Berndt2_Protocol
Project
protocol
—
Contents
Procedure
(non-invasive)
for
unrestrained
plethysmography
(UP)
Workflow
and
sampling
Equipment
Reagents,
supplies,
and
solutions
Data
Procedure
(invasive)
for
the
forced
oscillation
technique
(FOT)
Workflow
and
sampling
Equipment
Reagents,
supplies,
and
solutions
Data
References
------------------------------------------
Whole
Body
Unrestrained
Plethysmograph
(performed
at
The
Jackson
Laboratory)
Workflow
and
sampling
Workflow
| Step |
Procedure
performed |
Methacholine
(mg/mL) |
Age
(wk) |
Data
collected |
1 |
Mice
are
acclimatized
within
the
plethysmography
chamber
for
at
least
15
min |
- |
9-12 |
- |
2 |
For
baseline
measurements,
six
30-second
data
logs
are
collected
|
- |
9-12 |
Inspiratory
and
expiratory
measurements |
3 |
For
inhalation
control
(aerosolized
saline)
measurements,
8-10
30-second
data
logs
are
gathered |
saline
only |
9-12 |
Inspiratory
and
expiratory
measurements |
4 |
For
inhalation
challenge
(aerosolized
methacholine
(MCh)
measurements,
8-10
30-second
data
logs
are
obtained
|
MCh
5,
10,
20
|
9-12 |
Inspiratory
and
expiratory
measurements |
Equipment
•
Buxco
whole
body
plethysmograph
(Model
#PLY3211,
Buxco
Research
Systems,
Wilmington,
NC)
Figures
1A
to
1C.
Unrestrained
plethysmograph
layout
for
high-throughput
phenotyping.
Reagents,
supplies,
solutions
•
1
mL
syringe
•
Drierite
drying
agent
•
Garbage
bags
•
Yellow,
labeled
hazardous
waste
disposal
bags
•
Buxco-supplied
screwdriver
•
Nalgene
L900,
diluted
2
to
4
mL
per
liter
of
water,
in
a
squirt
bottle
•
Paper
towels
•
Vaseline
•
Saline
•
Sample
(customer-supplied
test
agent
or
vehicle
solution
such
as
PBS,
etc.)
•
Distilled
water
•
Disposable
plastic
cups
•
Methacholine
(MCh,
Sigma–Aldrich
Inc.,
St.
Louis,
MO,
USA)
•
Associated
document:
Procedure
log
book
Procedures
(non-invasive)
for
unrestrained
plethysmography
(UP)
a.
Mice
are
placed
from
the
home
cage
into
a
plethysmograph
chamber.
b.
Each
mouse
is
subjected
to
a
standard
protocol,
which
consisted
of
the
following
procedures:
1)
Mice
are
acclimatized
within
the
plethysmography
chamber
for
at
least
15
min.
2)
Baseline
measurements
are
collected
as
six
30
s
data
logs.
3)
Inhalation
challenge
measurements
in
response
to
aerosolized
saline
or
increasing
concentrations
of
MCh
(5,
10,
20
MCh
dissolved
in
saline)
are
collected
as
eight
to
ten
30
s
data
logs.
Investigator's
Notes:
"In
order
to
ensure
that
each
mouse
received
the
same
MCh
exposure,
we
tested
the
efficiency
of
the
nebulizer
at
the
beginning
of
each
test
week
and
adjusted
the
time
of
exposure
to
deliver
the
same
MCh
concentration
to
each
animal.
When
the
nebulizer
was
new,
the
time
was
generally
2.5
min,
but
as
the
nebulizer
got
older
and
became
less
efficient,
the
time
was
often
extended.
When
the
time
required
was
more
than
3.5
min
to
achieve
the
same
MCh
dose,
the
nebulizer
was
discarded."
c.
Inspiratory
(Pi)
and
expiratory
(Pe)
pressures,
as
well
as
inspiratory
(Ti)
and
expiratory
(Te)
times,
are
measured.
Likewise,
tidal
volume
(Vt)
is
obtained
for
the
derivation
of
minute
ventilation
(MV).
d.
Based
on
the
recorded
measurements
for
Pi,
Pe,
Ti,
and
Te,
enhanced
pause
(Penh)
is
calculated
according
to
the
following
equation:
Penh
=
(Pi/Pe)
*
((Te-Tr)/Tr)
Where
Tr
is
the
relaxation
time
(i.e.,
the
time
interval
that
includes
63%
of
the
integrated
expiratory
pressure
signal).
e.
Additional
UP
parameters,
including
total
respiratory
time
(Ti
+
Te=
Ttot),
duty
cycle
(Ti/Ttot),
and
various
ratios,
such
as
inspiratory
flow
rate
(Vt/Ti),
expiratory
flow
rate
(Vt/Te),
and
overall
respiratory
flow
rate
(Vt/Ttot)
are
also
calculated
for
each
mouse.
For
additional
information
regarding
assessment
of
pulmonary
function:
Whole
Body
Plethysmograph
For
Bench
setup,
machine
start-up,
and
calibration.
Data
collected
by
investigator
|
Parameters
measured |
Short
name |
Baseline
measurement |
MCh
dose
(mg/mL) |
Calculated
response
index |
breath
frequency |
F |
saline |
5,
10,
20
|
slope |
inspiratory
time |
Ti |
saline |
5,
10,
20
|
slope |
expiratory
time |
Te |
saline |
5,
10,
20
|
slope |
tidal
volume |
Vt |
saline |
5,
10,
20
|
slope |
minute
ventilation |
MV
(Vt*F) |
saline |
5,
10,
20
|
slope |
enhanced
pause |
Penh |
saline |
5,
10,
20
|
slope |
total
respiratory
time |
Ttot
(Ti+Te) |
saline |
5,
10,
20
|
slope |
duty
cycle |
Ti/Ttot |
saline |
5,
10,
20
|
slope |
inspiratory
flow
rate |
Vt/Ti |
saline |
5,
10,
20
|
slope |
expiratory
flow
rate |
Vt/Te |
saline |
5,
10,
20
|
slope |
respiratory
flow
rate |
Vt/Ttot |
saline |
5,
10,
20
|
slope |
*All
listed
parameters
are
available
as
MPD
measurements.
Definitions
and
calculations
Figure
2.
Schematic
and
graphic
illustration
of
lung
volumes
in
the
mouse.
Lung
volumes
and
lung
capacities
refer
to
the
volume
of
air
associated
with
the
various
phases
of
the
respiratory
cycle.
Note
that
in
Figure
2
above,
lung
capacities
are
inferred
from
lung
volumes
which
are
directly
measured.
Breath
frequency
is
the
number
of
breathing
cycle
(inspiratory
and
expiratory)
over
a
given
period
of
time.
Duty
cycle:
applies
to
UP
functions
which
are
intermittent
rather
than
continuous,
thus,
referring
to
the
ratio
of
the
time
UP
operates
to
its
total
cycle
time
(see
Chatburn
and
Mireles-Cabodevila,
2011).
Tidal
volume
is
the
amount
of
air
inspired
or
expired
from
the
lungs
under
normal
breathing
conditions.
Minute
ventilation
(minute
volume)
is
the
amount
of
air
inspired
or
expired
from
the
lungs
in
1
minute:
MV
=
Vt
x
F
---------------------------------------------
Forced
oscillation
technique
(performed
at
University
of
Pittsburgh)
Workflow
Workflow
and
Sampling
Step |
Procedure
performed |
|
|
Data
collected |
1 |
Mice
are
allowed
to
acclimate
in
their
new
facility
for
<3
wks |
0 |
9-12 |
- |
2 |
Mice
are
weighed,
anesthetized,
and
undergo
pre-treatment
testing |
0
(pre-treatment) |
9-12 |
pre-treatment
airway
resistance
and
elastance |
3 |
While
still
under
anesthesia,
mice
are
challenged
with
saline
and
tested
again
to
obtain
baseline
measurements |
0
(saline) |
9-12 |
baseline
airway
resistance
and
elastance |
4 |
Following
saline
challenge,
mice
are
challenged
with
4
incremental
and
consecutive
MCh
doses
(mg/kg
BW) |
1,
3,
10,
30
|
9-12 |
airway
resistance
and
elastance
|
Equipment
•
Balance
scale
•
Electric
hair
clippers
such
as
the
Oster
Finisher
Trimmer
(Cat.
#
76059-030,
Oster
Professional
Products,
McMinnville,
TN)
•
19
G
cannula
•
Small
rodent
dissecting/surgical
pack
(forceps,
scissors)
•
Computer-controlled
ventilator
(flexiVent,
SCIREQ,
Montreal,
QC,
Canada)
•
ultrasonic
nebulizer
(Aeroneb®
Lab
Nebulizer
System
by
Aerogen
Ltd.,
Galway,
Ireland)
Figures
2A
and
B.
Forced
oscillation
technique
setup
and
layout.
Figure
2C.
Schematic
diagram
of
the
flexiVent
system.
Reagents,
supplies,
solutions
•
5-0
USP
surgical
suture
•
methacholine
(MCh,
Sigma–Aldrich
Inc.,
St.
Louis,
MO,
USA)
•
pentobarbital
sodium
(Ovation
Pharmaceuticals
Inc.,
Deerfield,
IL,
USA),
Procedure
(invasive)
for
the
forced
oscillation
technique
(FOT)
a.
Mice
measured
with
the
invasive
FOT
are
age-matched
and
are
allowed
to
acclimatize
for
at
least
7
days.
b.
Body
weights
are
measured
for
the
calculation
of
anesthetic
dose.
c.
Mice
are
anesthetized
by
intraperitoneal
(i.p.)
injection
of
60
mg
per
kg
body
weight
pentobarbital
sodium.
d.
Following
adequate
anesthesia,
mice
are
positioned
ventro-dorsally
(supine)
for
tracheostomy
(see
Figures
3A-D
below).
e.
A
19G
cannula
is
gently
inserted
into
the
trachea
and
secured
using
5-0
USP
surgical
suture,
and
mice
are
attached
to
a
computer-controlled
piston
ventilator
(see
Figures
2A-D
above).
f.
Each
mouse
receives
a
tidal
volume
(Vt)
of
10
mL/kg
body
weight
at
120
breaths/min
with
0-cm
H2O
positive
end-expiratory
pressure
(PEEP)
(i.e.,
baseline
ventilation).
g.
Following
5
min
of
stabilization
one
deep
lung
inflation
(DI)
is
applied
to
ensure
uniform
lung
recruitment
and
to
normalize
to
the
same
volume
history
for
each
mouse.
This
DI
procedure
consisted
of
a
constant
1.0
mL
airflow
with
a
pressure
limited
to
20
cm
H2O
over
4
s
followed
by
a
passive
exhalation.
h.
Baseline
impedance
measurements
are
then
obtained
in
triplicates
during
regular
sinusoidal
ventilation.
i.
Following
baseline
measurements,
mice
are
maintained
under
baseline
ventilation
for
another
3
min,
after
which
a
2nd
set
of
impedance
measurements
are
taken
without
preceding
DI.
This
2nd
set
of
baseline
measurements
is
used
to
calculate
the
mean
baseline
values
per
strain.
j.
Saline
or
MCh
challenges
(1,
3,
10
and
30
MCh)
are
delivered
by
channeling
inspiratory
flow
from
the
ventilator
through
an
ultrasonic
nebulizer.
During
aerosol
challenge
the
piston
delivers
a
Vt
of
40
mL/kg
at
30
breaths/min.
k.
Following
consecutive
challenges
with
(1,
3,
10,
30
mg/mL)
MCh
the
piston
is
returned
to
delivering
a
Vt
of
10
mL/kg
at
120
breaths/min
and
impedance
measurements
are
taken,
which
is
followed
by
a
DI
to
re-open
the
lungs.
l.
Impedance
measurements
are
taken
every
5
s
within
the
period
of
30
to
60
s
after
each
aerosol
challenge.
Hence,
for
each
aerosol
challenge
6
to
12
impedance
measurements
are
reported,
of
which
is
calculated
as
the
mean
of
the
3
highest
values.
The
impedance
is
derived
from
the
signals
detected
by
the
ventilator
piston
volume
displacement
(i.e.,
flow)
and
cylinder
pressure
(i.e.,
pressure),
which
is
obtained
during
8
s
oscillatory
volume
perturbations
(i.e.,
Prime-8
perturbations).
The
perturbations
consisted
of
sine
waves
with
frequencies
ranging
between
0.5
and
19.75
Hz.
m.
The
impedance
is
then
fitted
to
a
constant-phase
model
of
the
viscoelastic
lung,
from
which
the
parameters
Rn
(i.e.,
central
airway
resistance)
and
H
(i.e.,
tissue
elastance)
are
obtained.
(Because
the
major
objective
of
the
Berdt2
project
is
to
compare
Rn
and
Penh,
the
detection
of
Rn
changes
due
to
MCh
exposure
is
of
primary
significance.)
n.
At
the
peak
of
Rn
response,
the
associated
H
parameter
is
also
recorded.
It
is
noteworthy
that
because
H
usually
peaks
after
Rn,
the
reported
H
values
may
not
reflect
peak
H
measurements.
Figures
3A
to
D.
Schematic
illustration
of
tracheostomy.
For
additional
information
regarding
invasive
assessment
of
pulmonary
function:
Flexivent
Data
collected
by
investigator
|
Parameters
measured |
Short
name |
Baseline
measurement |
Methacholine
(mg/mL) |
Calculated
response
index |
airway
resistance |
Rn |
saline |
1,
3,
10,
30
|
slope |
lung
elastance |
H |
saline |
1,
3,
10,
30 |
slope |
Definitions
and
calculations
Plethysmograph
is
a
device
for
measuring
changes
or
fluctuations
in
the
volume
of
air
within
lungs.
Pneumotachograph
(pneumotach)
is
a
device
for
measuring
the
instantaneous
flow
of
respiratory
gases
or
the
rate
of
air
flow
during
breathing.
Measurements
in
passive
subjects
For
passive
anesthetized
subjects,
the
single
compartment
model
is
commonly
fit
to
airway
opening
pressure
and
flow
obtained
from
a
body
plethysmograph
during
mechanical
ventilation
with
passive
expiration.
However,
it
is
advantageous
to
fit
the
model
to
data
obtained
during
a
Single-frequency
Forced
Oscillation
maneuver
where
amplitude
and
frequency
content
are
precisely
standardized.
This
is
the
technique
used
by
the
flexiVent.
In
both
cases,
measurements
represent
respiratory
system
mechanics
that
include
both
lung
and
chest
wall
components
(unless
the
chest
wall
is
surgically
opened).
Resistance
is
most
commonly
estimated
using
mathematical
model-fitting
techniques
such
as
Multiple
Linear
Regression
that
take
all
data
points
into
account.
1)
For
the
determination
of
airway
resistance,
a
constant
phase
model
is
fitted
to
the
data
from
multiple
frequencies
simultaneously
applied
at
the
airway
opening.
2)
After
each
of
the
5
challenges,
airway
resistance
(Raw)
in
12
cycles
of
30–60
s
is
recorded.
3)
Changes
in
Raw
as
a
function
of
the
log-transformed
MCh
concentration
administered
is
then
plotted.
R
=
Resistance
=
Dynamic
resistance
is
a
quantitative
assessment
of
the
level
of
constriction
in
the
lungs.
Calculation
of
the
slope
measures
for
Penh,
Rn
and
H
For
all
measures
Penh,
Rn
and
H,
the
strength
of
their
increase
is
determined
(Penh_slope,
Rn_slope
and
H_slope)
with
consecutive
increases
in
MCh
exposures.
For
each
mouse
respiratory
indexes
(Penh_slope,
Rn_slope
and
H_slope)
the
following
are
derived:
1)
Mean
Penh,
Rn
and
H
response
of
each
mouse
at
each
dose
(i.e.,
individual
value).
2)
Since
the
raw
data
together
are
not
normally
distributed,
each
dose
and
each
individual
value
are
log-transformed.
3)
To
the
log-transformed
data
of
MCh
concentrations
(5,
10,
and
20
for
Penh
and
1,
3,
10,
and
30
for
Rn
and
H)
the
response
index,
therefore,
is
defined
as
the
slope
of
the
line
of
best
fit.
|
Test age:
9-12wks
