- My name is Dr. Ethel Gordon.
I'm a member of Salem State University's Biology Faculty
and Treasurer of the Darwin Festival Committee.
It is my pleasure to welcome
Salem State University community,
and our guests from local area and beyond.
We're happy to welcome some of our area high school students
as well as STEM students of all ages.
It is my honor today to introduce
our Salem State University
alumnus speaker Mr. Jesse Moreira.
Jesse is an undergraduate with a BS in Biology
from Salem State.
Following his graduation in 2017,
he joined Boston University's Department of Health Sciences
where he completed a Master of Science and Physiology
for Research in Cardiac and Skeletal Muscle Biology.
Jesse applied for a pre-doctoral program
in the same department
and was awarded the Dean's Teaching Fellowship
to study the Neural and Neuro-immuno Mechanisms Contributing
to the Development of Neurogenic Salts Sensing Hypertension.
Because of his research,
Jesse was invited to give oral presentations
at the International Experimental Biology Conference
in both 2019 and 2020
and was invited to give oral presentations
at three consecutive Annual
International American Heart Association
Hypertension Scientific Sessions from 2018 to 2020.
In 2020, he was given the Top Trainee Investigator Award
by the American Heart Association's Council on Hypertension.
When not conducting research or writing scientific papers,
he serves as the primary lab instructor
for the Gross Human Anatomy Cadaver Lab,
and is co-instructor
for Advanced Cardiac Physiology Pathophysiology course.
Jesse Moreira's talk entitled, "Salty Microglia:
Gaining Mechanistic Insights
into Sodium-dependent Hypertension"
is sponsored by the Department of Biology
and the College of Arts and Science.
Please give a warm Salem State welcome to Jesse Moreira
- Thank you so much
for that wonderful introduction, Dr. Gordon.
Let me go ahead now and share my screen
so I can bring up my presentation for today.
So yes, thank you so much.
And thank you to the entire Darwin Festival Committee
for inviting me here to talk about
my dissertation work that I'm conducting
at Boston University.
As Dr. Gordon said,
I am in the Department of Health Sciences
and I work in the Laboratory of Dr. Richard Wainford
in collaboration with the Department of Pharmacology.
And without further ado, let me talk to you about
microglia and salt sensitive hypertension.
So to provide some background,
I'll begin with blood pressure.
What exactly is blood pressure?
Well, blood pressure is the pressure
inside your closed cardiovascular system
that is required to facilitate tissue gas exchange
at the level of different organs and drive blood
against gravity of the carotid arteries in your neck.
So your brain gets perfused
and it's a very critical component of physiology,
is blood pressure and with that,
the maintenance of normal blood pressure.
But what exactly determines blood pressure?
Well, the mean arteriole pressure or the average
between the systolic pressure that you receive
at the doctor's office,
that top number and the diastolic pressure,
the bottom number, is really determined
by two critical factors.
Primarily, we see cardiac output.
So you can see that here, and cardiac output
is just how much blood is being pumped
by your heart in a given amount of time.
And so that's determined principally
by how often your heart is beating, your heart rate,
and how much blood is getting pumped out
with every beat, your stroke volume.
Now, arteriole pressure is also determined
by peripheral resistance.
So how resistant is the cardiovascular system as a whole
to the flow of blood from the heart into the vessels?
And really impacting that heavily
are things like arteriole or radius.
So how constricted or dilated are these arterioles,
these very small little arteries that start to feed
into the organs as well as how thick is the blood?
How viscous is it?
Because a more viscous fluid,
one for example, with less water volume,
less plasma is more resistant to flow.
When we think about
what actually controls blood pressure, though,
there are many systems, both hormonal and structural
like the nervous system that impact blood pressure.
But for the purpose of my research
that I do with Dr. Wainford,
what I want to share with you has to do
with the autonomic division of the nervous system.
Now, the autonomic division or the nervous system
is a part of the nervous system at large
which controls all of your function.
But unlike things like the voluntary motor control
of skeletal muscle that allows us to do things
like lift our arm,
this operates below the level of awareness
and it's constantly helping us do things
like breathe or maintain a normal heart rate.
And functionally, it's divided into both the sympathetic
and parasympathetic divisions.
With the sympathetic division,
you may have heard of as your fight or flight response
actually activated far more frequently
than when you're doing something like running from a tiger.
The sympathetic nervous system has tonic activity
at a certain level that helps integrate
nervous system function and control of blood pressure
through its interactions with many target organs
like the adrenal gland at the medulla, the middle,
the arterioles directly being innervated by these nerves
as well as the kidney and the heart.
And really importantly, through its impacts
on all of these different organs,
it can control the various factors
that we were just discussing like cardiac output
by stimulating the heart directly
or peripheral resistance through inducing constriction
of those arterioles or sodium and water retention
at the level of the kidney to impact blood volume.
And with respect to the autonomic nervous system, of course,
we're going to zoom in a little more
and see that there are these cardiovascular control centers,
many of which integrate functions together
to ultimately affect responses,
but critically involving this one right here
in the middle, the PVN
which stands for paraventricular nucleus
which takes the signals from all of these different regions
that are receiving input about blood pressure
and fluid and electrolyte balance
and composes a unified response through the spinal cord's
sympathetic nervous system control center
which it's connected to
and out to the organs via the nerves.
To give you a little closer insight into the PVN itself,
this is actually a stain that was performed
by a colleague of mine who has since left our laboratory,
Dr. Parul Chaudhary, and she stained
for a G-protein coupled receptor alpha sub units RNA
using in C2 hybridization.
And what you can see is the distribution
throughout the bilateral paraventricular nucleus here
of this red stain for this protein highlighting,
in particular, of the various sub regions,
this middle region known
as the medial parvocellular sub nucleus
and the dorsal region known as the dorsal parvocellular.
And all of the various sub regions that you can see
that anatomically makeup the PVN,
the dorsal and medial parvocellular regions contain
those neurons which actually affect
the biological responses intended to control blood pressure
via their connections
to those other cardiovascular control centers
we were just speaking about and that sympathetic nucleus,
the IML and the spinal cord.
So I'll get in a minute why we're interested
in the GNAI2 RNA and resultant G alpha I two subunit protein
but we'll first talk a bit about
G-protein coupled receptors as a whole.
You may or may not have been exposed
to G-protein coupled receptors before,
but if not, that's all right.
These are seven pass transmembrane domain proteins embedded
in the plasma membranes of cells
that are then coupled intracellularly
to a variety of subunit proteins.
You have this G alpha subunit
and then there's this beta gamma sub unit.
But really the important part
is that upon ligand binding at the extracellular domain,
what you're going to get is an effect activated
by the G alpha sub unit.
So it's going to swap out its inactive GDP
for a more high energy GTP.
And then according to the subtype of alpha subunit you have,
whether that's a G alpha sub S,
sub I slash O sub Q or sub Z,
you're going to get a variety of effects.
And with respect to the PVN and blood pressure control,
what we're really interested in
are these G alpha I slash O sub units
and their ability to inhibit adenylyl cyclase activity
which reduces intracellular cyclic AMP production.
Because this signal pathway,
as I'll get to, has been demonstrated to be relevant
in blood pressure control through knockout studies.
Now giving you some clinical background
for all of this, why is this important?
Why, is the PVN important?
Why is blood pressure control important?
Well, we're going to step and look at sodium for a second.
You can see in the CDC infographic
that the average American is consuming
three times the daily recommended sodium
by the CDC at about 3,400 milligrams a day
in comparison to 1500 recommended.
And this is problematic when we consider
that a variety of people exhibit different phenotypes.
So if you are this blue line here,
salt resistant, might not be such a big deal
that you eat so much salt because salt resistant individuals
possess mechanisms that enable them
to offload excess dietary sodium intake into the urine.
So no big deal for you, but a significant proportion
of individuals, I'll get to how many, are salt sensitive
which means that they do not possess these mechanisms,
rendering them unable to offload that sodium
and driving their blood pressure up
both in the acute and the chronic setting
such that with an elevated dietary sodium intake over time,
they develop salt sensitive hypertension.
And our laboratory is particularly interested
in what the mechanisms are that account
for the phenotypic difference observed
between salt resistant and salt sensitive individuals.
And so at this point, and you might be asking this
and I said, I'd get to this.
How common is hypertension, right?
Well, according to the recent
American Heart association
Heart disease and Stroke Statistics,
you can see that approximately
one in two adults in the United States
over the age of 18 are categorized as having hypertension
which is particularly problematic because hypertension,
as you can see in the bottom, contributes
to about 10 and a 1/2 million deaths worldwide per year.
And it's in addition, the leading risk factor
for stroke, heart attacks,
as well as chronic kidney disease.
And with respect to how salt sensitivity plays in,
it's currently estimated
that 1/2 of hypertensive individuals, and even a 1/4
of people who don't have hypertension are salt sensitive.
So salt sensitivity of blood pressure
may actually be driving a significant component
of hypertension and its overall disease burden
in the United States and in the world.
Now to kind of bring these multiple factors
I've been discussing together
and investigate the actual mechanisms that, as I said,
account for the difference between salt sensitivity
and salt resistance in hypertension,
our laboratory has previously used rat models
such as the Dahl salt-resistant rat
or the Sprague-Dawley rat
which I'll talk more about later to study this.
And so what happens is you take these rats
and you give them a high salt intake.
And what you actually see is that in these naive rats
on high salt, there's upregulation
in the paraventricular nucleus
that critical cardiovascular control center
of the G alpha I two class protein.
So a G alpha I subunit protein version two,
and this ultimately leads to decrease
in sympathetic nervous system activity.
So you can turn down that tonic activity
of the fight or flight response so that the kidney
is not constantly being stimulated to retain sodium.
That sodium is offloaded into the urine
and you maintain a nice, steep relationship
here in the pressure and nature Risa slope
such that very small changes in X,
the mean arteriole pressure are required
to facilitate large changes
in sodium offloading into the urine,
leaving the animal with normal blood pressure.
Now in contrast, in a salt sensitive phenotype,
our laboratory has identified that there is no upregulation
of PVN G alpha I two proteins
in response to a high salt diet.
So then not only does the animal fail
to do the things that previously did that we just discussed
but there's also renal nerve dependent simpatico excitation.
So on its head,
excessive sympathetic nervous system activity
which induces massive sodium retention
at the level of the kidney,
instead of telling the kidney to disperse it
into the urine and this shifts
this pressure nature recess relationship right
and down so that now very large changes
in blood pressure are going to be required
to offload that excess of sodium into the urine resulting
in the development of robust salt sensitive hypertension.
And in addition to this work that we've done,
I've got for just two examples citations here,
there has been literature
in models of hypertension in animals
that has shown a really critical role of inflammation
in addition to all of the other physiological mechanisms
going on that drives salt sensitive hypertension,
as well as other models like angiotensin
two dependent hypertension.
And so this data being new in addition
to all the things we've previously done led us
to our beginning hypothesis.
And here's where I'll begin talking about what I've done.
And so my dissertation began with the thought
that maybe PVN inflammation would be correlated
with the development of our model,
G alpha I two protein dependent salt sensitive hypertension
in addition to all these others.
So to provide you some understanding of what we've done,
I'm going to walk you through my methods first
and you can see here, I've got Sprague-Dawley rat,
a classically salt resistant rat
and we put a radio telemetry in it.
So this is an acute surgery we do
where we instrument the femoral artery up
into the aorta with an indwelling cannula
that's connected to a telemetry probe
so that when the cage is near the probe,
I can chronically measure this rat's blood pressure.
And we leave that in there for the duration of the study.
Then to achieve knockdown of the protein of interest,
the G alpha I two protein,
what we're going to do is put an ICV cannula into the brain.
So we do stereotactic neurosurgery on these rats
and into the right lateral cerebral ventricle,
we insert a stainless steel cannula
that's connected to a mini pump that's on the back,
filled with antisense oligodeoxynucleotide,
a variety of flavors.
So as a control, a sham surgery,
we do a scrambled sequence antisense nucleotide
that we validated to not be complimentary
to any known mRNAs in the rat brain
such that it doesn't render any proteins knocked down.
And then we also use a targeted G alpha I two
oligodeoxynucleotide, another subset of rats,
so that they do get specific sequence binding
of this antisense ODN to the mRNA
for the G alpha I two protein rendering it untranslatable.
And then to study the impact of inflammation,
if there is one, we use a combination
of either of these ODNs with minocycline,
which is an anti-inflammatory drug,
also a tetracycline class antibiotic that has been shown
in other hypertension studies
to reduce inflammation in the brain.
We randomized these rats to receive either a normal diet
for control or a high salt diet with 4% sodium chloride.
And at the end of the study,
we're looking at things like mean arteriole pressure,
western blotting for the G alpha I two protein
to validate the knockdown,
plasma neuro epinephrin levels as an index
at how much sympathetic outflow there is.
Because if there's excessive sympathetic outflow,
there will be a lot of plasma and epinephrin in the blood.
Cytokine mRNA, looking at whether or not
there are transcripts occurring that suggests inflammation.
And then lastly, immunohistochemistry.
So antibody-based tissue staining to look at the morphology,
the structure of cells like microglia
as well as astrocytes respectively stained
by these two proteins, CD11b/c and GFAP,
and pro-inflammatory anti-inflammatory cytokines
as they've actually been expressed
and secreted into the tissue.
Just to give you one more concept
of how these ODNs work, the current hypothesis
for how an antisense complimentary oligodeoxynucleotide
induces protein knock down is this top one.
This is the leading theory
that there's ribonuclease H-mediated degradation
of this DNA RNA hybrid.
So the antisense oligodeoxynucleotide
is a single-stranded DNA complimentary to the mRNA,
you get cleavage and then degradation
of both of the fragments and so no translation,
and that can happen in the nucleus or in the cytoplasm.
And should this not be the case,
the next theory that we have
is that there's actually translation inhibition directly.
So the ribosome's not able to complete translation
into a functional, mature peptide
because of the blockage that's occurring
from the complimentary binding.
So the first thing we see, and this is a validation here,
was that with targeted G alpha I two ODN and fusion,
but not scrambled ODN infusion,
we get downregulation of G alpha I two proteins.
So western blot here, you can see
this is two lanes of ladder so that we could know exactly
where we were on the block.
And then S1G1, S2G2 represents animals
that were sham scrambled control ODN infused
versus receiving a targeted G alpha I two ODN.
And this here is a representative loading blot
that's been stained with Coomassie
for total protein loading normalization
as the field is actually moving a little bit away
from using stains for beta acting or GAPDH,
these housekeeping proteins, since they can saturate out
quite easily on a western blot imaging.
And so you get the normalization of protein levels
in your blot to total protein loaded
across all samples in several different proteins.
And what we see is that as previously demonstrated,
our G alpha I two ODN does result
in efficacious downregulation of the G alpha I two protein.
So we're sure this is working this time.
When we look at the blood pressure phenotype that we see,
you can see first to have a graph here
for the scrambled ODN and fused rats.
And you can see that when we switch those control rats
from a normal to their experimental diet
whether that's normal or high salt, right,
the high salt does not evoke an increase in blood pressure.
So they do maintain salt resistance
for the entirety of the duration of their high salt diet.
And moreover the minocycline co-infusion
had no baseline impact in these animals,
suggesting there's a lack of inflammation going on
in a salt resistant rat.
Now in contrast, things start to get interesting here.
We see as we've previously seen
that immediately following the switch from a normal
to high salt diet, animals lacking essential
G alpha I two protein actually have robust increases
in blood pressure that go up
and up and up over the first few days,
and then plateau out on a high salt diet.
And very interestingly, and our first key
that there might be something going on
with inflammation was the minocycline significantly reduced
the magnitude of the hypertension here.
So the blood pressure comes back down
when you take away inflammation
or whatever we think the minocycline at this point
might be doing.
Now to get an understanding of a few different indices
of blood pressure control systems,
we began, as I mentioned, with plasma norepinephrine.
So a good index of how much
sympathetic nervous system activity there is.
And on the left here, you can see
that with a scrambled ODN infusion, a high salt intake
actually suppresses sympathetic nervous system activity.
And so this goes back to that figure
I showed you before of our previous demonstrations.
It goes down with high salt in a normal rat,
meaning that they've turned off
their sympathetic nervous system
to maintain normal blood pressure.
And importantly, minocycline just as it didn't impact
the blood pressure in these rats, does not impact
their sympathetic nervous system at baseline.
So no impact going on.
And in contrast now, when we look at the knockout rats,
we see the animals that lack G alpha I two proteins
actually have an increase
in sympathetic nervous system activity as measured
by epinephrin, so a very robust sympathoexcitation
which was normalized by minocycline.
So that was really cool when we did these studies
and we actually found
that you could turn off excessive sympathetic activity
through this minocycline co-infusion.
And that was the first time we thought to ourselves
in addition to the blood pressure,
we might have something here.
Now we have two more indices here.
These are almost like controls that we performed.
There's another pathway that regulates blood pressure
that's not really something we look at
because as I'll show you, it's not particularly active.
And it's known as the renin-angiotensin-aldosterone system.
And this is another kidney-based system
of hormonal blood pressure regulation
that we can actually see when we look is suppressed.
So plasma renin activity is a good index
of how much renin enzyme activity
generating angiotensin one there is in these rats.
And what you can see is that regardless of ODN infusion,
so whether they have G alpha I two proteins or not,
high salt diet evokes significant suppression of this
as well as suppression of angiotensinogen in the urine.
So what you see is that with regards
to both of these assessments
of renin-angiotensin-aldosterone system activity,
that system appears to be A, suppress
and B, not related to G alpha I two proteins.
So what we're thinking is we mostly have
a sympathetic nervous system model
of blood pressure regulation here,
independent from rat's pathway activity.
When we look at cytokine expression levels,
which now is gonna be a direct assessment of inflammation,
what we see is that with respect to A, B and C
which are IL one beta, IL six
and TNF alpha pro-inflammatory cytokines
and D which IL10, an anti-inflammatory cytokine,
there's no impact of a high salt diet
in our control scrambled ODN infused rats.
So there's no pro-inflammatory signaling,
there's no anti-inflammatory signaling changes
and minocycline doesn't impact baseline
in these animals either.
So not really much going on here.
There's some pathway inhibiting,
they've got normal blood pressure, no inflammation.
When we look in the knockout rats, we see this seesaw effect
where pro-inflammatory signaling comes up
at the expression level of the transcripts
and anti-inflammatory signaling is suppressed.
So you actually get an imbalance
of pro and anti inflammatory signaling favoring
pro-inflammatory transcripts in these rats.
And all of these data were normalized
by minocycline infusion as well.
So this brought us to the notion that, okay.
Yep, we're definitely changing inflammation
when we give this minocycline on top of the blood pressure
and the sympathetic changes that are going back to baseline.
And here I have some preliminary immunofluorescent stains
in the PVN.
So the histological slides
that we've done to show upregulation of the protein
within the actual tissue
that's happening following these transcriptional changes.
And we're currently working out ways
to better assess the protein, but you can see here
between a scrambled rat and a G alpha I two knockdown rat
on a high salt diet, that transcriptional change
where there's elevation in IL six and IL one beta follows
through to the protein level it appears
because there's so much more robust immunostaining.
And now trying to understand
the cellular localization of these,
we go to a couple of different cells in the brain
which can contribute to inflammation.
And as I mentioned before,
those are microglia and astrocytes.
Now astrocytes, to start, is what you're looking at here.
You've got these images, astrocytes are neural cells.
So they derive from the same stem cells
that neurons do in the developing brain.
And they are a critical component
of the blood brain barrier,
as you can see them surrounding blood vessels here,
as well as playing a role
in interstitial glutamate homeostasis,
pH regulation of the brain tissue, and they can contribute
to a cytokine production when they're inflamed.
But what we see when we do a quantification
across all these groups,
whether they have G alpha I two proteins or not,
is that a high salt diet does not evoke an increase
in the density in that tissue
of these astrocytes, suggesting to us at this point
that there's no change going on in astrocyte reactivity.
They're probably not contributing
to this inflammatory process because they would change
on immunostaining and take up more space,
stain more intensely, and they're not doing that.
But when we look at microglia
which are innate immune cells derived
from precursor immune cells during development
that migrate to the nervous system
and play immune functions there, there's a different story.
Now, if we look at the percent
that are categorized as active or reactive
in the PVN in a scrambled control rat,
we see that we don't have any changes in immunoreactivity
from normal to high salt diet, no change in the actual shape
and structure of these microglia and minocycline, moreover,
doesn't really do anything here either.
You see, we have nice, branched to microglia
which suggests that they possess an anti-inflammatory
or surveillant phenotype.
And that's what the G alpha I two knockout rats look like
at rest at normal salt as well.
But as you can see,
there's a robust increase in the tissue expression
by these microglia of the CD11b/c marker
also known as the compliment receptor three
following a high salt diet.
So what it might be is that
these microglia are actually the cellular source
of the cytokines that we saw being up-regulated
because they possess a much more inflammatory morphology
at this point, as well as being upregulated in quantity
and having upregulated their compliment receptor.
And just like the rest of the indices we saw in these rats,
they were normalized by minocycline infusion.
This does go back as well to literature
that suggests minocycline can act
by inhibiting immune cells.
And so it's quite possible here
that the effective minocycline infused into the brain
was directly to inhibit microglial reactivity
in response to high salt
to prevent the production of cytokines
and the upregulation of sympathetic nervous system activity
which is what ultimately helps bring the blood pressure
partially back down.
Now, when we assess those microglia here,
we used a semi quantitative method
that was somewhat subjective of a manual cell counting,
but we wanted to really be sure we were right
about the morphologic changes that we were seeing
in these microglia from being nice
and branched out anti-inflammatory
to having their processes pulled in,
and their somas, what's known as hypertrophic or amoeboid.
And so we used an index Sholl Analysis.
And with Sholl Analysis,
what we ultimately did was we took each cell
for many cells from each tissue slice
and cropped images of them alone down, and subjected them
to a computerized process where a software program places
a dot in the center of the soma
and a set of equally distant concentric rings radiating
out from the soma.
And then the software counts
how many times the processes of these microglia
actually intersect these rings
and it formulates a chart like this.
And what you get is on the X axis,
how far away you're going from that dot
in the center of the soma and the Y axis,
how many intersections are occurring.
And so you get this fun peak that's occurring here.
And basically, the way you interpret this
is that the higher the peak is over a given distance,
the more branched and complexity this microglia has.
And so when you get average plots for each group,
what you can do is compare them
and see whether or not average branch in complexity
as an index of microglial inflammatory status changes.
And so first what we saw
in our scrambled control rats was that
all of the peaks for these different rats basically happened
at the same point and their standard deviations
amongst the groups overlapped significantly,
suggesting to us that what we had seen first
with no changes in our subjective morphology were correct.
There was no change in branch of complexity
of these microglia induced by high salt.
So they're maintaining their anti-inflammatory phenotype.
And in contrast, what you can see here
is this peak occurring in the normal salt
G alpha I two knockdown rats is significantly reduced
by about seven intersections per group overall.
So there's really less branching going on in this group,
a significant lesser amount of branching,
suggesting that they've pulled in those processes.
And they do have that hypertrophic morphology
that we were seeing on histology.
And so this kind of validated for us, oh yeah,
we were seeing activation.
And what you can see was that
towards the tail end of the branches,
they began to branch back out and significantly come up
from the high salt group when you gave minocycline.
So there was restoration of the branch in complexity
with minocycline infusion, suggesting that, yep,
the minocycline was acting through the microglia.
And altogether, these things led us
to our first conclusion and the first conclusion
of my studies as a PhD student,
which was kind of to remake that figure about
salt sensitive rats I showed you at the beginning
that in a salt sensitive rat,
a high sodium intake does not evoke changes
in PVN G alpha I two protein levels,
and that in a renal nerve dependent way,
you still get some pathogenic citation
and renal sodium resorption
and thus changes in pressure, nature, recess relationship,
meaning big changes in blood pressure required
to offset that sodium dysregulation.
But now we've added in a component
where there's a pro-inflammatory PVN microenvironment
featuring microglia that produce cytokines
like interleukin six and interleukin one beta
to drive the development of salt sensitive hypertension.
Now that was a lot, but I've got a little more for you.
And so these studies led us to think
a couple of different things
that formed the next chapter of my thesis.
And I'm going to go over the first one with you
because we were thinking to ourselves,
this happened in male rats.
But studies have shown robust sex differences.
So what about female rats?
And these phenomena are correlated
but asking a chicken and egg question,
which comes first, inflammation, hypertension,
some currently undiscovered factor.
And so to get into the possibility
that there are gonna be sex differences,
let me just talk about that for a little bit.
We've got sex differences
in multiple models of hypertension.
In humans and in animals, there has been a robust role
for sex steroid hormones, particularly estrogen
like estradiol, estrone in modulating cardiovascular risk.
And this is evident in females,
human females, people assigned female at birth
that have female sex steroid hormones
and their cardiovascular risk pre-menopause
is very, very low versus post-menopause
when it actually shoots up over two fold.
And so to kind of get a model of this and understand
whether or not this would hold true with respect
to the expression of G alpha I two proteins,
we started looking in female rats.
So using a similar paradigm as last time,
what you can see is that with the female Sprague-Dawley rat,
we inserted the same intracerebral ventricular cannula
into the right lateral ventricle.
And we infused these rats with either a controlled scrambled
or targeted G alpha I two ODN, randomized them to a normal
or elevated dietary salt intake
and looked at things like blood pressure,
G alpha I two protein expression to validate
whether or not the knockdown was still occurring.
Sodium balance, plasma norepinephrine
as an index of sympathetic nervous system activity.
And again, tissue markers of microglia and astrocytes,
the immune cells that can possibly contribute
to inflammation if there should be any in the brain.
What we first saw, and this is a very similar graph
to what you've seen last time,
was that the scrambled ODM infused rats
have a robust expression of the G alpha I two protein
at about 41 kilodaltons.
And our G alpha I two ODN infusion significantly brings down
that expression almost to zero.
So very, very robust decrease in tissue protein expression.
And that's again, a Coomassie stained blot
that was loaded simultaneously to the gel
from which this was transferred to a membrane
so that you can validate normal protein loading
across all lines.
And then we looked at blood pressure,
and really interestingly what we saw was,
again, at a scrambled control rat,
there's no high salt diet evoked increase in blood pressure.
And in our knockout rat,
there's interestingly only a mild increase.
And to give you some comparison here,
these are day seven acute blood pressures,
not chronic blood pressures over time like I showed you,
but just one day blood pressures.
And at the same time and point,
these female rats increase their average blood pressure
by about seven or eight millimeters of mercury,
whereas the males increased from about 125 to almost 150.
So there's a significant difference in the magnitude
of hypertension evoked
following G alpha I two protein downregulation.
So we're already seeing sex differences occurring here.
Now, that's interesting.
When we look at plasma norepinephrine as a measure
of sympathetic nervous system activity,
we first see that there's actually a significant reduction
in plasma norepinephrine levels
in the scrambled control rats, these female rats,
but what was really kind of a brain teaser
was that they still sympathoinhibited
even after you'd taken out essential G alpha I two protein.
So they actually don't develop sympathoexcitation
the way a male rat does.
And this was interesting because
when we sat down to think about it,
we thought, well, maybe these rats won't sympathoexcite.
Maybe there'll be protected from going up with respect
to neuro epinephrin, but they shouldn't still go down.
They've lost their regulatory mechanism responsible
for facilitating that.
And sometimes science is a mystery. (chuckles)
When we look at sodium balance now, because we want to see
if they even developed salt sensitivity,
do they retain sodium?
What you can see is we've got these nice baseline values
here on this longitudinal time graph
where they're on normal salt.
And when you switch these rats to a high salt diet,
there's only ever a single significant increase
in sodium retention calculated
as the difference between the sodium consumed
and the sodium excrete it in the urine.
And then you returned to non-significant difference
from baseline and the scrambled rats of course never deviate
from baseline in a statistically significant way.
So they only develop mild hypertension.
They maintain their ability to sympathoinhibit,
and their sodium retention is not very bad at all
especially when you consider it in comparison to the males
which display a greater salt sensitivity of blood pressure,
such that even the scrambled control rats
significantly retain sodium for some time
before coming to baseline, although not nearly as bad
as the G alpha I two knockdown rats
which have a very exacerbated sodium retention
over several days that doesn't return
to baseline values until the very end of the study,
seven days of high salt diet.
So they're protected from many things here, it appears.
When we looked in the PVN
at indices of inflammation, you can see up here,
these scrambled control rats,
no change in microglial cell count,
no change on average in the number or percent
that are active amongst these animals,
and the same holds true
following G alpha I two protein knocked down
in high salt diet.
There's a failure to evoke an increase
in microglial reactivity.
So the next big difference, obviously,
as I'm showing you here is a complete lack
of inflammatory cell activation
which may partially be responsible
for the lack of sympathoexcitation we see here
because as we saw with the male rats,
when you take down the inflammation, you completely remove
the sympathetic nervous system excessive activity.
So without it ever happening at baseline,
it may be that they are not developing
robust salt sensitive hypertension.
And lastly, we look at these astrocytes
and we can see the same thing.
There's a complete lack of an impact
of high salt diet on changing the immunostaining intensity
or reactivity of astrocytes, suggesting
that female rats are not going to develop inflammation
in the brain.
And this led us to an initial conclusion
for these studies that they develop blunted
salt sensitive hypertension, female rats,
which lack inflammation and sympathoexcitation overall.
So there are robust sex differences
which we may be able to attribute to sex steroid hormones.
Now, just to give you some quick future directions
for these studies, we want to investigate things
like the electrophysiological impacts
of G alpha I two proteins.
So how do they impact neuronal firing?
How do they impact neuronal resting membrane potentials
as well as what is the impact of these
on single cell parvocellular neuron transcriptomics?
How is the phenotype of a neuron, for example, altered
when these proteins are lost
and further studies impact investigating the impact of,
as I just mentioned, the sex steroid hormones
on this model of hypertension in the absence
and presence of G alpha I two proteins?
With that, I'd like to think these are just
some of the people that are listed here
but several of these wonderful individuals
as well as my laboratory and my mentor, Dr. Richard Winford,
some previous members are here.
Current members are here, but my dissertation committee,
they've all been fabulous
in helping me refine this project
as well as my department and our funders.
And with that, I'm very happy to take your questions.
- Thank you so much for such a great talk.
I really appreciate your time
and I appreciate you sharing with us
all this good stuff you've been up to.
So thank you.
All right, so we have a few questions here for you already.
So the first one is what tests can be done to find out
if you are salt sensitive or if you possess mechanisms
for salt resistance?
- So that's a really great question.
In humans, we're currently studying
the G alpha I two protein to look for easier biomarkers
for the identification of salt sensitivity.
We've done a couple of studies
with respect to a single nucleotide polymorphisms
in the GNAI2 gene as well as some other groups have,
showing its significant correlations
with essential hypertension.
And that goes back to looking for easier biomarkers
because it's a very robust study
that you have to do with humans over several weeks
where you sodium deplete them, feed them high sodium,
sodium deplete them and take ambulatory blood pressures
all throughout the way.
So it's very involved to determine at this point
if you're salt sensitive or not.
- We have another question.
What was the motivation for you to move
from muscle physiology to neuro immunology and neuroscience?
- Oh, I want to be as honest as possible.
It wasn't as direct of a motivation as it may seem.
I just love everything in physiology.
And I spent my fair share of time doing this
and then I wanted to do something else,
and then I did that and I wanted it to do something else.
And some of you who know me from Salem State
know that I was interested in microbiology
and pharmacology when I was there.
And so I've been very fortunate and had the opportunity
to just kind of pursue my interests on a whim.
And this was the most recent of those whims. (laughing)
- Honest is good, I like that.
So another question we have here
is how is this connected to evolution?
- So when I think about cardiovascular disease
and evolution, really interesting concepts come up,
like the notion that usually with respect to evolution,
traits that are higher
in inducing morbidity are not selected for.
You usually don't get selection for those things,
but that has to do mostly with its impact on fitness.
So what's really interesting about
things like cardiovascular disease
is that they don't necessarily have a direct impact
on the life span per se.
Most individuals make it well beyond reproductive age.
And so we kind of have to fight that evolution
isn't selecting against these negative traits
like salt sensitivity by coming up with our own solutions
through understanding the mechanisms that mediate them.
- We have another question.
What would be your advice
to any undergraduate students wishing to go on
to conduct a graduate level research?
- Oh, that's such a great question.
I didn't know I wanted to do research, honestly.
I got involved in research in undergrad,
but not super heavily.
And in pursuing a master's degree first,
as Dr. Gordon said before,
that was to enhance my qualifications for Medical School,
but I ended up incidentally falling in love
with the research process
and sticking with it along the way.
So I guess the advice in that is just to be open-minded
and you don't know where your passion may end up lying.
So try everything, dip your toes in the water.
You might like it.
- That's a great advice.
We have a question here from one of our colleagues
in the Biology Department is asking,
your female rats had less inflammation in response to salt,
but in humans, we know that females are more likely
than males to get inflammatory autoimmune diseases
that include inflammation.
Any thoughts?
- Oh, that is such a fabulous question.
Honestly, I don't know.
And that's a really powerful answer, I think,
even though it's a cop out
because the role of sex steroid hormones really evades us.
In some instances, we see that sex steroid hormones
directly exert like estrogens
on anti-inflammatory polarization on immune cells.
But then we see that as it was pointed out,
it's likely that females have more autoimmune disease
and dysregulation of the immune system featuring
these hyper inflammatory responses.
I think that that mechanism slightly remains a mystery
or at least to me at this point.
I'm not sure.
- We have another question that's sort of related
to a previous question, but what inspired your research?
Do you have advice for those who are looking to do research
and how do they go about looking for research topics?
- That's really a great set of questions
when we tackle them one at a time.
My research was inspired largely
by my personal interests in neuroscience
and my mentor, Dr. Wainford.
He had this decades long set of research
investigating the physiological impact
of the G alpha I two protein.
And I took it simply to what is its role in gating
these inflammatory pathways
in addition to sympathetic nervous system control?
So very largely inspired by his previous work
and his funding, which was to investigate this work further.
For those looking to go into research,
I think a critical component is following something
you're passionate about.
You're not going to be passionate about research
that's on a topic you don't care about.
And then finding a mentor with whom you really click
is so important because it's like a marriage.
You are gonna have your fights, but when you're working
with a research mentor, it's better when you like them.
And then looking for research topics,
that comes with the other two things I just said,
your interests and your mentor's interests aligning.
And that last part, the research topic
should just come a little more naturally.
Do something you're passionate about.
- Yeah, and speaking about finding a mentor
and the doctor path, one of the questions was
can you share with us some that was most challenging about
either your undergraduate path or your doctoral path?
- Yeah, a huge challenge, especially with respect
to graduate school is time management.
As Dr. Gordon mentioned, I served through a portion
of graduate school as a teaching fellow.
So I was teaching gross anatomy labs, neuro anatomy labs,
cardiovascular physiology lectures,
while conducting 40, 50 hours a week of my own research.
And then the part we all forget about,
being a person and having a life
and an interpersonal relationship with a partner
and a family to visit for birthdays.
And so really getting into a solid swing
of time management is super helpful.
That's the most important part,
and the hardest part. (chuckles).
- This is sort of out of your area
but I think it's an interesting question.
Why are women more prone to getting auto immune disease?
- (sighs) It's a fascinating question
and there is various is research
currently being done, I'm certain.
I would say it's got to have something to do
with the biological differences that reside in our genetics.
Almost everything is rooted there
in tissue-specific gene expression
and hormonal impacts on those things.
But I would encourage you to pub-med it.
(both laughing)
- Another question says, why did you choose
this animal model?
And can your new information be transferred
to human studies?
- Fabulous question.
We chose this animal model because there is 70 years
of research characterizing the physiology
of the Sprague-Dawley rat, it's extremely widely used
in studies on salt resistance and salt sensitivity
and with respect to transfer to human studies,
that's along with what I brought up earlier,
having to do with polymorphisms in the GNAI2 gene in humans.
So several studies published by us and other labs
have shown correlations between snips
and essential hypertension as a phenotype, salt sensitivity,
blood pressures as a phenotype in multiple cohorts,
like the GenSalt study performed in North China
in the 1900s, as well as Tissue from the UK Biobank
in western European individuals performed
much more recently, these analyses.
So we think that with the correlation
between protein expression and snips
and salt sensitivity holding true in humans,
the mechanistic information may provide useful
because of the conservation of things
like fluid and electrolyte balance pathways
across different mammalian species.
- I wanted to ask a question, why high blood pressure
would lead to inflammation?
I assume, partly because of leakage, I'm not a physiologist
but what is the possible reasoning
behind why that's happening?
Does that make sense?
- I've got some fun.
Yeah, I've got some fun insights on that.
We've recently been doing studies
along with other labs doing studies that have shown
that there's changes that occur
within the neuronal phenotypes in response
to inflammation and as well as just studies
that baseline show that cytokine production
as a predisposing factor
can then change neuronal firing rates.
So we think the inflammation is directly acting locally
on those projection neurons and increasing their firing rate
as well as exacerbating the number of neurons
that express excitatory phenotypes
in comparison to inhibitory phenotypes.
So you get uncoupling of excitation in addition
as well as exacerbated amount of signaling.
It's quite fascinating to unravel that.
That's a great question.
- I have a question here
that I wanted to elaborate further too.
So the question is, do you feel that SSU prepare you
to be successful in research?
So I want to add to that.
How did you use the tools that SSU has?
What kind of tools were not working?
What resources did you use from SSU to be today successful
in your research or you feel used additional tools
that are connected to SSU,
share a little bit about those as well, thank you.
- Yeah, I think with one thing with respect
to your professional career is you can't really be afraid
to ask the question and it was really
just kind of approaching Dr. Mwananyanda and saying,
I'm really interested in doing research
and he wanted to know what I wanted to do.
And so I think that the biggest hill to overcome
is that first question, finding a faculty member
who's doing something you're doing.
And that's what I did.
And so SSU significantly repaired me in that.
I got comfortable asking those kinds of questions
of people and asking to make space
for me in somewhere to grow.
And then once that happened,
working with him was a significant opportunity
for me to engage in the scientific method
and start thinking about how to think about experiments
and whether or not that would be interesting for me.
So SSE really did prepare me,
both the Biology and Chemistry Departments
had great opportunities available.
I know several people
who worked with other faculty like Dr. Tracy
where I had students working with her and Dr. Jason Brown.
And so there are plenty of opportunities
if you're interested at SSU to get involved.
And I really encourage you to pursue them.
- We'll end with one last question.
Did you notice lethargy or other physical issues
with the rats that were sensitive to sodium?
- They were a little tired of the day after surgery,
but (laughs) most people are, but no in all honesty
would leave the rats a five day recovery period
following the cannular implantation into their brain
to facilitate rest and relaxation
so that the study can proceed unobstructed in the rats,
display all normal behavioral parameters.
They eat the same amount of food.
They drink the same amount of water.
They aren't scoring higher on any pain scales
that we can assess them with.
So they appeared to be functioning entirely normally
but you never know,
there's always the undiscovered factor, right?
- So I'll pass you over to Dr. Gordon.
- And with that, I'd like to thank Jesse Moreira
for being our alumnus speaker.
I think you're a great model for other students.
I'm glad we had students asking questions about
how they get involved in research
and what motivated you and how did you become so successful?
So I think you've given them a lot to think about
and you've given all of us a lot to think about.
So we want to thank you for being our alumna speaker.
And we also want to wish you well in your career
and success in your upcoming dissertation defense.
Thank you very much for participating in this,
our 42nd Annual Darwin Festival
and our first online webinars series
for Darwin, and so historic moment for us.
And we are very proud of you
and we wish you well in the future.
Thank you.
- Thank you so much.