Accurately Measuring Mouse Urinary Voiding Frequency and Volume Using The UroVoid System

Learn about new technology that enables noninvasive measurement of mouse urinary voiding frequency and volume from a recognized scientist in the field, Dr. Nathan Tykocki.

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Gerry Herrera, PhD

Accurately Measuring Mouse Urinary Voiding Frequency and Volume Using The UroVoid System

Catamount R&DMED Associates Inc.gerry.herrera@med-associates.com

• MED-CMG Small Animal Cystometry System, developed in early 2000’s

• Allows detailed urodynamic assessment lower urinary tract function in mice

• Very good phenotyping tool

• However, it requires surgical implant of bladder catheter

Measuring Bladder Function in Mice

Urinary bladder instability induced by selective suppression of the murine small conductance calcium-activated potassium (SK3) channel

Need for Noninvasive, Chronic Measurements of Bladder Function in Mice

1. Requires a cage designed for the mouse to live in.

2. Requires access to food and water.

3. Need to prevent food and water from contaminating measurements.

4. Need to separate feces from urine.

5. Need to collect and measure voided urine. Each void time and volume need to be captured.

Mouse Metabolic Cages

www.tecniplast.it www.hatterasinstruments.com www.labproductsinc.com www.colinst.com

Mouse UroVoid

✓ Allows chronic non-invasive measurements of voiding frequency in mice (up to 40 days)

✓ Monitor up to 12 mice simultaneously

click to learn more

Mouse UroVoid

✓ Accurate capturing of time and volume for every void

✓ Full separation of feces/urine

Voiding Micturogram (VMG)

Nathan Tykocki, PhD

Accurately Measuring Mouse Urinary Voiding Frequency and Volume Using The UroVoid System

Research Assistant ProfessorThe University of Vermontntykocki@uvm.edu

What We’re Going to Cover Today

1. Bladder function testing

a. What we learn from these experiments

b. Pros and cons of current techniques

2. Designing complex experiments using knockout mice

3. Sample data, protocols, and applications

4. The UroVoid System: Best practices

Bladder Function Testing

Invasive

– Cystometry: Void volume, intermicturition interval (IMI), NVCs; also a clinical tool of bladder function

Non-invasive

– Void Spot Assay: Void volume, void occurrence

– UroVoid System: Void volume, void frequency, IMI, fluid intake over time

Void Spot Assay

• Allow rodents to urinate on filter paper for 2-6 hours

• After drying, urine spots fluoresce when exposed to UV light

• Spot area, location and quantity can be determined using several image analysis software packages (e.g., ImageJ)

• Void spot area: measure of void volume

Yu W, et al. Am J Physiol Renal Physiol 306(11): F1296–F1307, 2014

Void Spot Assay

• Non-invasive

• Inexpensive

• Unimpeded voiding behavior (no catheter)

• Repeatable in the same animal at multiple time points

• Semi-quantitative measure of void volume

• Void frequency difficult to interpret

• False-negatives (urine spot overlap, feces, eating the paper)

• Short duration

Pros Cons

UroVoid System

• Hybrid of spot assay and metabolic cage

• Allow rodents to urinate on a scale for 24-48 hours (maybe longer?)

• Food/water mass can be measured before and after experiment

• Void frequency, volume and urine production rate can be determined with included analysis software

• Void mass: measure of void volume

Experimental Design: Knockout Mice

• Tamoxifen-inducible knockout mice are an indispensable tool to explore basic physiology

• Proper control experiments are key to their power

• This gets tricky with regard to voiding behavior

Experimental Design: Knockout Mice

✓ Time Controls

✓ Vehicle Controls

✓ Genetic Controls

✓ Age Controls

Tim

e

The Biggest Problem?

voiding varies a lot!

Solutions

1. Multiple measures from same animal (or lots and LOTS of animals per group)

2. Maximize data collected from each animal

3. Minimize animal loss post-surgery

4. Maintain tissue viability for further exploration

Limitations with other Methods

Cystometry

• Difficult to do in same animal pre/post tamoxifen

• Risk of infection over time

• Surgical intervention complicates complementary experiments (e.g., isometric force recordings)

• Cost-prohibitive

Void Spot Assay

• Short duration of measurements (~4 h)

• Incomplete measurement of voiding functions

• Safety concerns (tamoxifen in urine)

• Reproducibility can be a problem

In a Perfect World…

✓ Long-term measurements of voiding function

✓ Measures taken before, during and after drug/vehicle administration

✓ reproducible and objective measures of void volume, frequency, and behavior

✓ Non-invasive, yet simple to analyze data

✓ Complementary experiments after voiding behavior is possible

What Do The Data Look Like?

• Unpublished findings using a tamoxifen-inducible knockout mouse

• Tamoxifen delivered by 21-day time release pellet (10 mg/kg)

• Measurements taken 3 time points/ages

– 4 weeks: pre-pellet implantation (implanted at 6 wks)

– 8 weeks: during tamoxifen treatment

– 14 weeks: 5 weeks post-tamoxifen completion

First Test: Example Data

16 weeks of age. Sham (top) or pellet implantation (bottom) surgery 8 weeks prior to recording.

Knockout Mice Voiding Micturogram (VMG)

4 weeks of age. Prior to sham (top) or pellet implantation (bottom) surgery.

14 weeks of age. Sham (top) or tamoxifen treated (bottom).

Knockout Mice Voiding Micturogram (VMG)

Water Intake

• Relatively constant over time

• Small increase in water intake during tamoxifen treatment

• This increase returned to sham levels after treatment

4 wks

(pre

-TM

X)

8 wks

(dur

ing

TMX)

13 w

eeks

(pos

t-TM

X)

0

2

4

6

8

10

12

14

Mass (

g)

Water Intake

KO-Sham

KO-Tamoxifen

Voids Per Day

4 wks

(pre

-TM

X)

8 wks

(dur

ing

TMX)

13 w

eeks

(pos

t-TM

X)

0

2

4

6

8

10

12

14

Void

s p

er

Day

Void Frequency

KO-Sham

KO-Tamoxifen

• Steady decrease with age in sham mice

• Tamoxifen treatment increased void frequency

• This increase never returned to sham levels

• Average volume voided steadily increases in sham mice

• This coincides with the decrease in void frequency

• This increase in volume is much less pronounced in tamoxifen treated mice

4 wks

(pre

-TM

X)

8 wks

(dur

ing

TMX)

13 w

eeks

(pos

t-TM

X)

0.000

0.100

0.200

0.300

0.400

0.500

0.600

Mass (

g)

Average Amount Voided

KO-Sham

KO-Tamoxifen

Void Volume

IntermicturitionInterval

• Steady increase with age in sham mice

• Tamoxifen treatment reduced IMI

• This decrease never returned to sham levels

4 wks

(pre

-TM

X)

8 wks

(dur

ing

TMX)

13 w

eeks

(pos

t-TM

X)

0

50

100

150

200

250

300

Tim

e (

min

ute

s)

Intermicturition Interval

KO-Sham

KO-Tamoxifen

Urine Production Rate

• Defined as amount voided per duration since last void (IMI)

• Measure with the highest variability

• Relative differences between sham and tamoxifen-treated mice is unchanged over time

• Suggests normal kidney function (i.e. the changes in voiding behavior are not due to increased diuresis) 4

wks

(pre

-TM

X)

8 wks

(dur

ing

TMX)

13 w

eeks

(pos

t-TM

X)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Rate

(m

g/m

in)

Urine Production Rate

KO-Sham

KO-Tamoxifen

What Next?

• Cystometry

• Histology/molecular biology

• Isometric force recordings

• Ex vivo afferent nerve recordings

• Any additional techniques to explore the scientific questions at hand

UroVoid System: Best Practices

✓ “Training” does not appear to be necessary

✓ Proper cleaning between experiments is imperative

✓ Cage-mates work best

✓ Quiet, climate-controlled place for entire duration

Given the duration (48+ hours), the little things count:

✓ A quiet humidifier and lighting controls

✓ Turning off the monitor and preventing computer sleep/ screen saver

✓ UPS power supply suggested (if your building is as old as mine)

UroVoid Systems for Rats

• Uses standard metabolic cages

• Cages for rats < 300 grams, and > 300 grams body weight

• Urine collected in special funnel – minimizes evaporation

click to learn more

✓ Very stable voiding patterns

✓ Clear circadian patterns

UroVoid Systems for Rats

Nathan Tykocki, PhDResearch Assistant ProfessorThe University of Vermontntykocki@uvm.edu

Gerry Herrera, PhDCatamount R&DMED Associates Inc.gerry.herrera@med-associates.com

Thank YouFor additional information on the products and applications presented during this webinar, please visit www.med-associates.com