Role of Animal Models - American Geriatrics

Role of Animal Models

Matthew O. Fraser, PhD

9th Annual Bedside-to-Bench Conference:

Urinary Incontinence in the Elderly: A Translational Research

Agenda for a Complex Geriatric Syndrome

U13 Conference Series, Bethesda, MD

October 17, 2016

Division of Urology, Department of Surgery, Duke University Medical Center

Institute for Medical Research, Durham Veteran’s Affairs Medical Center

Disclosures • Current Funding

– NIDDK LURN Network

– VA RR&D SPiRE

– VA RR&D SPiRE

• Other financial relationships

– SAB for Amphora Medical

– Invited Speaker for Allergan

– Consultant for Synergy Pharma and InVivo Pharma

– Patent royalties from Lipella Pharma

• Conflicts of interest

– None

Outline • Animal models for basic and translational research

– Species differences in LUT anatomy and physiology

– Rodents as research models

– Animal models of aging

– Age-related changes in LUT function

• Measurement of LUT function

– Cystometric Measurement of the Lower Urinary Tract • The Micturition Cycle

• Open Cystometry

• Closed Outlet

– Metabolism Cage/VSOP

– LPP

• Conclusions

Animal models for basic and translational research

• Species differences in LUT anatomy and physiology

Muriform Rodents Humans

Female Urethra

Pelvis

Levator Muscles Work the Tail Form Pelvic Floor

Autonomic Ganglia Extramural Intramural

Sleep Patterns Nocturnal Diurnal



Muriform Rodents Humans



Special Circumstances - SCI



In quadrupedal animals, gravity directs urine to the ventral abdominal wall.

In bipeds, gravity directs urine through the outlet.








• Open Cystometry

• Closed Outlet


– LPP

• Conclusions


• Rodents as research models

– Rodents are more closely related to humans than dogs, cats or pigs !!!

10

11


• Rodents as research models

– Rodents are more closely related to humans than dogs, cats or pigs

– However, rodents are not always reliable as preclinical models for human

disease and the scientific literature is littered with examples of drugs that

worked well in animals but turned out to be ineffective in clinical trials on

humans.

– This is in part due to differences in

• Anatomy and physiology

• Drug metabolism

• Structure Activity Relationships (SAR) between species-specific receptor

modifications and a constant structure drug candidate

• Off-target effects that may contribute to species-specific outcomes

– It is also due, in part, to methodology and interpretation of results failing to

account for species differences








• Open Cystometry

• Closed Outlet


– LPP

• Conclusions


• Animal models of aging

– Aging (health span) is similar across species when normalized to life span

Mitchell et al., 2015. Ann Rev Anim Biosci 3:283-303



– NIA Rodents Available for Aging Research

Rats

M

ice

NIA Website. https://www.nia.nih.gov/research/dab/aged-rodent-colonies-handbook/strain-survival-information



– Comparison of results of interventions in mice and humans

Vanhooren and Libert, 2013. Age Res Rev 12:8-21








• Open Cystometry

• Closed Outlet


– LPP

• Conclusions



– Affects of Aging on LUT Function – Conscious cystometry in F344 rats

Ito et al., 2016. J Urol 196:1575-1583

6 moa 25-28 moa



– Affects of Aging on LUT Function – Conscious cystometry in F344 rats

– Combined results suggest more of an overactive bladder condition, as

functional bladder capacity would be expected to be decreased, NVC are

increased and compliance is low

– This is consistent with their gene expression studies which demonstrated

increases immune and inflammation pathways in the bladder and DRG

Ito et al., 2016. J Urol 196:1575-1583



– Affects of Aging on LUT Function – Anesthetized cystometry in C57BL6 mice

Smith et al., 2012. Am J Phys Reg Int Comp Phys 302:R577-R586

These data are consistent

with ageing-induced

underactive bladder !!!

Species difference,

conscious vs. anesthesia

difference, cystometric

technique difference ???


Atropine Hexamethonium Isoproterenol

Blood Pressure

Bladder Pressure

Control

• Affects of Aging on LUT Function – Anesthetized cystometry in 18 moa SD rats








• Open Cystometry

• Closed Outlet


– LPP

• Conclusions

23

Cystometry - The Micturition Cycle

Compliance = V/ P

Pressure Threshold

Maximum Voiding Pressure

Common Descriptors

24

Cystometry - The Micturition Cycle

Compliance = V/ P

Pressure at Volume Threshold

Opening Pressure

Better Descriptors

Cystometric traces during conscious, restrained cystometry in a chronic SCI rat – The

top trace is from the vehicle control period, while the bottom trace is from the period

following 100 µg/kg of CL-316,243.

Where is “Pressure” Threshold?

26

What is Maximal Voiding Pressure?

Conclusions about the actual voiding

contraction are not so straightforward.

Need to understand the anatomy of

the voiding contraction:

Phase I – Isovolumetric Contraction

Phase II – Entire LUT open to external

environment during peak detrusor

contraction

Phase III – Isovolumetric Relaxation

Pressure-Flow relationships can be

explored during Phase II

Maggi et al, 1986

Human OP and CP also Discernable

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Chan

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2:16:20 2:16:25 2:16:30 2:16:35 2:16:40 2:16:45

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Ph I Ph II Ph III

Easy Bladder Contraction

OP CP VP

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Ph I Ph II Ph III

OP CP

Ambiguous Bladder Contraction – Tonic EUS gives False OP*

* *

VP

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5/23/2006 12:30:42.383 PM

Ambiguous Bladder Contraction – “Missing” OP

Ph I

OP

Ph III

CP VP








• Open Cystometry

• Closed Outlet


– LPP

• Conclusions

Open Cystometry Protocol

Saline Infusion,

Bladder Pressure

EUS EMG Jugular

Carotid

EUS EMG

Transvesical Approach Saline Infusion,

Bladder Pressure Transurethral Approach

33

What is Bladder Capacity

Continuous vs. Single Fill Cystometry

• Continuous open cystometry is the current method of choice by many

researchers

• Allows for the determination of functional bladder capacity (FBC), as defined as

infusion flow rate x ICI or IMI

• However, it often underestimates true bladder capacity (TBC), which is best

determined by single fill cystometrograms

• By combining the approaches, as shown above, one can determine voiding

efficiency easily by the equation: %VE = mean FBC/TBC x 100

cy130710_fr130606a_+_fr130606b_DoD_SA1b - A-NF-H.adicht

Rat 1

IVP

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Rat 1

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7/10/2013 2:45:57.456 PM

Atropine

Control

Response to Drugs

• FBC decreases with atropine

• TBC increases !!! Decreased FBC due to decreased voiding efficiency.

If had only performed continuous open cystometry, might misinterpret effect as

mild irritation or sensitization of reflex voiding !!!

35

Response of the Bladder to Filling:

Biomechanical Considerations

• Rate dependency – slow strain causes lesser increase in force

than fast strain – or - rapid filling results in decreased compliance

• Time dependency – It takes longer to reach equilibrium pressure if

strain is faster

• Hysteresis – the pressure-volume relationship (force curve) is

different – Viscoelasticity!

Flow rate affects the compliance measurements!

P

t

P

V

Fill

Empty

Equilibrium

Coolsaet 1985

36

Response of the Bladder to Filling: Measurement System Considerations

• Flow rates matter not only to tissue

biomechanics, but also to recordings

– Resistance of the filling and recording catheter affects

the pressure baseline as well as the fidelity of

recording during filling

– Effects become worse with increased fill rate

37

cy100219_fr10113b.adicht

Transvesic

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Transurethral (cm

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2/19/2010 2:54:39.992 PM

Transvesical Filling T

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Baselines Detail

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Transureteral Filling cy100219_fr10113b.adicht

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2/19/2010 3:08:58.075 PM

Tra

nsvesic

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Response of the Bladder to Filling: Measurement System Considerations

• Placement of catheters may affect

dynamic active measurements

– The top-down contraction of the dome may

occlude the catheter tip in transvesical filling

and recording

40

Transvesical Filling T

ran

svesic

al

Traces are from transvesical double-lumen catheters with a

static internal lumen for pressure recording.

Arrows Point to Apparent Closing Pressures

41

Transvesical Filling – False CP T

ran

svesic

al

Tra

nsu

reth

ral

False closing pressures (red arrows) may be due to bladder contraction

from top-down, creating transient seal around transvesical

filling/recording catheter tip








• Open Cystometry

• Closed Outlet


– LPP

• Conclusions

Simultaneous Isolated Bladder and Urethra

• Fraser MO, Flood HD, de Groat

WC, 1995, Journal of Urology, 153:

461A.

• Jung SY, Fraser MO, et al., 1999,

Journal of Urology, 162: 204.

• Kakizaki H, Fraser MO, de Groat

WC, 1997, American Journal of

Physiology, 272: R1647.

Bladder Pressure

Urethral Perfusion

Pressure

Rat UPP (3-Way System)

Urethral Saline Infusion

UPP Recording

Bladder Filling/Recording

EUS EMG Jugular

Carotid

Ureteral Drainage

Bladder Voiding

45

Isovolumetric IVP and UPP

Control NMB

UPP

IVP

• Allows for pharmacological dissection of Active State players in the

physiology of LUT function – External Urethral Sphincter contribution

• Note no change in the dynamic active responses of the bladder to

isovolumetric conditions (constant volume distension)

46

NO-Mediated Relaxation

Control L-NO-Arg L-Arg

• Allows for pharmacological dissection of active players in the

physiology of LUT function – Parasympathetic NO relaxation of

urethral smooth muscle.

• Note no change in the dynamic active responses of the bladder to

isovolumetric conditions (constant volume distension)








• Open Cystometry

• Closed Outlet


– LPP

• Conclusions

Metabolism Cage / VSOP


2 moa SD Rats, 3 days

Eriksson et al. 2004 Exp Physiol 89(4):427-433

7 woa ddy mice, 2 hr

Sugino et al. 2008 NUU 27:548-552

Metabolism Cage Voided Spots on Paper

Both measure functional bladder capacity, similar to a bladder diary for humans.

VSOP suffers from short sampling window and need to fix time due to diurnal variation.








• Open Cystometry

• Closed Outlet


– LPP

• Conclusions

Leak Point Pressure Measurements

• Leak Point Pressure Measurement

– Developed a method whereby bladder pressure could be increased

in an experimental animal without evoking a micturition reflex

• Does not require rat to cough or sneeze, animal can be supine or vertical

• Includes entire outlet (from bladder neck through meatus)

• Incremental increases in pressure

until bladder pressure exceeds

outlet resistance → Leak!

• Affected by both striated and

smooth muscle surgical and

pharmacological manipulations

50 Intravesical Pressure Clamp Trace

Bla

dd

er

Pre

ss

ure

Rat Leak Point Pressure








• Open Cystometry

• Closed Outlet


– LPP

• Conclusions

• Significant species differences in anatomy and normal physiology,

drug metabolism and SAR

• These differences must be addressed, understood and accounted

for in order to interpret experimental results properly

• All animal appear to age similarly (health span/life span)

• Species and/or approach differences may yield seemingly disparate

results, only by head-to-head comparison can these seeming

differences be parsed out

• A variety of techniques are available for measuring LUT function,

proper interpretation depends on in depth consideration of LUT

physiology and measurement technique interaction with it

Conclusions

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End

Role of Animal Models - American Geriatrics

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