.

In these mice we've shown that
this injury model accelerates the.

Development of beta so
here this is a sham injured miles and

this is a mouse after three of these
injuries spaced once daily and

then sacrifice one month later and

you can see that there's amyloid beta
seen in bread starting to emerge

quantifying the amyloid beta deposition
across all of our animals we can see

that there are significant opposition
after a month a month after injury

I just lost everything on this screen but
this one still here.

Can you see my mouse even though it's
here we go OK So so again what we

want to see is within this injured group
you know what's wrong with these three

guys who had such bad amyloid beta versus
this guy who basically looked like a sham.

So now we want to start talking about Who
are these mice how do we figure that out

so the first thing we did was we
started by using D.C.'s to assess

flow as potentially a biomarker of
these mice there's some literature

in the human condition that suggests
that blood flow at the time of cognitive

testing is a marker of injury so
we thought let's see if this is

true in mice we designed this custom
optical sensor so the great thing

that I love about these diffuse optical
techniques is I can use them in humans and

then I can scale down my sensor and
make the exact same measurements

that we're making in the clinical
scenario in the pre-clinical models.

So basically what we do is we rest our
little optical sensor over their head

everything's noninvasive we briefly
anesthetize of the now we're doing away

measurements and we validated these
measurements in mice as well so

here's our study protocol we actually
did two we looked at wild type mice in

the wild type mice we gave them five
closed head injuries spaced once daily and

we measured blood flow kind of at a couple
of Representative time points across

the injury protocol and
then we did testing this Morris water maze

testing seventy two hours after the final
injury and then we had an all time as

a group Alzheimer's mice were older they
were much more susceptible to injury so

we only had them three times and we
measured flow before and four hours after

the final injury and then we waited
a month and we sacrifice them and.

That acclamation accumulation of amyloid.

So just in the repetitive
hit my we can see that.

So here and so in the relative
change in blood flow from the.

Levels so in the injured group four
hours after the third injury and

after the fifth injury
blood flow was reduced but

it seemed to return to baseline
by seventy two hours later.

And like I said these mice are going to
develop significant cognitive deficits.

Seventy two hours up to that final injury
which blood flow has returned to normal so

it seems to suggest that these
deficits are not due to deficits

in blood flow at the time of assessment
but we thought that maybe blood flow early

on in these acute time points could
be a part Bostic marker of outcome.

And lo and behold let me just get to
this one when we looked at the four hour

after the third hit injury time point we
saw a significant correlation between

blood flow and their average Morris
water maze performance so the mice with

the lowest blood flow were the ones who
did worse on on their cognitive testing.

And then also within our
all timers My We saw that

blood flow at this four
hours up to the third hit.

Time point was correlated with

the amount of amyloid beta accumulation
and one month after injury.

You know we only did six mice today but

I think there's definitely a trend there
with the mice who have lower sort of

a blood flow having more
accumulation of amyloid beta so

we seem to have identified this
biomarker sensitive acutely sensitive

to the long term cognitive effects of and
pathological effects of

repetitive mild traumatic brain injury and
if this finding does translate to humans

then we could have an acute marker
that could tell you know athletes or

soldiers when it's safe to return to
play or return to the battlefield.

Or potentially identify those who are in
risk of having multiple concussions.

So next we want to look at why these
particular my with low cerebral blood flow

are more susceptible to injury so
we actually have now have this like pretty

unique platform to study mechanisms
because we've got this acute biomarker of

how my surgeon to do you know a long
term cognitively or pathologically.

So what we do is we first measure blood
flow we predict how they're going to do

and then we can harvest the brains
of these animals at any time point

you know that we're interested
in to investigate molecular

pathological changes that are going on and
we can then relate these.

You know underlying physiological changes
to blood flow predicted outcomes.

So what mechanisms could be responsible
for changing cerebral blood flow and

ultimately leading to worse outcome
because this model doesn't induce

significant neuronal injury like I said
the next kind of logical mechanism

to look at is neural inflammation so
in severe traumatic brain injury.

Neurons limitation has been shown to.

Contribute to blood flow changes and

also to be associated with worse
outcome and so what we have.

What's happening is repetitive.

Mild T.B.I. is leading isn't due seen
mechanical stress on the neurons release.

Or signal or increase.

In signal pathways and and
increase expression of inflammatory

cytokines which lead to micro Clio
information and reduce blood flow and

ultimately to worse long term outcomes.

So I'm going to start by talking about OK.

But.

Activation so
micro Glee are just kind of a gross.

Estimator of neurons laboratory
response they serve as.

Kind of regulators of the immunity
response they breathe and

they're known to.

Promote remodeling and
repair after T.B.I..

So at this point out four hours after
a third injury we basically measure blood

flow and then we looked at the microbial
response right there this is a sham animal

with the micro clear and red stain with
I.B.A. wine and you can see in our two

injured animals there's increased Amalie
there's increased I.V. A one uptake.

It looks like more micro glee as well so

within the injured animals we used
Western Blot to quantify I.B.A. one and

all these animals and what we saw was that
the animals with the lowest low again

were the ones who had the worsening or
inflammation and remember those low C.B.S.

animals are going to be the ones
who go on to have worse outcomes so

it suggests that you know could be
playing a role in long term outcome and

also is interesting to me just that blood
flow is not only a marker of long term

outcome but it seems to be a marker
of underlying pathological changes.

So we think that nitric oxide may provide
a functional link between microcode.

Elective ation then reduce blood
flow nitric oxide plays a well known

role in mediating vascular tone and
also the inflammatory response.

And so nitric oxide in the brain
is synthesized by three times.

Which.

If I was by nitric oxide since which
takes the form of three ice of forms I'm

going to focus on one of them and

to feel ill nitric oxide sent this which
has been implicated in more severe T.B.I..

And what we saw basically was
that the mice with reduced.

Feel and nitric oxide availability were
the ones with the lowest cerebral blood

flow so again.

You know because C.B.S.
was correlated with I.B.A. one.

Or inversely correlated that.

Was also positively or
negatively associated with I.B.S. one.

We'll see next we can focus on cytokine
expression after T.B.I. and specifically

what cytokines are expressed acutely
in animals with longer worst turnout.

So to do this we use Lumina illumine X.

assaye to assess twenty seven
different cytokines each.

Kind of confusing but
basically each column denotes a different

side a kind that we looked at and each row
denotes one of our different animals so

we've got our eight injured animals on the
top in our sham animals at the bottom you

can already see that there are some
cytokines that are regulated.

In the injured animals.

So next we use partially squared
regression again through blood flow within

the injured animals to identify
the top cytokine correlates with.

Cerebral blood flow and
with worse cognitive outcome and

you know the individual.

As relevant but
basically Randy's was our top correlate so

we did him you know his to
chemistry looking at Costain with

a bunch of different cell types and
we actually saw that.

Coke lies with the.

Neurons suggesting that.

This is correct that it's these injured
neurons that are releasing these.

Cytokines.

And finally we wanted to look at
interest signaling that could be

disrupted after T.B.I. these
are the key signaling pathways in the.

To be.

Katie that regulate transcription of
inflammatory factors like cytokines and

here in showing just the fossil proteins
associated with these pathways which

I mean they're like so small and
you can't read them but

the point is the orange ones are the ones
we measured you can see we just

kind of took a representative
sample across all these pathways.

Again another heat which I don't love but

it's showing all the fossil proteins that
we measured for each of our animals and.

Just by I already you can see
that the map kinase from the F.

Kappa B.

pathways have significant
regulation in the.

Injured animals so similarly we can do
a part of partially squared regression

within this injured group against cerebral
blood flow and find fossil proteins

that are highly correlated with cerebral
blood flow I mean with worse outcome.

We didn't you know his chemistry on
a couple of these elevated proteins and

again we're seeing them localized to the
neuron suggesting this mechanical stress

is causing the neurons to really up
regulate these signaling pathways and

here I'm just highlighting in with
yellow stars the top correlates.

Here and you can see they mostly
fall within the N.F. Kappa B.

and map kinase pathways so

potentially what I like about this result
is that if we could hit these pathways

we could potentially improve outcome so
there's already.

F.D.A. approved blood brain barrier
permeable inhibitors of these.

Pathways so I think that they have
promising therapeutic potential but

potentially addressing this
question of what can we do.

So in summary these results demonstrate
how our diffuse optical technique D.C.S.

can be a powerful tool to.

Are a powerful biomarker not
only long term outcome but

also of underlying path of
physiological changes after T.B.I..

We saw that and T.B.I. leads to.

Acute microbial activation
cytokine expression and

percolation of interest or
signaling pathways and

through partially regression we write
able to identify which of these

kind of the hallmarks features are going
to be most predictive of outcome.

And hopefully the next steps will be
to start leading these systems and

seeing if we can improve
outcome in our mice so

with that I'd like to thank you for
your attention and

my wonderful group members if
anybody has any questions I'm open.

Just gross like looking at.

Not you know nothing nothing really stood
out to me these were relatively the.

Climbers My but they were a little older
and they were like six months as opposed

to our wild type mice were two to three
months but they weren't really old yet.

Yes

I'm sorry they were just submerge
slightly into the water and

the water is like filled with milk
basically so you just if it's

not transparent they can't see it but they
can feel it once they hit it you know but

they That's a good question they also
tested to just to make sure that

the injury wasn't causing some
kind of visual impairment and

they can see if you raise the platform
up they're all very good at finding the.

Yes.

We didn't.

You know we didn't really
see that's not my mouse.

There it we didn't see many changes in
that pathway right there was just not.

Exactly and I've got to be.

Right.

This was four hours after injury and
again we chose that

time point because we knew that our
blood flow biomarker was predictive of

outcome at that time point so
we thought let's just look at what's going

on right at that time point but our next
thirty is going to be to really like.

The kinetics of all these
parameters because.

They definitely change over time.

Exactly and I think the key then is
going to be really hitting how those

transients in my translate to humans
which is a whole nother can of worms.

But you know if we find the blood flow is
a nice surrogate of those changes then

you know hopefully that will cause blood
flow we can measure in humans easily.

Yeah.

No.

I mean there are certainly many things
that scatter late into shoe but the moving

things are what Worst were sensitive so
if you take that away we would.

We would have been trouble.

If you injected like I see two years.

So you.

Know what I'm excited to hear you're
talkin here about what we could do yeah.

Yeah I mean so we we talked.

We were thinking about maybe doing that

we've actually been talking with Michelle
because they have a lot of you know.

Once we get the brains they can do a lot.

Lifted.

The top one likes and stuff like that.

So she actually has
a bunch of our samples.

Well.

So.

Yes.

It's.

Sort of those.

No I mean you know right so

you know no they're the because
it diffuses so rapidly.

You know you don't get
a lot of heat deposition.

We are certainly limited by the standards.

And I don't know if you're from
the government puts standards on you

know what safe levels for
tissue exposure with D.C.'s we're kind of

pushing the boundaries because we
are measuring just one single speckle So

we're really light starved so
we try to pump in as much as we can.

I've never had a problem with with burns.

Or.