An essential webinar for preclinical scientists that wish to learn how to integrate hemodynamic, respiratory and neurological measurements to study multiple biological systems simultaneously while benefiting from more efficient data collection and workflow in the laboratory. In this case study webinar sponsored by Data Sciences International, Dr. Brian Roche of Charles River Laboratories and Jason Payseur of GlaxoSmithKline discuss advantages and challenges pertaining to the combination of physiologic monitoring technologies to collect respiratory, cardiovascular and neurological endpoints from a single animal subject. Specifically, Dr. Roche presents an evaluation of the AllayTM restraint technology utilized in DSI Respiratory solutions versus other commonly used methods. Complimented with implantable telemetry, Dr. Roche shows how he examined the effects of each method on various cardiopulmonary parameters and discusses the benefits and challenges associated with the use of the AllayTM restraint. Jason Payseur presents his assessment of a novel rodent model that examines cardiovascular, respiratory and neurobehavioral endpoints at the same time. He investigates the surgical feasibility of this model and tests its reliability in measuring multiple physiologic endpoints using tool compounds with known physiological effects, caffeine and chlorpromazine.

1. Combining Cardiovascular, Respiratory and
Neurobehavioral Endpoints for Efficient
Study Design
Brian M. Roche
PhD, DSP, DABT
Director of Safety
Pharmacology,
Charles River Laboratories
Jason D. Payseur
Senior Scientist – IVIVT, MSD
Safety Pharmacology US
GlaxoSmithKline

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3. Combining plethysmography, telemetry and Allay™
restraint technology for the use in safety pharmacology
and inhalation toxicology environments
Brian M. Roche PhD, DSP, DABT
Director of Safety Pharmacology,
Charles River Laboratories,
Ashland, OH
Copyright 2016 B.M. Roche, Data Sciences International and InsideScientific. All Rights Reserved.

4. Agenda
1. Allay Restraint Technology
• Overview of device
• Acclimation process
2. Allay restrainers vs. nose only
cone restraint tubes
• Acclimation data
• Hemodynamic telemetry
data
3. Plethysmography and
Telemetry: Allay vs Head Out
• Hemodynamic assessment
• Respiratory function
• Resistance and compliance
4. Dosimetry approaches with
Allay plethysmography
5. What we’ve learned from
conducting this pilot study

5. Allay Restraint Acclimation
The Process

6. Allay Restraint Technology
1 Allay restraint tube
2 Neck clip
3 Plethysmography bell
4 Nose seal1
2 3
4

7. Important to allow for adequate time
prior to study to acclimate the rats to the
restraint device
Introducing the allay restraint tube and
the rat either through placing the
restraint tube or “sled” portion of the
device into the home cage and/or
manually placing and holding the rat
inside the device.
Allay Restraint and Acclimation

8. After the initial familiarity with the device,
the next step was to introduce the restraint
portion of the tube.
This was conducted by manually placing and
holding the rat in the device without the
neck clip.
Allowing the rat to extend its head and neck
out through the front of the device while it
explored its surroundings.
Allay Restraint and Acclimation

9. The next step is to determine the
appropriately sized “clip” for the size of the
rat’s neck. This process starts as an
extension of the manual restraint of the rat
within the device.
Each time the rat extends it’s neck through
the “clip” area, slowly place the neck clip
over the rat’s neck. This step requires
patience as restraining the rat too early may
set you back a step in acclimation.
Allay Restraint and Acclimation

10. Once the appropriate sized neck clip is
in place the rat will begin acclimating to
this step of the procedure.
Allay Restraint and Acclimation

11. Once the rat has been introduced and the
appropriate neck clip has been determined,
acclimation to the allay restraint technology
for respiratory function beings. Depending
upon your study design, the next step in
acclimation is to add the plethysmography
bell.
This step is required if collecting respiratory
function. At this point the restraint is
similar to a head out chamber without a
neck seal. This step is critical in the
acclimation process for tail placement and
temperature regulation.
Allay Restraint and Acclimation

12. Nose and mouth seal.
The nose seal can also be placed on the
restraint tube without the
plethysmography bell for inhalation
delivery of test articles/chemicals.
The rat will react once the face whiskers
are stimulated but will calm down.
Additionally, the nose will be positioned
directly in the center of the nose seal.
Allay Restraint and Acclimation

13. Putting it all together.
The device is functioning as a nose only
plethysmography chamber for inhalation
delivery. This piece is also critical for
placement on the inhalation tower.
The acclimation will be conducted in steps,
with increasing duration. Once the entire
Allay restraint device is assembled around
the rat, start building up acclimation time to
cover the time that the rat will be restrained
on study.
Allay Restraint and Acclimation

14. Acclimation: Allay restrainers
vs. nose only restraint cone
HD-S21 Telemetry – Hemodynamic data

15. Allay Restraint vs. Nose Only Restraint Cone

16. Heart rate
reduced
initially in Allay
restrainers

17. No change in
blood pressure
between the
two methods
of restraint

18. EVERY STEP OF THE WAY16
Slight increase
in body
temperature,
expected with
no bias flow
and tail
enclosed in
Allay restraint

19. Plethysmography and Telemetry:
Allay vs. Head Out Restraint
Hemodynamic Assessment, Respiratory Function, Resistance and Compliance

20. Allay Restraint vs. Head Out Plethysmograph

21. Reduction in
heart rate
through first
60 minutes for
Allay restraint
Allay Head Out

22. No change in
systolic blood
pressure
Allay Head Out

23. Slight
reduction in
diastolic blood
pressure for
initial 60
minutes in
Allay restraint
Allay Head Out

24. Slight
reduction in
mean arterial
blood pressure
for Allay
restraint
Allay Head Out

25. Slight
reduction in
body
temperature
for the Allay
restrain
Allay Head Out

26. Decrease in
respiratory
rate for Allay
restraint
Allay Head Out

27. Increase in
tidal volume
with Allay
restraint
Allay Head Out

28. No change in
minute volume
Allay Head Out

29. No change in
lung
compliance
Allay Head Out

30. No change in
lung resistance
Allay Head Out

31. Dosimetry Approaches With Allay
Plethysmography
Inhalation drug delivery

32. Standard fit for the Allay restraint
system with a conventional nose only
inhalation system.
Inhalation tower designed with a
support ring to handle the size and
weight of the Allay restraint.
Allay Technology and Inhalation Tower

33. Telemetry hemodynamic and intrathoracic
pressure data were collected in combination
with respiratory function by placing a DSI
RPC-1 small animal receiver in close proximity
to the animal instrumented with HD-S21
transmitter.
Adequate separation of the animals
throughout the inhalation tower, reduces the
risk of cross-talk.
Allay Restraint and Telemetry

34. What we’ve learned from conducting
this pilot study…

35. Pros…  Ability to combine inhalation and dosimetry
 Ability to dose via inhalation (nose only cone)
 Ability to collect dosimetry (head out chamber)
 Potentially less stressful (nose cone and head out)
 Plethysmography - no thoracic compaction of rat vs head out
 Tight seal of device when mounted on inhalation tower
 Spacing collar for proper placement of head chamber and nose seal
 No physical obstruction against the throat (head out chamber)
 Dual chamber (reference) reduces variability of ambient pressure
changes in the collection environment (head out chamber)

36. Cons…  Nose seal
o Destruction by rat
o Ability to replace nose seal (loss of data)
 Acclimation process is time consuming
 Design
o Heavy (ring design of inhalation tower)
o Rolls (acclimation)
o Position of pneumotach and transducer ports
(Obstructed by tail/urine/feces)

37. Acute Respiratory Disorders
• Respiratory Depression
• Respiratory Syncytial Virus (RSV)
• Acute Respiratory Distress Syndrome (ARDS)
• Mucociliary Clearance and Dysfunction
• Pneumonia
• Cough
• Tuberculosis (TB)
• Bronchiolitis/ Bronchitis
COPD
• Emphysema
• Chronic Bronchitis
• Model Development
• Treatment Assessment
Asthma
• Model Development
• Treatment Assessment
Lung Fibrosis
• Pulmonary Fibrosis
• Cystic Fibrosis
Safety Assessment
• Safety Pharmacology
• Toxicology
Select any of the links below to
learn more about Respiratory
Solutions offered by DSI.
www.datasci.com

38. Combined Cardiovascular, Respiratory, and
Neurobehavioral Telemetry Model in the Conscious Rat:
Jason D. Payseur
Senior Scientist – IVIVT, MSD
Safety Pharmacology US
GlaxoSmithKline
Copyright 2016 J.D. Payseur, Data Sciences International and InsideScientific. All Rights Reserved.
A Novel Approach to Study the Acute Physiological Effects of
Caffeine and Chlorpromazine Following Oral Administration

39. Background
• Rodent studies historically focused on a single physiological system
• Integration of cardiovascular, respiratory, and neurobehavioral assessments
would allow for more insight into how changes in one physiological system
impact the others
• Advances in recent years have allowed combined models in large animals (i.e.
JET/EMKA jacketed models)
• The release of a dual pressure catheter in a small animal model implant (model
HD-S21) by Data Sciences International (DSI) opens up an opportunity to test a
combined rodent model

40. Surgical Procedure
• Prior to study, rats were implanted with
a telemetry device (Model No. HD-S21,
Data Sciences International (DSI), St. Paul
MN).
• 1 pressure catheter was inserted into the
abdominal aorta and advanced to a
position caudal to the renal bifurcation.
Another pressure catheter was inserted
under the serosal surface of the
esophagus in the thoracic cavity.

41. • 4x4 Latin square cross-over design [7 days between each treatment]
• Male rats (CRL:WI(HAN)) instrumented with telemetry devices for the measurement of
arterial pressure, heart rate, ECG, respiratory rate, respiratory pressure, activity, and body
temperature
• Each rat received oral doses of vehicle (distilled water) and caffeine at 3, 12, and 24 mg/kg
• Each rat received oral doses of vehicle (distilled water) and chlorpromazine at 2, 8, and 16
mg/kg
• Arterial pressures, ECG waveforms, respiratory rate, respiratory pressure, and activity were
monitored continuously for 2 hours prior to dosing and up to 24 hours post-dose
Study Design

42. Dose dependant
increase in heart rate at
all dose levels up to 2
hours post dose.
AbsoluteDataChangeFromPre-dose
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
HeartRate
(beats/minute)
250
300
350
400
450
500
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
HeartRate
(beats/minute)
-50
0
50
100
150
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
24mpk: 67.8 bpm; 19.3%
12mpk: 18.7%
3mpk: 10.2%
Effect of Caffeine on Heart Rate

43. Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
RespiratoryRate
(breaths/minute)
30
60
90
120
150
180
210
240
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
RespiratoryRate
(breaths/minute)
-50
-25
0
25
50
75
100
125
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
24mpk: 61.9 bpm; 54.9%
12mpk: 30.0%
3mpk: 22.3%
Dose dependant increase
in respiratory rate at all
dose levels up to 2 hours
post dose.
AbsoluteDataChangeFromPre-dose
Effect of Caffeine on Respiratory Rate

44. Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
RespiratoryPressure
(mmHg)
-10
-5
0
5
10
15
20
25
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
RespiratoryPressure
(mmHg) -6
-4
-2
0
2
4
6 Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
No statistically significant
changes in pressure at
any dose level.
Effect of Caffeine on Respiratory Pressure
AbsoluteDataChangeFromPre-dose

45. Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Activity
(AUC)
0
2000
4000
6000
8000
10000 Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Activity
(AUC)
-4000
-2000
0
2000
4000
6000
8000
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
24mpk: 400%
12mpk: 489%
3mpk: 257%
Significant increases in
activity in a dose
dependant manner up to
3 hours post dose.
Effect of Caffeine on Activity
AbsoluteDataChangeFromPre-dose

46. Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Activity
(AUC)
0
2000
4000
6000
8000
10000 Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Activity
(AUC)
-4000
-2000
0
2000
4000
6000
8000
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
24mpk: 400%
12mpk: 489%
3mpk: 257%
Significant increases in
activity in a dose
dependant manner up to
3 hours post dose.
Effect of Caffeine on Activity
AbsoluteDataChangeFromPre-dose

47. Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
BodyTemperature
(DegC)
32
34
36
38
40
42
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
BodyTemperature
(DegC) -5
-3
-1
1
3
5 Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Small increases in body
temperature in the mid
and high doses up to 2
hours post dose, most
likely due to the large
increase in activity at
those times.
Effect of Caffeine on Body Temperature
AbsoluteDataChangeFromPre-dose

48. Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
HeartRate
(beats/minute)
250
300
350
400
450
500
Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (16 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
HeartRate
(beats/minute)
-50
0
50
100
150
Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (16 mg/kg)
16mpk: 66.1 bpm; 54.9%
8mpk: 66.7%
An unexpected increase
in heart rate in both the
mid and high doses for up
to 8 hours post dose (the
duration of the light cycle
after dosing). Heart rate
was comparable to
vehicle once lights went
out and rats became
more active.
Effect of Chlorpromazine on Heart Rate
AbsoluteDataChangeFromPre-dose

49. Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
RespiratoryRate
(breaths/minute)
30
60
90
120
150
180
210
240
Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (12 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
RespiratoryRate
(breaths/minute) -50
-25
0
25
50
75
100
125
Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (12 mg/kg)
No changes in respiratory
rate at any dose level.
Very unexpected!
Effect of Chlorpromazine on Respiratory Rate
AbsoluteDataChangeFromPre-dose

50. Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
RespiratoryPressure
(mmHg)
-10
-5
0
5
10
15
20
25
Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (12 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
RespiratoryPressure
(mmHg) -6
-4
-2
0
2
4
6 Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (12 mg/kg)
No significant changes in
respiratory pressure
immediately post dose,
though we do start to see
some depressed
pressures during the
night cycle (hours 9-21
post dose), especially in
the high dose.
Effect of Chlorpromazine on Respiratory Pressure
AbsoluteDataChangeFromPre-dose

51. Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Activity
(AUC)
0
2000
4000
6000
8000
10000 Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (12 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Activity
(AUC)
-4000
-2000
0
2000
4000
6000
8000
Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (12 mg/kg)
16mpk: -231%
Once again we see no
changes in activity until
the night cycle, were we
see some depression in
the high dose.
Effect of Chlorpromazine on Activity
AbsoluteDataChangeFromPre-dose

52. Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
BodyTemperature
(DegC)
32
34
36
38
40
42
Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (16 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
BodyTemperature
(DegC) -5
-3
-1
1
3
5 Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (16 mg/kg)
A dose dependant
decrease in core body
temperature from hours
2-14 post dose.
Effect of Chlorpromazine on Temperature
AbsoluteDataChangeFromPre-dose

53. Results (Caffeine)
• Rats dosed with 24 mg/kg of caffeine
showed the expected transient increase in
heart rate (67.8 bpm; 19.3%) as well as
increases in respiratory rate (61.9 bpm;
54.9%), and activity (400%). There was no
apparent change in respiratory pressure.
• Rats given 3 and 12 mg/kg of caffeine also
showed increases in heart rate (10.2%;
18.7%), respiratory rate (22.3%; 30.0%),
and activity (257%; 489%) in a dose
responsive manner
• Rats given chlorpromazine showed an
increase in heart rate (66.1 bpm; 19.9%)
and the expected decrease in activity (-
231%) at a dose of 16 mg/kg.
• There was no apparent change in
respiratory parameters following
administration of chlorpromazine at any
dose.
Results (Chlorpromazine)

54. Conclusions…
• Surgically feasible model
• Continuous 24 hour respiratory monitoring (time consuming)
• Reliable cardiovascular and neurobehavioral results for both caffeine and
chlorpromazine
• Expected changes in respiratory parameters seen in caffeine dosed rats
• Unexpectedly, a decrease in respiratory parameters was not seen in chlorpromazine
treated rats, thus further studies must be conducted to determine if the this
combined model is able to detect decreases in respiratory rate with confidence.

55. Acknowledgements & References
• Dennis J. Murphy
• Safety Pharmacology staff at GlaxoSmithKline
• All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment
of Laboratory Animals and were reviewed by the Institutional Animal Care and User Committee
either at GSK or by the ethical review process at the institution where the work was performed.
1. Sgoifo, A., et al., “Electrode positioning for reliable telemetry ECG recordings during social stress
in unrestrained rats.” Physiology and Behavior. 1996 Mar; 60(6):1397-1401. LINK
2. Lynch III, J. et al., “Comparison of methods for the assessment of locomotor activity in rodent
safety pharmacology studies”, Journal of Pharmacological and Toxicological Methods, 64 (2011)
74-80. LINK

56. Thank You:
Brian M. Roche
PhD, DSP, DABT
Director of Safety
Pharmacology,
Charles River Laboratories
Jason D. Payseur
Senior Scientist – IVIVT, MSD
Safety Pharmacology US
GlaxoSmithKline
For additional information on the
solutions presented in this webinar
please visit www.datasci.com