Approved.

1. INSTITUTIONAL
ANIMAL
CARE
AND
USE
COMMITTEE
(IACUC)
ANIMAL
USE
PROTOCOL
I. General
Information
A.
Project
Title:
Perturbation of mouse intestinal microbiota through broad-spectrum antibiotic intervention,
high-fat diet: effects on glymphatic regulation, colonic epithelial permeability and sleep
pattern correlations.
B. Type
of
Application:
X
New
protocol
C. Source
of
Funding:
-­‐UWL
Undergraduate
Research
and
Creativity
-­‐Biotechnology
Grant
D. Principal
Investigator:
Name
(Last,
First,
MI)
Seebach,
Bradley
Mailing
Address
0042
Health
Science
Center,
Department
of
Biology
Office
Phone
#
608.785.6966
Cell
or
Home
Phone
#
608.406.0642
E-­‐mail
bseebach@uwlax.edu
PI
Certification
If
the
IACUC
approves
my
application,
I
agree
to
execute
this
work
as
described;
request
approval
from
the
IACUC
for
changes;
comply
with
the
guidelines
set
forth
by
the
IACUC
and
be
responsible
for
the
training,
supervision
and
work
of
my
staff.
I
realize
that
failure
to
adhere
to
policies
related
to
animal
care
and
use
may
result
in
suspension
or
revocation
of
permission
to
perform
animal
research
in
UW-­‐L
facilities.
The
activities
described
in
this
study
do
not
unnecessarily
duplicate
previous
experiment.
Date
Signature
of
PI
June
1,
2015
Date
Signature
of
support
from
Department
Chair
or
College
Dean
June
1,
2015
IACUC
USE
ONLY
PROTOCOL
NUMBER:
EXPIRATION
DATE:
USDA
REPORTING
CODE:
EXCEPTIONS
TO
THE
GUIDE:

2. E. Personnel
who
will
have
animal
contact
(Including
PI).
Copy
and
paste
additional
tables
if
needed
:
Name
Bradley
Seebach
Cell
or
Home
Phone
#
608.785.6966
E-­‐mail
bseebach@uwlax.edu
Role
in
Project
Principal
Investigator
Experience
with
proposed
procedures
Over
20
years
of
neuroscience
research.
Personnel
Certification
I
have
read
and
am
familiar
with
all
of
the
approved
animal
procedures.
I
agree
to
execute
this
work
as
described
and
realize
that
failure
to
adhere
to
policies
related
to
animal
care
and
use
may
result
in
suspension
or
revocation
of
permission
to
perform
animal
research
at
UW-­‐L.
Occupational
Health
and
Safety
Program
I
have
submitted
an
Animal
User
Risk
Assessment
Form
Training
Requirements
I
have
passed
all
the
applicable
training
tutorials
Date
Signature
June
1,
2015
Name
Sumei
Liu
Cell
or
Home
Phone
#
608.799.6145
E-­‐mail
sliu@uwlax.edu
Role
in
Project
Co-­‐Principal
Investigator.
Will
perform
euthanasia,
tissue
collection
and
analysis
of
colonic
epithelial
permeability.
Experience
with
proposed
procedures
20
years
experience
with
gastrointestinal
physiology,
gavage,
rodent
euthanasia
and
other
related
protocol.
Personnel
Certification
I
have
read
and
am
familiar
with
all
of
the
approved
animal
procedures.
I
agree
to
execute
this
work
as
described
and
realize
that
failure
to
adhere
to
policies
related
to
animal
care
and
use
may
result
in
suspension
or
revocation
of
permission
to
perform
animal
research
at
UW-­‐L.
Occupational
Health
and
Safety
Program
I
have
submitted
an
Animal
User
Risk
Assessment
Form
Training
Requirements
I
have
passed
all
the
applicable
training
tutorials
Date
Signature
June
1,
2015
Name
Jonathan
Lendrum
Cell
or
Home
Phone
#
920.850.7883
E-­‐mail
lendrum.jona@uwlax.edu
Role
in
Project
Investigator.
Administering
gavage
treatments,
monitoring
pain/distress,
collecting
fecal
samples
for
PCR
amplification
of
16S
rRNA
for
DGGE
and
next
gen
sequencing,
custom
cage
installation
of
video
monitoring
system,
assisting
with
euthanasia
and
tissue
collection,
analysis
of
colonic
epithelial
permeability,
etc.
Experience
with
proposed
procedures
Jon
will
be
trained
to
administer
gavage
by
Amy
Cooper
and
Sumei
Liu.
Personnel
Certification
I
have
read
and
am
familiar
with
all
of
the
approved
animal
procedures.
I
agree
to
execute
this
work
as
described
and
realize
that
failure
to
adhere
to
policies
related
to
animal
care
and
use
may
result
in
suspension
or
revocation
of
permission
to
perform
animal
research
at
UW-­‐L.
Occupational
Health
and
Safety
Program
I
have
submitted
an
Animal
User
Risk
Assessment
Form
Training
Requirements
I
have
passed
all
the
applicable
training
tutorials
Date
Signature
June
1,
2015

3.
Name
Amy
Cooper
Cell
or
Home
Phone
#
608.790.4157
E-­‐mail
acooper@uwlax.edu
Role
in
Project
Animal
care
facility
manager.
Will
train
and
provide
training
and
assistance
with
euthanasia
and
gavage
procedures.
Experience
with
proposed
procedures
Over
25
years
performing
gavage
and
euthanasia
on
rodents.
Personnel
Certification
I
have
read
and
am
familiar
with
all
of
the
approved
animal
procedures.
I
agree
to
execute
this
work
as
described
and
realize
that
failure
to
adhere
to
policies
related
to
animal
care
and
use
may
result
in
suspension
or
revocation
of
permission
to
perform
animal
research
at
UW-­‐L.
Occupational
Health
and
Safety
Program
I
have
submitted
an
Animal
User
Risk
Assessment
Form
Training
Requirements
I
have
passed
all
the
applicable
training
tutorials
Date
Signature
June
1,
2015
F. Summary
of
Project:
In
straight-­‐forward,
nonmedical,
nontechnical
language
that
would
be
understandable
to
a
layperson
(aim
for
a
high
school-­‐senior
reading
level),
outline
the
specific
scientific
goal(s)
and
significance
of
this
research.
Be
convincing
as
to
why
this
work
is
important
for
advancement
of
knowledge,
improving
human
or
animal
health,
or
for
the
good
of
society.
Spell
out
all
acronyms
at
first
occurrence.
If
this
is
a
renewal
submission
please
provide
a
brief
(2-­‐3
sentences)
description
of
your
progress
and
productivity
in
the
past
three
years
to
help
the
Committee
evaluate
animal
usage.
Since
this
summary
may
be
made
available
to
the
public
if
requested,
it
is
imperative
that
you
carefully
consider
its
content.

4. Abstract
In
2012
a
previously
unknown
waste
clearance
pathway
for
the
mammalian
central
nervous
system,
coined
the
glymphatic
system,
was
discovered
by
neuroscientists
at
University
of
Rochester
Medical
Center.
Analogous
to
conventional
lymphatic
vasculature,
which
is
found
in
all
parts
of
the
body
except
the
brain,
the
glymphatic
system
relies
on
changes
in
cerebrospinal
fluid
circulation
to
function
like
a
sink
for
removing
excess
waste
products
from
the
highly
sensitive
brain
tissue.
Each
night
sleep
drives
a
specific
network
of
microscopic
fluid
channels
to
open,
allowing
for
the
brain’s
sink
to
‘turn
on’
the
cerebrospinal
fluid,
which
is
restricted
to
the
brain’s
exterior
while
awake.
It
then
quickly
enters
deep
brain
tissue
where
it
washes
away
harmful
extracellular
waste
products,
such
as
beta-­‐amyloid,
that
progressively
accumulates
the
longer
you
remain
awake.
In
the
last
decade,
mounting
evidence
suggests
complex
interactions
between
hosts
and
their
microbial
communities,
known
as
the
gut-­‐microbiome,
play
important
roles
maintaining
and
regulating
our
health.
The
interactions
between
microorganisms
of
the
gut
and
host
physiology
are
a
result
of
complex
chemical,
neural
and
hormonal
influences.
We
depend
on
our
gut
microbiota
for
digestion
and
drug
detoxification
and
these
complex
exchanges
with
our
microbial
communities
influence
a
variety
of
important
host
functions
ranging
from
metabolic
activity
and
immune
response,
to
perhaps
most
remarkably,
behavior
and
cognition.
Our
study
uses
high
throughput
digital
video
tracking
software
to
accurately
determine
sleep
pattern
correlations
in
response
to
intestinal
dysbiosis
induced
through
broad-­‐spectrum
antibiotic
intervention
and
high-­‐fat
nutritional
dietary
changes
in
mice.
A
dysbiotic
state
adversely
affects
the
biochemical
conditions
responsible
for
healthy
communication
between
host
central
and
enteric
nervous
systems
and
the
100
trillion
microorganisms
encompassing
the
gut-­‐microbiome,
referred
to
from
here
as
the
brain-­‐gut-­‐microbiota
axis.
It
is
the
objective
of
this
pilot
study
to
acquire
preliminary
data
involving
relationships
between
the
diversity
of
gut
microbiota,
colonic
trans-­‐epithelial
permeability
and
sleep-­‐wake
patterns
in
C57BL/6
male
mice.
We
want
to
make
a
contribution
towards
the
perspective
and
understanding
of
these
intriguing,
yet
unexamined
relationships
to
the
biomedical
community
in
order
to
assist
with
the
development
of
clinically
relevant
therapeutics
involving
the
physiopathology
of
neurodegenerative
diseases
and
other
conditions
commonly
associated
with
congruent
brain
and
gut
dysfunction.
II.
Animal
Species
and
Numbers
Species
Total
number
of
animals
needed
for
3-­‐year
protocol
Source
(e.g.
vendor,
breeding
colony)
Housing
Location
Any
special
husbandry/housing
needs?
C57BL/6
Mouse
20
Harlan
(Madison,
WI)
HSC
15
individually
housed,
open
top
cages
(60.7cm
l
x
40.4cm
w
x
31cm
h)
custom
fitted
with
video
monitoring
capable
of
simultaneous
and
continuous
recording
all
15
mice
for
the
duration
of
the
experiment
(19
days).

5.
III.
Justification
for
the
Use
of
Animals
A.
Consideration
of
Alternatives
to
Live
Animal
Use,
Painful
Procedures
and
Unnecessary
Duplication
The
United
States
Department
of
Agriculture
(USDA)
requires
that
you
specify
at
least
two
sources
or
databases
used
to
determine
that
the
model
and
methods
described
in
this
protocol
do
not:
• Unnecessarily
duplicate
previous
experiments
• Unnecessarily
use
animals,
or
• Unjustifiably
expose
animals
to
potentially
painful,
uncomfortable
or
distressful
procedures
A.
Database
Searches.
Place
an
X
in
the
checkboxes
that
apply
to
indicate
which
databases
were
used:
National
Agricultural
Library
Web
of
Science
PsycINFO
(AGRICOLA)
AltWeb
TOXLINE
BIOSIS
Previews
CORDIS
X
Other
–
Google
Scholar
X
MEDLINE
via
PubMed
NORINA
Other
-­‐
B.
Date(s)
the
database
search
was
performed:
(Must
be
within
the
last
3
months)
5/20/15
C.
Years
covered
by
the
search
(e.g.,
1985
to
present):
2010-­‐2015
D.
Keywords
used
in
the
search.
The
more
"keywords"
you
use
the
more
specific
your
search
will
be;
however,
being
too
specific
may
lead
to
no
results
being
found.
In
that
case,
reduce
the
number
of
terms
used.
1.
In
vivo+rodent+glymphatic
system+model
2.
In
vivo+rodent
model+gut-­‐brain
axis
3.
In
vivo+rodent+gut
microbiome

6. E.
Database
Search
Narrative
Description.
Specifically
discuss
the
results
of
what
was
found
during
the
search.
Describe
the
steps
you
have
taken
to
replace
the
use
of
animals
with
in
vitro
procedures,
reduce
the
number
of
animals
used,
and
to
refine
the
experimental
design
and
procedural
techniques.
If
similar
experiments
are
found,
describe
the
aspects
of
your
research
project
that
are
novel
and
are
not
unnecessarily
duplicative
of
other
published
work.
If
alternative
methods
or
procedures
representing
refinements
to
your
procedures
were
found,
discuss
why
those
alternatives
cannot
be
used.
Discussion
of
Search
Results:
Due
to
the
recent
discovery
of
the
glymphatic
system,
we
are
familiar
with
each
of
the
top
20
search
results
involving
rodent
glymphatic
system
literature.
The
articles
found
in
the
database
search
were
integral
to
the
development
of
our
experimental
design.
The
most
recent
experiments
evaluating
the
glymphatic
system
involve
in
vivo
brain
imaging
techniques,
however,
these
methods
require
magnetic
resonance
imaging
and
the
methodology
of
such
is
just
now
becoming
established
for
additional
research
applications.
Replacement:
Replacement
of
animals
with
in
vitro
procedures
is
not
possible
at
this
time
because
the
waste
clearance
mechanism
responsible
for
the
glymphatic
system
function
operates
at
an
organ
level
of
organization,
not
tissue
or
molecular,
and
we
are
at
an
early
stage
of
understanding
of
this
function.
Additionally,
taking
into
consideration
the
necessity
for
the
anatomical
brain-­‐gut-­‐microbiota
axis
for
our
study
to
be
possible,
it
becomes
significantly
more
difficult
to
completely
replace
the
animal
component
of
the
experiment.
Reduction:
Only
using
a
single,
maximum
time
point
for
our
n=5
mice
groups,
rather
than
3
time
points
of
brain
tissue
fixation
for
each
mice
group
significantly
reduces
the
number
of
animals
used
(5
mice
instead
of
15
required
per
group).
Additionally,
the
tissue
of
our
mice
will
be
utilized
for
multiple
collaborative
experiments.
Refinement:
It
has
been
demonstrated
previously
that
medications
can
be
incorporated
into
palatable
diets
or
treats.
Custom
medicated
food
was
considered
as
a
potentially
less
stressful
alternative
to
gavage;
however,
Research
Diets
Inc.,
Bioserv
Medicated
Solutions
and
Harlan
Teklad
were
not
able
to
incorporate
this
antibiotic
cocktail
into
a
diet-­‐drug
intervention.
B.
Rational
for
the
Use
of
Animals
Federal
regulations
require
that
investigators
provide
a
narrative
describing
the
rationale
for
using
animals,
the
appropriateness
of
the
species,
and
the
methods
and
specific
sources
used
to
determine
that
alternatives
(e.g.,
replacement,
reduction,
refinement)
to
the
use
of
animals
and
to
the
procedures
have
been
considered.
1.
Explain
why
animals
are
required
for
these
studies,
and
why
non-­‐animal
model
replacements,
such
as
cell
culture
or
computer
modeling,
cannot
fully
replace
animals:
Mice
are
the
predominant
animal
models
used
in
similar
experiments
involving
the
brain-­‐gut-­‐microbiota
axis
and
is
the
species
from
which
most
is
known
about
the
recently
discovered
glymphatic
system.
Additionally,
due
to
our
interest
in
the
pathways
in
which
antibiotic
intervention
affects
different
organ
and
system
levels
of
organization,
mice
cannot
be
replaced
by
any
non-­‐animal
model
system.
This
is
because
of
our
experimental
design’s
dependence
on
the
macroscopic
brain-­‐gut-­‐
microbiota
axis.
Mice
have
been
the
source
of
most
of
the
existing
neurobiological
and
neurochemical
information
on
the
central
and
enteric
nervous
systems
as
well
as
neural
control
of
gut-­‐microbiota
function
for
the
past
50
years.
There
are
no
known
non-­‐animal
models
or
systems
that
encompass
the
complexities
of
the
nervous
system
in
the
digestive
tract.

7. 1. Describe
the
features
of
the
species
(e.g.,
anatomic,
physiologic,
genetic,
etc)
that
make
it
desirable
for
the
model.
Contrast
with
other
available
models,
if
any.
Cost
considerations
are
not
justifications.
Currently,
rodents
are
the
only
animals
known
to
possess
the
glymphatic
system
(though
known
anatomy
and
physiology
similarities
across
mammal
species
suggests
broad
application
of
findings);
therefore,
the
research
utility
of
rodents
is
of
utmost
value
in
this
exciting
and
quickly
expanding
branch
of
neuroscience.
Furthermore,
mice
are
informative,
dynamic
animal
models
that
have
continuously
demonstrated
their
research
utility
by
allowing
investigators
to
have
precise
control
over
manipulating
their
experimental
variables.
For
example,
“germ
free
like”
mice
(from
antibiotic-­‐induced
microbial
depletion)
are
not
only
relevant
to
research,
but
have
been
shown
to
contribute
to
clinical
diagnostics
by
allowing
researchers
to
make
inferences
about
the
relationships
between
the
human
gut
microbiota
and
associated
brain
function
in
ways
that
were
previously
nonexistent
(Hintze,
2014).
We
have
every
reason
to
believe
the
human
glymphatic
system
operates
similarly
to
that
in
mice
models.
2. How
are
the
number
of
animals
requested
scientifically
justified
for
this
species.
Include
all
control
animals
and
breeding
colony
animals
in
this
discussion.
A
table
may
help
clarify
different
experimental
groups
or
studies
and
the
specific
numbers
needed
for
each.
Include
any
statistical
analysis
used
(e.g.
power
calculations)
in
determining
the
animal
numbers.
Our
pilot
study
requests
the
application
of
20
mice.
The
sample
size
(n=15)
composes
three
groups
of
five
mice,
the
extra
five
mice
requested
will
be
used
to
improve
techniques
for
gavage,
tissue
handling
or
to
mediate
premature
removal
of
another
mouse
due
to
unanticipated
complications.
Selection
of
group
size
is
based
on
the
minimum
number
needed
for
statistical
significance
analysis
as
described
in
similar
studies
(Martinez
et
al.,
2004;
Yuan
et
al.,
2009).

8. IV.
Details
of
Animal
Use
A.
In
this
section
describe
the
animals’
roles
in
your
experiments
including
the
treatments
and
procedures
the
animals
will
receive
outside
of
normal
husbandry,
from
the
first
experimental
manipulation
to
the
final
outcome.
This
response
should
provide
a
clear
understanding
of
what
specifically
happens
sequentially
to
each
animal
or
group
of
animals,
and
over
what
time
period
the
procedures
occur,
including
but
not
limited
to:
§ definitions
of
all
materials
given
to
animals,
including
dosage
range,
routes,
and
frequency
of
administration;
§ the
expected
sequence,
frequency,
and
duration
of
procedures;
§ method,
frequency,
volumes,
and
numbers
of
biological
samples
taken;
experimental
diets
General animal care information. Nine to ten-week old male C57BL/6, inbred mice acquired from Harlan (Madison,
WI) will be randomized, and weight matched (to have a weight range of maximum 20% of medians) into three
experimental groups upon arrival at HSC, and then given a week to acclimate to the HSC facility and ensure
palatability of experimental diets. The three experimental groups include: the control group fed a calorically and
nutritionally matched low fat diet (10% kcal), antibiotic-treated group also provided the low fat diet, and a diet-
induced obesity group provided with a high-fat (60% kcal) purified diet (Harlan Teklad). All animal housing will be
maintained at a standard 12h light/12h dark cycle.
Animal housing. Upon arrival at HSC, the mice will be randomized and age-matched into three group-housing cages
for the initiation of their acclimation period. At this point, one mouse from each experimental group will be
removed from group housing and individually placed into one of the fifteen custom open-top cages previously
fitted with a HD infrared security camera and infrared LED illuminator. Our automated sleep monitoring system
produces high quality video recordings for post analysis of sleep pattern circulations using idTracker, a software
coding program developed on MATLAB for high-throughput determination of sleep-wake duration. The sleep
monitoring system will not negatively affect the quality of cages used to individually house each mouse. All mice
will be provided water and their prospective low or high fat formulated diets immediately upon arrival, ad libitum.
*Deviation from guidelines* The food will be provided ad libitum on the floor of the cages. There is sufficient room as
to discourage soiling of the food and because the mice will be gavaged every 12 hours for the entirety of the
experiment, these times will be used to provide fresh food, water or bedding as needed.
Experimental group one. The first group is the control consisting of five specific-pathogen-free (SPF) mice, which
have been subject to microbial pathogen screening. The mice will be fed a purified standard, low-fat control diet
and provided with autoclaved water ad libitum. The control group represents healthy, conventional gut-microbial
composition in both number and diversity of microbial species typically inhabiting the colon of mice. This group
will receive equal volume sterile water solution by gavage every 12 hours for 14 days to control procedural variables
introduced.
Experimental group two. The antibiotic-treated group consists of five mice housed individually in open top cages
custom fitted with a video monitoring system. These mice will be provided the identical purified low-fat diet as the
control group, however, this group of mice will receive broad spectrum antibiotic-cocktail by gavage every 12 hours
consisting of: 14 days of 50mg/kg vancomycin, 100mg/kg neomycin, 100mg/kg metronidazole and 1mg/kg
amphotericin B. Finally, 1 g/L of ampicillin will be provided in autoclaved drinking water. This cocktail is based off
established pain-free methods to safely induce “germ free-like” phenotypes in mice through depletion of intestinal
microbiota. (Reikvam, et al.)
Experimental group three. The diet-induced-obesity (dio) group consists of five mice to be housed individually in open-
top cages custom fitted with a video monitoring system. Alternatively, these mice will be provided with a high-fat
diet (60% kcal fat), formulated to induce obesity phenotypes in mouse models. This group is important to our
comparative study due to the alteration it has on gut-microbiota composition. Recently, using similar diet

9. manipulations, it was discovered that the gut-microbiome is pivotal for maintenance of host circadian clock
rhythmicity (Kohsaka et al., 2007) (Leone, 2015). The diet-induced obesity group has significant value; its
comparison with group two should be informative regarding potential consequences of sleep deprivation, such as
an increased susceptibility of obesity, especially following intake of endemic high-fat, Westernized diets.
Fecal sample collection and molecular processing. We want to approximate the time course and efficacy of diet/antibiotic
manipulations of mouse gut microbial composition. This will require estimations of the taxonomic diversity
encompassing the gut microbiome. We will accomplish this by collecting freshly voided fecal samples at three time
points from each of the mice: once before the (antibiotic) treatment course (day 0), midway through treatment
course (day 7), and upon completion of treatment intervention (day 14).
Animal euthanasia and tissue collection. Twelve hours following the final antibiotic gavage and fecal sample collection,
the mice will be euthanized after a four-hour fasting period- by CO2 asphyxiation and exsanguination. The brain
and proximal colon will be harvested promptly and used for immunohistochemistry, enzyme-linked immunoassay
and measuring trans-epithelial permeability.
Schedule, timeline and conclusion of experimental cycles. See figure below. An important point to consider is due to time and
resource limitations, we are not able to euthanize, collect tissues and run experiments on all 15 mice in a single day
following conclusion of one 14 day treatment cycle. To solve this problem, we will stagger day 1 administration of
gavage treatments among five days so upon conclusion of each 14 day cycle we will euthanize and run experiments
on 3 mice a day (one per each treatment group), for 5 consecutive days. See figure below.

10.
B.
Complete
the
following
table
if
dosing
with
any
substance:
Substance
Used
Species
Dose
mg/kg
Route
Frequency
Pharmaceutical
Grade?
Water
Mouse
0.5ml/kg
Gavage
Twice
daily
for
14
days
Yes
Ampicillin
Mouse
1g/L
Oral
Provided
in
water
ad
libitum
Yes
Vancomycin
Mouse
50mg/kg
Gavage
Twice
daily
for
14
days
Yes
Neomycin
Mouse
100mg/kg
Gavage
Twice
daily
for
14
days
Yes
Metrondiazol
Mouse
100mg/kg
Gavage
Twice
daily
for
14
days
Yes
Amphoterian
B
Mouse
1mg/kg
Gavage
Twice
daily
for
14
days
Yes
C.
Appendices
Checklist.
Check
all
that
pertain
to
your
project,
complete
the
appropriate
appendices,
and
attach
as
part
of
your
application.
Delete
all
non-­‐applicable
appendices.
X
Dietary
Manipulations
or
Fluid
Restriction
Appendix
E
V.
Potential
Animal
Pain
and
Distress
A.
Briefly
summarize
all
possible
adverse
effects
that
may
present
in
the
animals
as
a
result
of
study
procedures.
Adverse
Effects
include
any
reaction,
expected
or
unexpected,
that
may
occur
in
the
animals
as
a
result
of
any
experimental
procedure
or
manipulation.
Examples
include
drug
toxicity,
injury,
surgical
complications,
lameness,
lethargy,
anorexia,
tumors,
etc.
Although
rare,
adverse
effects
that
may
present
in
animals
as
a
result
of
our
study
include:
allergic
drug
reaction
from
antibiotic
exposure
or
dyspnea,
aspiration
or
unintentional
injury
due
to
complications
administering
gavage.
Any
animals
showing
adverse
drug
reactions
or
uncommon
behavior
as
a
result
of
human
gavage-­‐error
will
be
promptly
removed
from
the
study
and
euthanized.
B.
How
will
pain
and/or
distress
be
monitored?
Provide
specific
clinical
signs,
which
will
be
monitored
as
well
as
frequency,
including
provisions
for
off
hours.
Clinical
signs
may
include
tumor
growth,
lack
of
appetite,
lack
of
normal
grooming
behavior,
lethargy,
excessive
weight
loss,
abnormal
posture,
licking
or
biting
of
the
wound
area,
etc.
An
understanding
of
normal
species-­‐specific
behavior
is
crucial
in
evaluating
potential
abnormal
clinical
signs.
Signs
of
adverse
antibiotic
reactions
include
erratic
behavior,
diarrhea,
and
hunched
posture.
The
mice
will
be
closely
monitored
(especially
during
initial
introduction
of
antibiotic
and
dietary
provisions)
including
a
visual
assessment
of
both
physical
and
behavioral
health
characteristics
every
12-­‐hours
throughout
treatment
cycles
by
the
individual(s)
responsible
for
gavage.
These
individuals
will
take
careful
records
of
their
observations,
reporting
animals
experiencing
atypical
pain,
distress
or
behavior
to
Amy
Cooper
who
is
responsible
for
determining
an
appropriate
plan
of
action,
on
a
case-­‐by
case-­‐basis.
A
convenience
of
our
video
monitoring
system
is
that
because
we
utilize
security
system
cameras
there
exists
phone
and
computer
apps
allowing
for
24/7
monitoring
of
all
15
DVR
video
channels
for
any
obvious
signs
of
pain
and/or
distress.
This
is
an
efficient
method
to
closely
monitor
and
prevent
any
potential
complications
that
may
arise.

11. C.
Describe
the
management
plan
that
will
be
used
to
assess
and
treat
pain,
distress
and
discomfort
in
the
animals.
Include
any
special
procedures
that
will
be
used
(e.g.,
periodic
weighing
of
animals)
and
any
interventions
that
will
be
performed
to
relieve
pain,
distress
and
discomfort
in
the
animals
(e.g.,
analgesics,
antibiotics,
special
housing/bedding,
etc.).
The
animals
will
be
monitored
and
weighed
every
day
by
investigators.
If
observation
of
excessive
bloody
stools
or
anaphylaxis
occurs
as
a
result
of
antibiotic
treatments,
the
animal
will
be
immediately
removed
from
the
experiment
and
euthanized.
Amy
Cooper
and
Sumei
Liu
will
be
responsible
for
making
final
decisions
regarding
any
questions
involving
animal
discomfort.
D.
Describe
how
the
monitoring
of
animals
(e.g.,
daily
observations,
treatments
performed
by
research
staff)
will
be
documented.
Monitoring
records
must
be
readily
available
to
inspectors
and
IACUC
members
at
all
times.
Detailed
methodology
and
documentation
of
all
procedures,
animal
care
and
experimental
analysis
will
be
recorded
by
all
investigators
in
their
lab
notebooks
and
will
be
available
electronically
upon
request.
All
video
data
will
be
stored
for
post-­‐analysis
and
documentation
of
animal
monitoring
and
treatments
performed
by
researchers.
VII.
Euthanasia/Disposition
of
Animals
A.
Describe
the
specific
criteria
for
termination
of
animals
if
experiments
could
induce
chronic
disease,
tumors,
etc.
These
criteria
should
be
described
in
terms
of
tumor
size,
specific
animal
characteristics
or
behaviors,
weight
loss
changes,
observed
clinical
signs,
etc.
The
natural
endpoint
of
the
study
is
euthanasia
for
blood
and
tissue
harvest.
Experimental
protocol
should
not
cause
additional
pain
or
distress
to
the
animals,
nor
do
we
expect
any
complication
due
to
experimental
manipulations.
However,
should
any
of
the
mice
become
inactive,
lose
hair
coat,
not
respond
to
mild
stimuli
or
other
similar
behaviors
exhibited
on
the
day
of
the
experiment,
we
will
remove
the
mouse
from
our
experiment.
The
mouse
will
be
euthanized
by
the
procedures
described
previously.
Humane
endpoint
is
>30%
body
weight
compared
with
the
starting
weight
in
each
experiment.
B. Will
the
animal
be
euthanized
at
the
end
of
the
study?
X
Yes
–
Complete
the
tables
below
(Euthanasia
must
be
in
accord
with
the
AVMA
Guidelines
for
Euthanasia
of
Animals:
https://www.avma.org/KB/Policies/Documents/euthanasia.pdf
Specie
Drug/Method
Dose
of
Agent
if
applicable
Route
Mouse
CO2
Asphyxiation
NA
Inhalation
Second
method
of
euthanasia
for
assurance
of
death:
Specie
Drug/Method
Dose
of
Agent
if
applicable
Route
Mouse
Exsanguination
NA
Cut
through
the
diaphragm
to
open
the
thoracic
cavity
C. How
will
the
carcasses
be
disposed
of?
Carcasses
will
be
placed
in
opaque
bags
and
placed
in
the
freezer
in
room
0104
SHC
awaiting
disposal.

12. Appendix
E
Dietary
Manipulations
or
Fluid
Restriction
1.
Describe
any
dietary
manipulations
or
special
feeding
requirements:
T.D.
06414i
High-­‐fat,
Caloric
Adjustment
(60%
fat
kcal)
T.D.
08806i
Low-­‐fat,
Purified
Control
Diet
(10%fat
kcal).
In
many
cases
a
chow
(cereal
based
diet)
is
used
as
a
low-­‐fat
control
diet
for
a
purified
high-­‐fat
diet.
Chows
diets
contain
plant-­‐derived
ingredients,
thus
formulas
may
change
based
on
the
nutritional
composition.
Purified
ingredients,
on
the
other
hand,
are
highly
refined
and
contain
just
a
single
nutrient;
these
ingredients
have
little
variability
and
therefore
provide
consistency
between
batches.
There
are
numerous
differences
between
chows
and
purified
diets,
creating
countless
variables,
thus
making
it
difficult
to
interpret
the
results
when
these
diets
are
used
together
in
a
study.
In
addition,
chows
contain
plant-­‐based
compounds
such
as
phytoestrogens,
which
have
been
shown
to
reduce
the
degree
of
weight
gain.
For
these
reasons,
a
low-­‐fat
purified
diet
containing
the
same
ingredients
with
a
closely
matched
composition
to
the
high-­‐fat
formula
should
be
used
as
a
control
diet.
2.
Describe
length
of
time
animals
will
be
on
experimental
diet:
For
the
entirety
of
their
stay
at
the
HSC
animal
care
facility.
3.
Will
animals
be
provided
less
than
ad
lib
fluids
or
drinking
water
for
experimental
reasons?
X
No
Yes
(provide
details
below
including
amount/day,
monitoring
of
animals,
criteria
used
to
determine
the
well-­‐being
of
animals
and
scientific
justification)
References
1)
Benveniste,
H.,
et
al.
2012.
A
paravascular
pathway
facilitates
CSF
flow
through
the
brain
parenchyma
and
the
clearance
of
interstitial
solutes,
including
amyloid-­‐beta.
Science
Translational
Medicine.
4
(147):
111-­‐122.
2)
Bonaz,
B.
2013.
Brain-­‐gut
interactions
in
inflammatory
bowel
disease.
G
astroenterology.
144:
36-­‐49.
3)
Cryan,
J.
2010.
From
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to
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the
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rain,
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A.,
et
al.
2007.
Pattern
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225-­‐233.
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Foster,
J.,
and
Neufeld,
K.
2013.
Gut–brain
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How
the
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influences
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and
depression.
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in
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Nedergaard,
M.
2013.
Garbage
truck
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Stilling,
R.
2014.
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Please
direct
additional
comments,
concerns
or
requests
to
either
Amy
Cooper
(acooper@uwlax.edu)
or
Jonathan
Lendrum
(lendrum.jona@uwlax.edu,
920.850.7883)